THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 260, No. of 10720 ... · 10720 . Lipoprotein Lipase and Hepatic Triglyceride Lipase 10721 latter enzyme. None of these antibodies reacted

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 19, Issue of September 5, pp. 10720-10727.1985 Printed in U. S. A.

Purification and Characterization of Human Lipoprotein Lipase and Hepatic Triglyceride Lipase REACTIVITY WITH MONOCLONAL ANTIBODIES TO HEPATIC TRIGLYCERIDE LIPASE*

(Received for publication, September 28, 1984)

Chao-Fu ChengS, Andre BensadounSB, Thomas Bersotll, Jean S. T. HsuS, and Kristan H. MelfordS From the $Division of Nutritional Sciences and the Division of Biological Sciences, Cornell Uniuersity, Ithaca, New York 14853 and TThe Gladstone Foundation Laboratories for Cardiovascular Disease, Cardiouascular Research Institute, Department of Medicine, University of California, Sun Francisco, California 94140

Human lipoprotein lipase and hepatic triglyceride lipase were purified to homogeneity from post-heparin plasma. These enzymes were purified 250,000- and 100,000-fold with yields of 27 f 15 and 19 f 6%, respectively. Molecular weight determination by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents yielded M, of 60,500 f 1,800 and 65,200 f 400, respectively, for lipoprotein lipase and hepatic triglyceride lipase. These lipase preparations were shown to be free of detectable antithrombin by measuring its activity and by probing of Western blots of lipases with a monospe- cific antibody against antithrombin. In addition, probing of Western blots with concanavalin A revealed no glycoproteins corresponding to the molecular weight of antithrombin. Four stable hybridoma-producing distinct monoclonal antibodies (mAb) to hepatic triglyceride lipase were isolated. The specificity of one mAb, HL3-5, was established by its ability to immunoprecipitate hepatic triglyceride lipase catalytic activity. In- teraction of HL3-5 with this lipase did not inhibit catalytic activity. The three other mAb interacted with hepatic triglyceride lipase only after denaturation of the enzyme with detergents. The relatedness of these two enzymes was examined by comparing under the same conditions the thermal inactivation, the sensitivity to sulfhydryl and reducing agents, amino acid composition, and the mobility of peptide fragments generated by cyanogen bromide cleavage. The results of these studies strongly support the view that the two enzymes are different proteins. Immunological studies confirm this conclusion. Four mAb to hepatic triglyceride lipase did not interact with lipoprotein lipase in Western blots, enzyme-linked immunosorbent assay, and immunoprecipitation experiments. These immunological studies demonstrate that several epitopes of the hepatic triglyceride lipase protein moiety are not present in the lipoprotein lipase molecule.

Intravenous injection of heparin in humans and other ver- tebrates releases in the plasma at least two lipolytic enzymes:

* This work was supported by Grants HL-14990 and HL-24873 and was carried out in part at the General Clinical Research Center, San Francisco General Hospital with support (Grant RR00083) from the Division of Research Resources, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should he addressed.

lipoprotein lipase and hepatic triglyceride lipase. Lipoprotein lipase is the sole enzyme responsible for the hydrolysis of di- and triacylglycerol constituents of very low density lipopro- teins and chylomicrons (1, 2). I t functions as an exoenzyme in extrahepatic tissues on the lumenal surface of capillary endothelial cells (3). In cell culture experiments, it has been shown that lipoprotein lipase binds to endothelial cell heparan sulfate proteoglycans with an association constant of 0.7 X lo7 M” (4,5). The exact function of hepatic triglyceride lipase is still a matter of controversy. Intravenous injection of antibody against this enzyme into rats results in an increase of cholesterol and phospholipids in high and low density lipoprotein fractions (6, 7). These results led to the hypothesis that hepatic triglyceride lipase may play a role in the clearance of phospholipid and cholesterol associated with these lipopro- teins. Some investigators have also suggested a role of this enzyme in the metabolism of intermediate density lipopro- teins (8, 9).

The purification of human lipoprotein lipase and hepatic triglyceride lipase from post-heparin plasma has been a dif- ficult task. The difficulty arises from the low concentration of the two enzymes in the starting material and the presence of plasma proteins with similar physical and chemical properties. A report claimed that lipoprotein lipase and hepatic triglyceride lipase possessed identical amino acid composition, terminal amino acid residues, and tryptic peptide maps (10) and differed only in their carbohydrate moiety. However, Ostlund-Lindqvist and Boberg (11) clearly demonstrated that plasma lipase preparations purified from plasma by heparin- Sepharose chromatography are contaminated with antithrombin. In a subsequent report, Ostlund-Lindqvist (12) showed that these enzymes have significantly different relative amino acid composition and could be inhibited differentially by polyclonal antibodies. The recent development of hybridoma technology by Kohler and Milstein (13) has made it possible to obtain monoclonal antibodies (mAb’) which are excellent tools for revealing both distinctive and common structural features of proteins.

We report here methods for the purification to homogeneity of human lipoprotein lipase and hepatic triglyceride lipase and the characterization of four distinct mAb against the

The abbreviations used are: mAh, monoclonal antibody; SDS- PAGE, polyacrylamide gel electrophoresis in sodium dodecyl sulfate; ELISA, enzyme-linked immunosorbent assay; CHAPS, 3-[(3-cholam- idopropyl)dimethylammonio]-l-propanesulfonate-2H~0; PBS, phosphate-buffered saline (0.15 M NaC1, 10 mM phosphate, pH 7.4); HL- 1 and HL-3, identification code for two fusions of spleen cells from mice immunized with hepatic triglyceride lipase and myeloma cells; LPL, lipoprotein lipase; H-TGL, hepatic triglyceride lipase.

10720

Lipoprotein Lipase and Hepatic Triglyceride Lipase 10721

latter enzyme. None of these antibodies reacted with lipoprotein lipase. Furthermore, amino acid composition, cyanogen bromide mapping, thermal inactivation, and interaction with reducing reagents and sulfhydryl reagents confirm and extend the results of Ostlund-Lindqvist (12) demonstrating that these enzymes are distinct glycoproteins.

EXPERIMENTAL PROCEDURES'

RESULTS

Purification of Human Lipoprotein Lipase The results of a purification on 2500 ml of post-heparin

plasma are presented in Table I. Means f S.D. given in the text for various purification parameters were calculated from values obtained in five or six purifications.

Step Z: Heparin-Sephurose 4B Chromatography-Frozen post-heparin plasma samples were thawed in a 30 "C water bath and centrifuged at 22,000 X g for 20 min in 250-ml polycarbonate bottles. The precipitate was discarded. To each 100 ml of plasma, 67 ml of 0.65 M NaC1, 30% glycerol, 5 mM phosphate, pH 7.0, were added. The antiproteolytic agent, Trasylol (FBA Pharmaceuticals), was also included to a final concentration of 100 units/ml. This diluted plasma was loaded on two heparin-Sepharose 4B columns (5 X 15 cm) in parallel at a combined rate of 200 ml/h. The frozen plasma samples were thawed in small batches and loaded onto the columns within 4 h. The inclusion of NaCl in the diluted plasma decreases nonspecific binding of proteins (10). Glycerol was included since it has been shown to be an effective stabilizer of lipoprotein lipase (29). To enhance the effective capacity of the columns, the columns were washed after loading of 500 ml of solution with 200 ml of 0.3 M NaC1,30% glycerol, 5 mM phosphate, pH 7.0. Loading of diluted plasma was then re- sumed. After loading, the affinity matrix was transferred with 0.3 M NaC1, 30% glycerol, 5 mM phosphate, pH 7.0, to a liter jacketed funnel fitted on a 5-liter vacuum flask. Temperature was maintained at 4 "C. The gel was washed sequentially with the following buffers: 2.5 liters of 20% glycerol; 8 liters of 20% glycerol, 0.2% Triton X-100; 3 liters of 20% glycerol; 1 liter of 0.3 M NaC1, 30% glycerol. All of the above solutions contained 5 mM phosphate and were adjusted to pH 7.0. In the above washing protocol, it is essential that the column be free of NaCl when the Triton X-100 wash is initiated. In the presence of low NaCl molarities, Triton X-100 will elute significant amounts of lipolytic enzymes. The gel bed was washed at a rate of 2.5 liters/h. Following this wash procedure, the gel was transferred to a 5 X 30-cm column and eluted with a linear NaCl gradient between 0.5 and 1.5 M NaCl in 30% glycerol, 5 mM phosphate, pH 7.0, with a total volume of 1000 ml. As shown previously (lo), heparin-Sepharose 4B chromatography of post-heparin plasma yields two distinct peaks with lipolytic activity; lipolytic activity (hepatic triglyceride lipase) in the first peak is resistant to 1 M NaCl and elutes at 0.75 M NaC1, lipolytic activity (lipoprotein lipase) in the second is sensitive to 1 M NaCl and elutes a t 1.5 M NaCl (data not shown).

Step ZZ: Hydroxylapatite Chromatography-The catalyti-

"Experimental Procedures" are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-3021, cite the authmrs, and include a check or money order for $2.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

TABLE I Purification of human lipoprotein lipase (LPL) and hepatic

triglyceride lipase (H-TGL) from post-heparin plasma Enzymes were purified from 2500 ml of pooled post-heparin

plasma.

Purification step

LPL Plasma Heparin-Sepharose 4B Hvdroxvlapatite

H-TGL Phenyl-Sepharose 4B

Plasma Heparin-Sepharose 4B DEAE-Sephacel AcA 34 gel permeation

Protein Specific Re- Purif"

activity cov- cation erv factor

209,590 0.14 26.0 1,040 2.1 8,957 0.4 36,075

209,590 0.12 16.0 782 0.5 14,266 0.5 13,659

~~~

% -fold

100 95 7,430 64 63,980 46 257,680

100 48 6,520 30 118,880 26 113,830

cally active fractions with lipoprotein lipase activity from Step I were applied on a hydroxylapatite (Fast Flow, Calbi- ochem-Behring) column (2.6 X 11 cm) equilibrated with 1 M NaCI, 30% glycerol, 5 mM phosphate, pH 7.0. The column was washed sequentially with 500 ml of a buffer containing 30% glycerol, 5 mM phosphate, pH 6.8, then with 500 ml of 200 mM phosphate, pH 6.8, and finally with 500 ml of 30% glycerol, 5 mM phosphate, pH 6.8. The column was developed with a linear gradient between 5 and 200 mM phosphate, pH 6.8, in 0.5 M NaC1, 30% glycerol with a total volume of 500 ml. The column was eluted at a flow rate of 30-40 ml/h. Recoveries for this step for different preparations varied between 57 and 94%. All of the enzyme was bound during loading and none appeared in the washing.

Step ZZZ: Phenyl-Sepharose Hydrophobic Chromatography- The active fractions from the previous step were pooled, and solid (NH,),SO, (ultrapure, Schwarz/Mann) was added to a final molarity of 1 M. This fraction was applied to a phenyl- Sepharose CL-4B (Pharmacia Fine Chemicals) column (0.8 X 2 cm) equilibrated with 2 M NaCl, 30% glycerol, 5 mM phosphate, pH 7.0. The column was washed with a decreasing gradient of NaCl between 2 and 0.2 M in 30% glycerol, 5 mM phosphate, pH 7.0, in a total volume of 400 ml. A variety of eluting conditions were investigated. The best recoveries were obtained by elution with 4 mM SDS, 50 mM octyl-P-D-glucopyranoside in 30% glycerol. At these concentrations of detergents, lipoprotein lipase was not stable, and therefore 1-ml fractions were collected in tubes containing 1 ml of 50% glycerol. Recovery for this step averaged 64 f 24%. Active fractions were stored at -70 "C. At this temperature in the presence of 2 mM SDS, 25 mM octyl-P-D-ghcopyranoside, 40% glycerol, the enzyme is stable for over 1 year. The behavior of 800 pg of highly purified human antithrombin in this purification step was investigated. Antithrombin was monitored by direct protein assay (21). Eight-five per cent of the antithrombin was recovered in the column washing with the NaCl gradient, and no protein could be detected in the eluting phase. The mean overall recovery for the purification of lipoprotein lipase was 27 f 15%.

Purification of Hepatic Triglyceride Lipase Step Z: Heparin-Sepharose 4B Chromatography-The de-

tails for this purification step are described above. The first activity peak eluting at a molarity of 0.75 M NaCl was pooled and dialyzed against 4 liters of 3.6 M (NH&SO,, 5 mM phosphate, pH 6.5, for 18 h with one change of buffer. The precipitated hepatic triglyceride lipase was recovered by cen-

10722 Lipoprotein Lipase and Hepatic Triglyceride Lipase

trifugation for 1 h at 4 "C at 25,000 rpm in an SW 27 rotor (Beckman Instruments).

Step II: DEAE-Sephacel Chromatography-The precipitated hepatic triglyceride lipase was solubilized in 2 ml of 50 mM Tris-HC1, 30% glycerol, pH 7.2, and traces of (NH4),S04 were removed by dialysis in the same buffer for 3 h with buffer changes every 30 min. The enzyme fraction was applied on a DEAE-Sephacel (Pharmacia Fine Chemicals) column (2.6 X 10 cm) equilibrated with 30% glycerol, 50 mM Tris- HCl, pH 7.2. The sample was applied at a flow rate of 10 ml/ h. The column was washed first with 250 ml of equilibration buffer and then with 250 ml of Tris-HC1, pH 7.2. The enzyme activity was eluted with 0.2% Triton N-101,50 mM Tris-HC1, pH 7.2. Recovery for this step was 44 f 12%. Elution with salt gradients in the absence of non-ionic detergents resulted in lower purification factor and poor recovery of catalytic activity.

Step III: Ultrogel AcA34 Gel Permeation Chromatography- To the active fractions from Step I1 solid NaCl was added to a final concentration of 2 M. The fraction was concentrated by ultrafiltration with a UM-10 membrane (Amicon) to a volume of 2 ml. The sample was applied on a 1.6 x 100-cm column of Ultrogel AcA34 equilibrated with 2 M NaC1, 0.2% (v/v) Triton N-101, 0.1 M D(+)-galactose, 0.1 M 1-0-methyl- a-glucopyranoside, 50 mM Tris-HC1, pH 7.2. Fractions of 1.5 ml were collected at a flow rate of 5 ml/h. Active fractions were stored a t -70 "C. In several instances, inclusion of this purification step resulted in no improvement in specific activity. Step I11 was included since, in some purifications, very minor contaminants could be detected on overloaded (10 pg) polyacrylamide gels. The mean overall recovery for the purification of hepatic triglyceride lipase was 19 f 6%.

Purity of Lipase Preparations

Both lipoprotein lipase and hepatic triglyceride lipase ap- pear to be homogeneous by SDS slab gel electrophoresis (Fig. 1) utilizing the discontinuous Laemmli procedure (22) and the sensitive Fairbanks stain (20) or the silver stain procedure of Merril et al. (23). The apparent molecular weights were 60,500 f 1800 and 65,200 f 400, respectively, for lipoprotein lipase and hepatic triglyceride lipase. Antithrombin, a major plasma protein, has a high affinity for heparin-Sepharose columns and consequently is a common contaminant of lipase preparations. Antithrombin activity (28) was not detectable in either the highly purified lipoprotein lipase or hepatic triglyceride lipase.

The purity of the lipase preparations was also examined by detecting glycoproteins in nitrocellulose transfers of electrophoretically (24) separated samples of purified lipases. Gly- coproteins were located with the procedure of Clegg (26) by sequentially incubating the nitrocellulose blot with concanavalin A and catalytically active horseradish peroxidase. Bands corresponding to the molecular weights of lipoprotein lipase and hepatic triglyceride lipase were clearly visible (Fig. 2). No staining was present a t a migration corresponding to the molecular weight of human antithrombin which has a molecular weight intermediate between these enzymes. Western blot analysis of the purified lipase preparations with a goat antiserum against antithrombin confirmed the absence of antithrombin in the sample examined (Fig. 3). Similarly, probing of Western blot of antithrombin with HL1-1, HL1-2, HL3-5, and HL3-6 mAb to hepatic triglyceride lipase revealed no interaction (data not shown).

eK.7 1 2 3 4

1 2 3 4

FIG. 1. Polyacrylamide gel electrophoresis of human antithrombin, lipoprotein lipase, and hepatic triglyceride lipase. Left, Coomassie Blue-stained SDS-PAGE of protein standards (bovine serum albumin, ovalbumin, and lysozyme, 3 pg each) (lane I), human antithrombin (5 pg) (lane 2), human lipoprotein lipase (8.5 pg) (lane 3) , human hepatic triglyceride lipase (15 pg) (lane 4 ) . Right, silver-stained (22) SDS-PAGE of human hepatic triglyceride lipase (1 pg) (lane I ) , human antithrombin (1 pg) (lane 2), human lipoprotein lipase (0.8 pg) (lane 3), and protein standards (phosphorylase b (0.6 pg), bovine serum albumin (0.8 pg), ovalbumin (1.5 pg), carbonic anhydrase (0.8 pg) soybean trypsin inhibitor (0.8 pg), and n-lactalbumin (1.2 pg)) (lane 4 ) .

Properties of Liprotein and Hepatic Triglyceride Lipases Thermal inactivation of these enzymes a t 40 "C demon-

strates clearly different first order rates of inactivation (Fig. 4). The catalytic activities of both enzymes are sensitive to a variety of sulfhydryl-reactive compounds (Table 11). Sensitiv- ity to p-hydroxymercuribenzoate and to 5,5'-dithiobis-(2-nitrobenzoic acid) was similar. However, lipoprotein lipase was much more sensitive to inorganic mercurials like mercuric chloride than to the organic mercurial tested. Fifty per cent inhibition of lipoprotein lipase occurred at 0.5 p~ mercuric chloride uersus 1 mM with p-hydroxymercuribenzoate. Such a dramatic increase in sensitivity when comparing organic and inorganic mercurials was not observed with hepatic triglyceride lipase.

Lipoprotein lipase and hepatic triglyceride lipase catalytic activities were inhibited to different extent by four reducing agents (Table 11). The greatest differential reactivity was observed with cysteine. Fifty per cent inhibition of catalytic activity was observed with 3 p~ for lipoprotein lipase and 116 p~ for hepatic triglyceride lipase.

Both enzymes' catalytic activities were sensitive to diiso- propylfluorophosphate. Fifty per cent inhibition occurred a t 6 and 10 p~ for lipoprotein lipase and hepatic triglyceride lipase, respectively.

The relatedness of the two lipases was also examined by separating the peptides generated by cyanogen bromide cleavage by polyacrylamide gel electrophoresis (12 or 13.75% polyacrylamide) in the presence of 8 M urea and 0.1% SDS. Close inspection of the chromatograms obtained by scanning of the Coomassie Blue-stained gels shows that the finger prints for hepatic triglyceride lipase and lipoprotein lipase are clearly distinct (data not shown).

Amino acid analysis was conducted on three samples of each enzyme. Compositions are presented in Table 111. Sta- tistical differences in composition were observed for 10 amino


- 94,000 t.

- 43,000

- 30,000

FIG. 2. Western blot of hepatic triglyceride lipase, anu- thrombin, and lipoprotein lipase probed with concanavalin A. Hepatic triglyceride (H-TGL), antithrombin (AT), and lipoprotein lipase (LPL) (500 ng each) were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose sheets. The blot was then reacted sequentially with concanavalin A and horseradish peroxidase by the method of Clegg (26). Molecular weight standards are indicated on the right from top to bottom (phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and n-lactalbumin).

acids. The compositions are similar to those obtained by Ostlund-Lindqvist (11) for lipoprotein lipase and hepatic triglyceride lipase bands excised from polyacrylamide gels after electrophoresis.

Monoclonal Antibodies to Heaptic Triglyceride Lipase

A critical step in the selection of hybridoma producing mAb specific to hepatic triglyceride lipase was the developmeld of a sensitive ELISA assay which utilizes minimal amounts of the lipase antigens. The effects of the pH of the antigen coating buffer and the length of coating at 4 "C were evalu- ated. At a concentration of 50 ng of hepatic triglyceride lipase/ ml (200 pl/well), the optimum antigen binding efficiency was observed between pH 9 and 10. It was also observed that hepatic triglyceride lipase binding to the wells increased with length of incubation a t 4 "C up to 5 days. With these coating conditions, net absorbance reading of 0.20 at 490 nm could be observed with as little as 1 ng of the mAb with high affinity for hepatic triglyceride lipase. Throughout the screening proc- ess, controls without antigen and with control medium were included to correct for nonspecific interaction of antibodies with polyvinyl chloride plates.

Frozen hybridomas which were further characterized were those which following thawing would grow readily and exhibited 100% ELISA-positive wells upon cloning by limited di-

FIG. 3. Western blot of hepatic triglyceride lipase, antithrombin, and lipoprotein lipase probed with antibodies to antithrombin. Protein samples were resolved by SDS-PAGE and electrophoretically transferred to a nitrocellulose sheet. The nitrocellulose blot was reacted with goat antiserum (1:lOO dilution) and horseradish peroxidase-labeled rabbit anti-goat immunoglobulin by the procedure described under "Experimental Procedures." Lane 1 , hepatic triglyceride lipase (100 ng) after heparin-Sepharose 4B step; lane 2, hepatic triglyceride lipase (50 ng) after AcA34 gel permeation; lane 3, human antithrombin (50 ng); lane 4, lipoprotein lipase (50 ng) after phenyl-Sepharose CL-4B step; lane 5, lipoprotein lipase (100 ng) after heparin-Sepharose 4B step.

lution. Four distinct and stable hybridoma are described in this report, HL1-1, HL1-2, HL3-5, and HL3-6.

Gel Electrophoresis and Isoelectric Focusing To verify that the mAb were distinct immunoglobulins, the

affinity-purified mAb were analyzed by two electrophoretic procedures. Agarose gel electrophoresis of four mAb at pH 8.6 indicated that the mobilities of the mAb were distinct. The mAb HL1-2 migrated toward the cathode, whereas HL1-1, HL3-5, and HL3-6 migrated toward the anode with different mobilities. The HL3-5 mAb gave a very diffuse band which was observed with all preparations of this clone (data not shown).

Isoelectric focusing of the four clones between pH 3.5-10 and pH 4-7 revealed distinct and reproducible patterns of isoforms for each clone (data not shown). Evidence of micro- heterogeneity has been reported by others for mAb to other antigens (30).

Immunoprecipitation and Inhibition of Hepatic Triglyceride Lipase Catalytic Actiuity

The four distinct mAb were examined for their ability to inhibit and immunoprecipitate hepatic triglyceride lipase cat-


4 1

I ‘ I 10 20 3 0 40 50 60

TIME (mm) FIG. 4. Thermal inactivation of lipoprotein lipase and he-

patic triglyceride lipase. Lipoprotein lipase (LPL) and hepatic triglyceride lipase (H-TGL) were incubated a t 40 “C in the presence of 0.5 M NaC1, 30% glycerol, 10 mM phosphate, pH 7.0.

TABLE I1 Reactivity of lipoprotein lipase and hepatic triglyceride lipase with

sulfhydryl reagents and reducing agents Lipoprotein lipase (LPL), after the phenyl-Sepharose step, and

hepatic triglyceride lipase (H-TGL), after the DEAE-Sephacel step, were preincubated for 20 min a t 20 “C in 120 pl of the respective column eluting buffers with the reagent indicated below. The specific activities of the two lipases were 14,500 peq/h/mg (lipoprotein lipase) and 13,600 peq/h/mg (hepatic triglyceride lipase). With the respective control buffers, this preincubattion resulted in less than 5% loss of catalytic activity. Two 5O-pl aliquots were assayed. Concentrations of the respective reagents are final concentrations in the 50O-pl assay mixture.

Reagents 50% inhibition

LPL H-TGL mM

p-Hydroxymercuribenzoate 1.1 0.7 5,5’-Dithiobis-(2-nitrobenzoic acid) 0.8 0.5 HgCL Dithiothreitol 0.0005 0.2 2-Mercaptoethanol 0.4 0.08 Cysteine 4 1.2 Reduced glutathione 0.003 0.116

0.002 0.057

alytic activity. In order to study the interaction of mAb with lipases under conditions close to equilibrium, the mAb and antigens were incubated for 15 h at 4 “C. TO prevent irrever- sible inactivation of lipase activity, the amphoteric detergent CHAPS was included in the incubation mixture. None of the four mAb studied could inhibit catalytic activity. Monoclonal antibody HL3-5 in combination with a second antibody (rabbit anti-mouse immunoglobulin) immunoprecipitated 90% of

TABLE 111 Amino acid composition of human hepatic triglyceride lipase and

lipoprotein lipase Three distinct preparations of lipoprotein lipase (LPL) (120, 75,

and 100 pg) and hepatic triglyceride lipase (H-TGL) (120, 75, and 100 pg) were hydrolyzed in 6 M HC1 a t 110 “C for 24 h and analyzed as described by Spackman et al. (32) with a Beckman analyzer. Mean values f S.D. for three determinations are given as well as the probability level for significant differences (p) in amino acid composition.

Amino acid H-TGL LPL P

molJ100 mol amino acids Aspartic acid/asparagine 7.13 f 0.3 9.28 f 0.3 0.001 Threonine 6.70 f 0.2 5.22 f 0.4 0.01 Serine 7.75 f 0.3 9.14 -t 0.3 0.01 Glutamic acid/glutamine 11.25 f 0.5 11.47 -t 0.5 Proline 4.98 f 0.7 5.29 ? 0.09 Glycine 9.43 f 0.2 10.08 f 1.1 Alanine 5.68 -t 0.1 6.57 f 0.01 0.001 Valine Methionine

5.84 f 0.9 5.95 +- 0.5 3.19 f 1.3 2.20 f 0.6

Isoleucine 7.02 f 1.3 5.10 +. 0.9 Leucine 9.54 f 0.3 7.64 -t 0.3 0.005 Tyrosine 2.26 f 0.02 3.16 -t 0.6 0.05 Phenylalanine 4.46 f 0.09 4.01 & 0.3 0.05 Histidine 3.95 z i 0.3 2.79 k 0.4 0.02 Lysine 5.87 f 0.2 6.86 +- 0.6 0.05 Arginine 4.97 f 0.05 5.21 +- 0.09 0.02

L H - TGL

> V

30

0- 0 1 2 3 . 5

HL3-5(vMl HL3-5(pMI FIG. 5. Immunoprecipitation of hepatic triglyceride lipase

and lipoprotein lipase by HL3-5. Highly purified hepatic triglyceride lipase (H-TGL) or lipoprotein lipase (LPL) were preincubated with HL3-5 as described under “Experimental Procedures.” After addition of rabbit anti-mouse immunoglobulins, the complexes were removed by centrifugation, and the supernatants ( O ” 0 ) and pel- lets (o”-o) were assayed.

the catalytic activity at a molarity of 4 pM. Fifty per cent inhibition occurred at less than 5 X M (Fig. 5). When HL3-5 was incubated with lipoprotein lipase in the presence of 1 M NaCl, no immunoprecipitation occurred. However, if the NaCl molarity was reduced to 0.1 M, nonspecific interaction occurred and 13.8% of lipoprotein lipase activity was immunoprecipitated a t a concentration for HL3-5 of 4 pM. In the presence of 0.1 or 1 M NaCl, rat hepatic triglyceride lipase, purified by a method similar to that described for the human enzyme, was not immunoprecipitated by HL3-5.

Probing of Western Blots of Heaptic Triglyceride Lipase and Lipoprotein Lipase

Purified lipoprotein lipase and hepatic triglyceride lipase were subjected to SDS-PAGE, electrophoretically transferred to nitrocellulose, and probed with HL1-1, HL1-2, HL3-5, and HL3-6. The blots were treated with peroxidase-conjugated rabbit anti-mouse IgG and processed as described under “Ex- perimental Procedures.” The staining patterns in Fig. 6 demonstrate that the four mAb studied reacted strongly with hepatic triglyceride lipase and not a t all with lipoprotein


HL-3-5 HL-3-6 HL-1-2 HL-1-1

FIG. 6. Western blots of hepatic triglyceride lipase and lipoprotein lipase probed with mAb to hepatic triglyceride lipase. Purified hepatic triglyceride lipase (If-TGL) (100 ng), lipoprotein lipase (LPL) (100 ng), and reference proteins were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose sheets. The blots were probed with four mAb to hepatic triglyceride lipase. The reference proteins were stained with Coomassie Blue. The stained bands in the lanes which received hepatic triglyceride lipase correspond to a molecular weight of approximately 65,000.

lipase. The observed stained bands corresponded to a molecular weight of approximately 65,000, identical to that of purified hepatic triglyceride lipase. The absence of reactivity of mAb to hepatic triglyceride lipase with lipoprotein lipase was confirmed in ELISA assays where microtiter plates were coated with either enzyme (data not shown).

DISCUSSION

This report describes for the first time the purification of human hepatic triglyceride lipase and lipoprotein lipase to homogeneity and the production of mAb to the former enzyme. The purification of human lipases in post-heparin plasma has been arduous because of the low concentrations of these enzymes in post-heparin plasma and the occurrence at relatively high concentrations of other plasma proteins with molecular properties similar to those of hepatic triglyceride lipase and lipoprotein lipase. There are more than 20 plasma proteins which have been reported (31) to bind tightly to heparin-Sepharose 4B, a matrix commonly employed as a single purification step for the purification of plasma lipases. Antithrombin present in plasma at a concentration of 0.2 mg/ ml has been a common contaminant in purified plasma lipase preaprations. Ostlund-Lindqvist and Boberg (11) were the first authors to call attention to the contamination with antithrombin of plasma lipases purified by a single step on heparin-Sepharose columns. In this report, we have verified

that hepatic triglyceride lipase and lipoprotein lipase preparations were not contaminated with antithrombin by measuring its activity and by Western blot analysis of purified lipases with antibodies to antithrombin. In addition, probing of West- ern blots with concanavalin A revealed no glycoproteins corresponding to the molecular weight of antithrombin. The novel chromatographic step employed in the purification of lipoprotein lipase was the hydrophobic step on phenyl-Seph- arose CL-4B. In this step, traces of antithrombin left after the hydroxylapatite chromatography were removed. The phenyl-Sepharose matrix exhibits a high capacity and tight affinity for lipoprotein lipase. As little as 1 ml of matrix could be employed to quantitatively bind the enzyme purified from 2500 ml of post-heparin plasma when employing the protocol described. The addition of salts in the loading solution en- hanced binding of lipoprotein lipase to phenyl-Sepharose CL- 4B columns, confirming that hydrophobic interaction is the basis for the binding of the enzyme. Low ionicity phosphate or Tris-HC1 buffers or distilled water were not effective in eluting the enzyme. The best recoveries of catalytic activity were obtained with elution with 4 mM SDS, 50 mM octyl-P- D-glucopyranoside in 30% glycerol and immediate dilution of the eluate with an equal volume of 50% glycerol. The purification procedure for hepatic triglyceride lipase was improved over those previously published (10, 12) by the inclusion of a DEAE-Sephacel chromatographic step. The purification factor for this step was 17 h &fold and the recovery was 44 f 12%. When the protocol described was employed, binding was quantitative. A relatively low pH, pH 7.2, for the equilibrating buffer was chosen to limit binding of contaminating proteins. Elution of enzyme activity occurred with 0.2% Triton N-101, 50 mM Tris-HC1, pH 7.2, in the absence of added salt, sug- gesting that at pH 7.2 hydrophobic interactions are significant in the binding of hepatic triglyceride lipase to DEAE-Sepha- cel. The enzyme could also be eluted with 0.075 M NaCl, 50 mM Tris-HC1, pH 7.2, in the absence of detergent. In this instance, the elution profile exhibited tailing and poor recoveries were observed.

The relatedness of hepatic triglyceride lipase and lipoprotein lipase was systematically examined by comparing amino acid composition, the thermal inactivation, the sensitivity to sulfhydryl and reducing reagents, and the electrophoretic mobility of peptide fragments generated by cyanogen bromide cleavage. Statistically significant differences between the two lipases were observed for the per cent composition of 10 amino acids. The present results confirm the observations of Ostlund-Lindqvist (12) for acidic and neutral amino acids and demonstrate that the basic amino acids, histidine, lysine, and arginine, which were not analyzed by these authors are also present in different proportions in the two lipases. By these criteria, the results strongly support the notion that the two enzymes are different protein moieties. Utilizing polyclonal antibodies to human hepatic triglyceride lipase and bovine milk lipoprotein lipase and inhibition of catalytic activity as a criterion for antigen-antibody interaction, Ostlund-Lind- qvist (12) observed no cross-reactivity between the two lipases. This conclusion was also strengthened by the cross- reactivity studies of hepatic triglyceride lipase and lipoprotein lipase with four distinct mAb to hepatic triglyceride lipase. The specificity of mAb HL3-5 for this lipase was soundly established since it could immunoprecipitate catalytic activity. The three other mAb, HL1-1, HL1-2, and HL3-6, did not immunoprecipitate catalytic activity. However, they strongly interacted with hepatic triglyceride lipase in Western blot assays following denaturation of the enzyme with SDS. Prod- ucts of the horseradish peroxidase-catalyzed reaction yielded


bands with mobilities identical to that given by HL3-5 and corresponded to the molecular weight of highly purified hepatic triglyceride lipase, The epitopes recognized by HL1-I, HL1-2, and HL1-3 must not be accessible in the native catalytically active enzyme, and denaturatization of hepatic triglyceride lipase is necessary for interaction with these mAb to occur. The four mAb did not interact with lipoprotein lipase in Western blot and ELISA assays. In Western blot studies with endoglycosidase F-treated enzyme, we have shown that deglycosylated hepatic triglyceride lipase main- tains its reactivity with the four mAb ~tudied .~ Therefore, the epitopes for these mAb are present in the protein moiety and are not dependent on the carbohydrate moiety for their expression. The immunological studies with four distinct mAb demonstrate that several domains of the protein moiety of hepatic triglyceride lipase are not present in the lipoprotein lipase molecule.

Acknowledgments-We express our appreciation to Thomas Sori- ano for excellent technical assistance and to Connie Ingraham for her help with t.he manuscript.

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Supplementary material to

PURlFlCATlON AND CHARACTERIZATION OF HUWN LIPOPROTEIN LIPASE AN0 HEPATIC TRIGLYCERIDE LIPASE: R E A C T I V I T Y YITH MONOCLONAL ANTlBOOlES TO HEPATIC TRIGLYCERIDE LIPASE

Chaa-Fu Chenq. findre Benradaun. Thomas Berrot . Jean 5.1. HSU and K r i s t a n H. l l e l f o r d

EXPERIMENTAL PROCEDURES

E n z p p u l i f i c a t i o n s - Post-heparin plasma was o b t a i n e d a f t e r O b t a i n i n g i n f o m d Consent frm m l e and female healthy volunteers. After an overn ight fast , subjects received an i n t r a - venous i n j e c t i o n O f 100 un i t s o f hepar in (po rc ine i n tes t i ne , E l k ins -S inn I n c . C h e r r y H i l l . N J I l k g o f body welght . F ive minutes la ter approx imate ly 250 ml O f plasm; was i o l l e c t e d by plasmapheresis and s t o r e d i n p l a s t i c bags a t -7O'C. P u r i f i c a t i o n s were c a r r l e d Out on 2000 rnl t o 2500 m l o f par t -hepar in plasma.

phosphate, pH 7.4 (PBS) UaS emu ls i f i ed w i th an equal volume O f complete Freund's adjuvant I m u n i r a t i o n ~ H l g h l y p u r i f i e d H-TGL (IO ug O f p r o t e i n 1 i n 100 u1 O f 0.15 M NaCl. 10 m

(DifCO Laborator ies) and i n j e c t e d i n t r a p e r i t o n e a l l y i n t o BALBIc mice (Jackson Laboratories). Th i s i n jec t i on was fo l lowed by b iweekly booster in ject ions wi th 10 ug i n i n c m p l e t e F r e u n d ' s ad juvant over d two m n t h p e r i o d . E i g h t , f i v e and one day before fusion the mice received 10 ug of H-TGL i n PBS.

muse spleen Cel ls w i t h m y e l m P3X-63 Ag8 for the HL-1 f u s i o n and w i t h t h e n y e l m SP210 f o r Cel l fus ion. h y b r i d m p r o d u c t i o n and c l o n i n g - Hybr idma were produced by the f u r i o n of

the HL-3 fusion. Fusion WaS Derfo-d by the 0Cner.l DrOcedure d e r c r i k d bv Kwan e t a l . 1141 w i t h c r i t i c a l m d i f i c a t i o n r i n t h e p r o t o i o 1 i n i i c a t e d bela. s p l i i n c e i l s ~ k - &i&- c e i l s ' were fused a t a Fat io o f 1011 i n 1 a1 of 502 polyethylene glycol 1000. I" the HL-3 fusion, t h e r a t i o o f c e l l s t o m y e l m a c e l l s was 2 l l . The polyethylene glycol treatment w s l i m i t e d to 90 Sec. The fusion was stopped by addit ion O f 20 m l Of Dulbecco's modif ied Eagle's medium as f011011: One ml i n 30 Sec; three ml i n 30 Iec; and 16 ml i n 1 mi". Fused c e l l s were p l a t e d i n t o f i v e . 9 6 - 4 1 1 p l a t e r which had received 24 hours before a feeder layer O f m u s e i n t r a - per i tonea l Mcrophages a t a d e n s i t y of 4 a 103 c e l l s / w e l t . en days aftcr. fusion, media were tes ted fo r PrOdUCtiOn Of anti-H-TGL Speci f ic I g G by an ELISA assay described b e l a . Hybridoma frm P o s i t i v e w e l l s were c l o n e d b y l i m i t i n g d i l u t i o n ( 1 5 ) u n t i l a11 c e l l c o n t a i n i n g w e l l s were

Hybridma which weTe character ized were those which could be Cul twed readi ly af ter thawing p o s i t i v e i n two successive c lon ings. Hybridoma were stored frozen in l i q u i d nitmgen,

of an t ibody-conta in ing asc i tes f lu id , p r is tane (A1dr ich) -p r imed BALBlc n i c e were i n j e c t e d and which exhib i ted 1002 p o s i t i v e we111 i n c l o n i n g by l i m i t i n g d i l u t i o n . FOP the product ion

w i th 1 .5 x lo ' c e l l s i n 1 nl o f D u l b e c c o ' s m d i f i e d ~ a g l e ' r medium.

o f an i m u n o a d r o r k n t col~mn. fen nu^ o f r a b b i t a n t i - m u s e i m u n o g l o b u l i n r (Cappel Labolato-

7.0, by the method o f M r c h e t a l . ( 1 6 ) . h e nl o f a s c i t e s f l u i d was dia lyzed against a 0.5 M r i e s ) we* coup led to 20 ml packed Sephamse 48 gel i n a 102 g lycero l -0 .2 I) NaHCO, b u f f e r . pH

NaC1-0.2 W Tr is buf fer . pH 8.0 and a p p l i e d a t a ra te o f 5 m l l h r . The e luate was r e a p p l i e d t o the co lunn twice to enhance recovery. The c o l v m was washed ex tens i ve l y w i th t he l oad ing

w i t h 0.5 M NaC1-0.2 M glycine-HCL buffer. pH 2.8, and c o l l e c t e d i n 1 nl f r a c t i o n s i n t u b e r b u f f e r u n t i l no more p r o t e i n was detected i n t h e e l u a t e . The bound h n u n o g l o b u l i n was e l u t e d

conta in ing an equal v o l m of 0.2 M T r i s b u f f e r pH 8.0. t o r a i s e t h e pH t o 7.0. Tubes w i t h s i g n i f i c a n t p r o t e i n were pooled and i m d i a t e l y d i a l y z e d a g a i n s t PBS and then concentrated by u l t r a f i l t r a t i o n w i t h a O i a f l o c e l l f i t t e d w i t h I "10 membrane ( h i c o n Corp.). The a f f i n i t y

b u f f e r . pH 8.0. column was r e c y c l e d i m d i a t e l y a f t e r e l u t i o n by e q u i l i b r a t i o n w i t h 0.5 M NaCl-0.2 W T r i s

1m1YnOglobUlin p u r i f i c a t i o n - 1munOglobUl inr were pur i f ied f m a s c i t e s f l u i d by the use

ELISA s p e c i f i c f o r muse a n t i b o d i e s t o h u m n h e p a t i c t r i g l y c e r i d e l i p a l e - A nonconpeti- t i v e ELISA assay was developed. Imwlon 2 r e m v a w e l l s t r i p s ( D y n a t e c h ) f i t t e d i n m i c r o t i t e r p l a t e s were used i n t h e d e v e l o p l e n t o f t h e assay. Wells were c o a t e d w i t h p u r i f i e d H-TGL by incubat ion fo r 3 t o 5 days a t 4-C w i t h 200 p1 a t a concen t ra t i on o f 50 nglml i n 0.1 M sodium carbonate buf fer pH 9.5. There condi t ions were se lec ted a f te r eva lua t i on Of t he f o l l ow ing buf fers : C i t ra te buf fers between pH's 3 t o 5; phosphate buffers between pH 7 and 7.5; T r i s HCl b u f f e r between pH's 8 . 0 and 9.0 and carbonate buf fer between pH 9 and 10. A l l b u f f e r s were employed a t a m l a r i t y O f 0.1 and contained 0.022 Nan2. The coa t ing so lu t i on was remved by aspivat ion. and the we l l s were washed three t imes wi th 0.052 Tween 20, PBS. Residual bind- i n g s i t e s were blocked wi th 3W u1 o f 12 bovine serum albumin, 0.051 T e n 20. PBS fo? 2 hours a t 37'C. Tissue cul ture supernatant ( 5 0 u l ) was added t o w l l r conta in ing 150 p l o f 12 bov ine rerum albumin. 0.052 Tween 20 i n PES. There samples were incubated Overnight a t 4-C OF f o r two hours a t 37-C. The sample r e m v a l and washing procedure was s i m i l a r t o t h a t d e s c r i b e d above. De tec t i on o f t he bound i m u n o g l o b u l i n s was Carr ied Out by a d d i t i o n O f 200 "1 O f a p p r o p r i a t e l y d i l u t e d r a b b i t a n t i - m u r e IgG covalent ly conjugated to horseradish peroxidase (Cappel Labooratoriesl. This incubation was conducted f o r 2 hours a t 37% i n t h e presence o f 1% bovine serum albumin. 0.051 Tween 20 i n PBS. The w e l l s were washed three t imes wi th 0.052 Tween 20. PBS and the subst rate, 0.012 H&. 0.4 mgtml 0-phenylenediamine, 0.1 W c i t r a t e phosphate buffer pH 5.0 added. A f t e r a 30 minute incubat ion a t 20'C the reac t i on was Stopped by

M i c r o e l i l l Auto Reader IR 580 (Oynatech). Samples were read against a chemical blank. The the add i t ion o f 50 "1 o f 2.5 M H,SO, t o each w e l l . The p l a t e s were read a t 490 nm on a

c u l t u r e medium madings were cowected fo r non-spec i f i c b ind ing o f muse imunog lobu l ins to

w e l l s t r e a t e d w i t h 0.1 M sodium carbonate b u f f e r pH 9.5 conta in ing no H-TGL. the wel ls. This was estimated f m the readings obtained wi th the same s i z e samples added t o

I m n a p r e c i p i t a t i o n Of h e p a t i c t r i g l y c e r i d e l i p a s e C a t a l y t i c a c t i v i t y - Three t o f i v e ng o f p u r i f i e d H-TGL or LPL were preincubated with "?.Tied l e v e l s Of a f f i n i t y p u r i f i e d mAb f a r 15 h r a t 4'C i n the presence of 21 nu Tris-HCl. pH 7.0, 12 bovine sewm albumin, 0.1 M NaCl or 1 M NaCl and 4 M CHAPS i n a t o t a l v o l m o f 50 u1. The a m m t o f mAb was v a r i e d between 0 and 30 ug150 "1. The t o t a l a m u n t o f muse immunoglobulin i n each incubat ion was maintained Constant by the addit ion Of p u r i f i e d muse i nnunog lobu l in (Mi les Labora tor ies) . The so lub le ant ibody-ant igen complexes were assayed f w C a t a l y t i c a c t i v i t y or prec ip i ta ted by the add i t lon O f 150 ug O f r a b b i t a n t i - m u s e i m u n o g l o b u l i n (Cappel L a b o r a t o r i e s ) i n 150 u1 of PBS. This

incubat ion was c a r r i e d Out f o r 2 h r a t 4'C. The immune complexes were p e l l e t e d a t 4°C i n an Eppendorf cent r i fuge. A l iquot6 of the supernatant were ar rayed fo? ca ta ly t i c ac t i v i t y . I n some instances the pe l le t uas washed w i t h 200 u1 O f PBS and resuspended i n 0.15 M Nat l . 20 m T r i s HC1. pH 7.4. An BO u1 a l i q u o t was assayed fop c a t a l y t i c a c t i v i t y .

a l l t h r e e p o s i t i o n s was e m u l s i f i e d i n t h e presence O f gum arabic . The assay for LPL contained L l p i d assays - For l i p o l y t i c a c t i v i t y m e a w r e r e n t s , t r i o l e i n c o n t a i n i n g [ l - L q C ] o l e a t e i n

i n a t o t a l volume O f 0.5 m l : 1.25 ml O f l + C - l a b e l e d t r i o l e i n w i t h a s p e c i f i c a c t i v i t y o f 45.000 cpmlwal of f a t t y a c i d ; 0.01 ml of heat- inact ivated (56'C. 30 .in) r a t sewm I S a

0.05 -1 O f NaCl. 5 wl o f CaCl, and 0.1 m o l o f Tri l-HC1, pH 8.6. H-TGL a c t i v i t y was source of apo-CII ICtiYatO?. 2.5 mg O f gum arabic ; 5 mg O f c r ys ta l l i ne bov lne serum albumin;

nedwred w i th 1 s im i la r a l l ay w i th t he excep t ion t ha t t he ra t rerun was a m t t e d and the N a C l mo ls r l t y inc reased t o one molar. FOP both l ipase assays f ree fa t ty ac ids were extracted by a l i q u i d / l i q u i d P d r t l t l o n System (17) . There w e ~ e ar rayed fo r rad ioac t i v i t y i n ACS x l n t i l l a - t i o n c o u n t i n g s o l u t i o n (Amerrham).

C N B l deqradatlon - D iges t ion w i th CNBr (181 Was Conducted on 40 ug O f H-TGL and LPL. Enzymes were concentrated by prec ip i ta t lan wi th 9 volumes O f absolute e thanol lacetone (1 : l .

mlnuter . The p r e c i p i t a t e d e n z m was washed wi th the same so lven t mx tu re and dr led by vacuum "/VI a t -2O'C for 1 hour and c e n t r i f u g a t i o n a t 10.000 rpm ~n a SS 34 S o r v a l l r o t o r f o r 30

dessICatlOn. Over 95 percent recovery was Obtalned when d l l u t e s o l u t i o n s ( 5 to 50 ug/rnl) Of

glucopyranorlde. 50 mM Tris-HC1, pH 1 . 2 or 2 d SDS. 25 mH OCtyl-R-O-gl~c~pyr.noslde 401 bavlne rerum albumin i n 2 M N a C l . 0.2% Tr i ton N-101. 0.1 M O(t1 galactose 0.1 M l -O-methy l -x-

Were Incubated a t 30°C f o r 24 hours w i t h 318 ug O f CNBr In 200 u l O f 70% ( v l v ) formic ac id g l y c e r o l . were concentrated by the above pro toco l . A f te r Precipitation the e n z w & l e r

o p h l l l i a t i o n of the d l luted ranples to dryness. The cyanogen bromrde fragments Yere sepa la ted f r esh l y sa tu ra ted w i th m t rogen . The re iCt lOn H I S terminated by add i t ion o f water and l y -

a t OH 6 . 8 on 12 and 13.75% cylindrical Dolwcry lamide qe15 lb lsacry lam?de aCrYlamlde r a t 7 0 O f 1: lOl contalning 0.1% SOS and 8 M urea by the method o f Swank and Munkrel ( 1 9 ) . The gels were stained w t h Coomaisie blue by the method of Fairbanks e t a l . ( 2 0 ) and scanned a t 550 nm.

L d e m T ~ l l 122). The PVOtelnr Yere f ixed and Stdlned by the Falrbdnkl procedure 1201 01 v l t h a I11ver i t d l n ( 2 3 1 (0loRad L d b o r l t o r l e $ ] .

SUS-PAGE O f enzyme ~ P e p a r l t l o n l was performed ~n 10% acrylamlde qelr by the method of

"Western B l o t s " we* c a r r i e d Out by the method Of Towbin e t a l . ( 2 4 1 w i t h a few modi f ica- t ions . E lec t rophore t ica l l y separa ted p ro te ins were t rans fer red to n i t roce l lu lose sheets (BA85S597. 0.45 lm, Schle icher and S c h u e l l l w i t h a c m w r c i l l t r a n s f e r apparatus (TE 42, HOefer Sc ien t i f i c Ins t ruments ) . T rans fer was Conducted f o r 15 hours a t 200 mA i n 25 m T r i r -

was Stained f o r 5 min i n 0.22 C o m s s i e b r i l l i a n t b l u e R-250, 402 methanol. 101 ace t ic ac id HC1. pH 7.5, 192 d glyc ine, 20% m t h a n o l . A p o r t i o n o f t he b l o t conta in ing protein standards

and destained for 5 nin i n 902 methanol, 2% acet ic ac id (251. The remain ing par t Of t h e n i t r o - ce l lu lose sheet was r insed w i th shak ing fo r 15 m i " i n 10 nll Trir-HC1, pH 7.5, 0.15 M NaCl. 1 d EDTA. and 0.1% Tr i tonX- lOO(buf fer A ) . Prote in b ind ino s i tes were blocked bv incubation w i t h 3% bovine serum albumin i n the same r i n s i n g b u f f e r A s f o r 90 min. The b l o c W e F i - k a c t e d fo r th ree hours a t 20'C w i t h a s c i t e s f l u i d or a n t i s e r a a p p r o p r i a t e l y d i l u t e d i n b u f f e r A con- t a i n i n g 0.3% bovine serum albumin. The n i t r o c e l l u l o s e sheets were then washed w i t h b u f f e r A

body d i r e c t e d a g a i n s t t h e f i r s t antibody i n b u f f e r A con ta in ing 0 .31 bv ine serum albumin f o r (three changer. 60 min, t o t a l ) , and incubated wi th a horse lad ish perox idare-conjugated ant i -

pmces red f a r t he co lo r reac t i on as described by Towbin e t a l . (24). 3 hours a t ZO'C. A f t e r r i n s i n g w i t h b u f f e r A (6 chanqes d u r i n g 60 min. t o t a l ) t h e b l o t s were

Glycopwte ins on n i t r o c e l l u l o s e b l o t s were detected bv the OmCedUre of C l e m l l h l u i f h

ce l lu lase sheet was incubated wi th concanaval in A (20 ug lm l ) i n bu f fe r B (PBS Containing 50 VW some m d i f i c a t i o n r . A f t e r b l o c k i n g O f p r o t e i n b i n d i n g ~ i i t e s a; i e & i b e i ;bo;e;7;h;-"it;oI

each of CaCl,. WgCl,. IhC1, and 0.5% T r i t o n X-1001 a t ZO'C f o r 3 hours w i t h shaking. The b l o t was washed ( three changes, 60 min t o t a l ) w i t h b u f f e r B and incubated wi th horseradish oeror i - dare (50 yglml Of b u f f e r 8 ) f o r 3 hours. The b l o t was washed wi th buf fer B. The g l y 6 p r o - t e i n s were v i sua l i zed by i ncuba t ing w i th 3-..ino-g-ethylcarba2ole ( 5 0 uglml l . 0.012 H,O,, 50 m sodium acetate. pH 5.0.

method of Gidez e t a l . ( 2 7 ) . Sanples were appl ied i n 4 M urea on d i s c ge ls conta in ing 7.52 I s o e l e c t r i c focusing of mnoclonal ant ibodies was p e r f a m d by a m d i f i c a t i o n O f the

acrylamide. 0.161 N.N'-methylene-biS-~Crylamide. 22 mpho l ine pH 3.5-10 or pH 4 t o 7. 101 g lycero l . 5 M w e e , 0.0182 ammonium p e r s u l f a t e and 0.422 ("/VI TEMEO (n,n.n' n'-tetramethyl- ethylenediamine). The gels were prefocused f o r 1 hour a t 5.5 watts and the ;ampler focused a t 5.5 watts (400 V l i m i t i n g ) f o r 20 houFs. Agarose gel electrophoresis Of mnoclanal ant ibodies was conducted a t pH 8.6 f a r 35 min on precast gels containin 1% agarose 52 sucrose and 0.035% EDTA d i s o d i u m S a l t i n 0.065 M b a r b i t a l b u f f e r pH 8.6 ?Corning. Universal elecirophore- l i s f i l m ) .

the rnethod of Mamh e t a1 (16). The coupl ing r a t i o was 1250 mg Of heparinlZ50 ml o f packed Heparin (unbleached from Inolex), 128 USP uni t r lmg, vas coupled t o Sepharose 48 gel by

gel .

conducted i n Dr. Isidore Oanishefsky's laboratory (New York l ledical Col lege Valhal la New Analyses o f f r a c t i o n s f o r a n t i t h r o m b i n a c t i v i t y and p u r i f i c a t i o n of a n t i t h m b i n w w e

Ywk 10595) as descr ibed previously 1201. Antibodies against human p l a s m k i t h m b i n were r a i s e d i n a goat by in jec t ing 1 mg of ant i thrombin wi th caplete Freund's ad juvant Iubcutane- OUIly i n many do rsa l s i t es . Boos te r i n iec t i on r of 250 YO i n the same adivvent wem aiven every other week for a per iod o f th ree kan thr . A large '6 lood sample was";oil&tid-a'ii;i a f t e r the last imunizat ian. Th is ant iserum produced a s i n g l e p r e c i p i t i n band on double- imunodi f - f us ion p la tes when reacted wi th the imuniz ing ant igen.

Oata Cmputat ion and expression - Data are expressed as means. and where i n d i c a t e d * 1 s tandard dev ia t i on . S ta t i s t i ca l s i gn i f i cance was determined by student's t t e s t s .

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 260, No. of 10720 ... · 10720 . Lipoprotein Lipase and Hepatic Triglyceride Lipase 10721 latter enzyme. None of these antibodies reacted