THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 11, Issue of June 10, pp. 7005-7016, 1983 Printed in U.S.A.

Amino Acid Sequence Studies on Lactate Dehydrogenase C4 Isozymes from Mouse and Rat Testes*

(Received for publication, May 19, 1982)

Yu-Ching E. Pan, Farida S. Sharief, Masaru Okabe, Shyuan Huang, and Steven S.-L. Lis From the Laboraton of Genetics. National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709

Carboxymethylated sperm-specific lactate dehydro- genase isozyme C4 (LDH-C4) proteins from mouse and rat testes were cleaved with cyanogen bromide and trypsin. Proteins were also citraconylated and digested with trypsin. In the case of mouse LDH-C4 isozyme, all 7 CNBr and 11 limited tryptic (arginine) peptides were isolated and sequenced. Some of the CNBr peptides were further fragmented with trypsin and chymotryp- sin and their compositions and/or sequences charac- terized. Also, 34 of the 36 expected tryptic peptides were purified, and their compositions and sequences determined. Amino acid sequences of these peptides purified from mouse LDH-C4 were overlapped into a complete covalent structure of the 330 residues. For rat LDH-C,, 5 of 6 expected CNBr peptides, 5 of 8 expected arginine peptides, and 28 of the 34 expected tryptic peptides were isolated, and their compositions and se- quences were determined. Some of the CNBr and argi- nine peptides were further fragmented with chymo- trypsin, thermolysin, or V8 protease, and their compo- sitions and/or sequences characterized. The amino acid sequence of 85% of the 330 residues from rat LDH-C subunit has been unambiguously determined, and the sequences of the remaining regions were tentatively aligned on the basis of peptide compositions and se- quence homologies with the other known lactate de- hydrogenase sequences, including mouse LDH-C. A comparison of the proposed rat LDH-C sequence with the complete covalent structure of mouse LDH-C indi- cates that 27 differences are located in the established rat LDH-C sequence of 280 residues and that 5 addi- tional differences are in the tentative sequence of the remaining 50 amino acids.

L-Lactate dehydrogenase (EC 1.1.1.27) catalyzes the inter- conversion of lactate and pyruvate with nicotinamide adenine dinucleotide as coenzyme. Biochemical and genetic studies of this enzyme have elucidated that the lactate dehydrogenase isozymes in mammals and birds are encoded by three different genes (1). The Ldh-a and Ldh-b gene products (LDH-A and LDH-B subunits) randomly combine in vivo to give rise to five forms of tetrameric lactate dehydrogenase isozymes in somatic tissues, whereas the homotetrameric LDH-C41 iso-

* 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.

‘:he abbreviations used are: LDH-C,, sperm-specific lactate de- hydrogenase isozyme previously designated as LDH-X; PTH, phen- ylthiohydantoin; MC, mouse LDH-C,; RC, rat LDH-C,; AA, amino acid; LC/HPLC, high performance liquid chromatography.

o whom all correspondence should be addressed.

zyme is present only in mature testes and spermatozoa (2-4). The physical and enzymatic properties of the mouse testicular LDH-C, isozyme have been studied extensively (4-7). The three-dimensional structure of mouse apoenzyme LDH-C4 has also been determined at 2.9 b, resolution using the technique of molecular replacement (8-10).

Species-specific antibodies produced against mammalian LDH-Ca isozymes have been developed as a marker for de- tecting mutations induced by mutagens and carcinogens. A rat-specific antibody reacts only with rat sperm and not with mouse sperm. By the use of fluorescent antibody technique, this rat-specific antibody is then used to detect rat-kind mu- tant sperm in mice. The induced mutation frequency has been shown to increase proportionally to the dose of mutagen administrated to the mice (11).

In order to correlate the sequence differences between LDH-C, isozymes of mouse and rat with species-specific an- tigenic determinants as well as to relate the chemical sequence of mouse LDH-C, with x-ray diffraction data, we have under- taken amino acid sequence determinations of LDH-C, iso- zymes from mouse and rat. This paper presents the detailed results of amino acid sequence determinations of the 330 residues each from mouse and rat LDH-C, isozymes. The second paper describes the molecular structure and immuno- logical properties of LDH-C4 isozymes from mouse and rat (12). The third paper discusses the evolutionary relationship of vertebrate lactate dehydrogenase isozymes A4 (muscle), B4 (heart), and C4 (testis) (13).

MATERIALS AND METHODS’

RESULTS AND DISCUSSION

Mouse LDH-C, Sequence-The sequences and/or compo- sitions of peptides purified from mouse LDH-C4 isozyme are summarized in Fig. 1. All 7 expected CNBr peptides from mouse LDH-C subunit were isolated and 6 of them were sequenced. Peptide B1 was presumably derived from the blocked NH2 terminus of LDH-C since Edman degradations failed to show any identifiable amino acid, as was the case with intact LDH-G protein. Two peptides B1-A1 and Bl-A2a were isolated from the limited tryptic digestion of peptide B1, and the sequence of peptide Bl-A2a was determined. The composition of the arginine-containing peptide A1 was the

“Materials and Methods” and the detailed results of peptide purification and sequencing are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magni- fying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 82M-l311A, cite the authors, and include a check or money order for $31.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

7005

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7006 Mouse and Rat LDH-C4 Sequences

S T V K E E L l Q N L V P D K L S R C K l T V V G V G D V G M A C A l S l L L K G L A D E L A L V D 10 20 30 40 50

8 1 8 2

81 -A1

1 . . T . l . . . . . . . . . . . . * + . l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 1 A 2

1 6

82-C1 c . * * * * * + * * *

I. . . . . . . 60 70 80 90 100

A D T D K L R G E A L O L L H G S L F L S T P K I V F G K D Y N V S A N S K L V I I T A G A R M V ~ 8 2

A 2 A 3 A 4

T 6 1 7 T ID T l i 112

* * + * * * * + * * * * + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ . + * + * * * * * * * * * + * + * * * + * * + * . . . . . . . . . . . . . . j I * * * * * * . 11- -1 . . . . . . 4 I + * + + + * * " I I " * , T 1Da ,,TlOb

FIG. 1. Amino acid sequence of the peptides purified from mouse LDH- C,. CNBr, arginine, tryptic and chymo- tryptic peptides are designated by B, A, T, and C, respectively. These peptides are numbered according to their posi- tions in the sequence of mouse LDH-X. The arrow indicates the residues which were positively identified. The reuerse arrow shows the residue released by car- boxypeptidase digestion. The hyphen means residue assignment based on the specificity of enzymatic and chemical cleavage. The dot represents peptide composition.

1. 8 2 4 2 . . .

I. . . . . . .I I. 4 B2-C3 82-C4 82-C5 . . . . . . . . . . . . . .I(. . . . . . . .

110 G Q T R L D L L Q R N V A l M K A I V P G V I Q N S P D C K I I V V T N P V D I L T Y V V Y K I S G

120 130 140 150

8 4

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 6

T 14 T 15. 84-115 B4-116 TI 7 . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

160 F P V G R V I G S G C N L D S A R F R Y L I G E K L G V N P T S C H G Y V L G E H G D S S V P I W S

170 180 190 200

8 4 + * * * * * * * * * + * * t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B4-AB B4-A9a

A 6 A 7

T 18, B4-TI8 . . . . . . . * . . . . . . . . . . . . . . . . .

84-C7 . . . . . . . . . . . . . . . . . 1.

210 G V N V A G V T L K S L N P A I G T D S N K q H W K N V H K Q V V E G G Y E V L D M K G Y T S W A i

220 230 240 250

8 4 I I 5 ............................................ """

* * * * + * * + * .......................................... B4-A9a B5-A9b

A 9 * * * . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T 21 T 22tT23. 84-T22+123 T 26

84-124 B4-C9

. ~. 84-CIO 84-Cl l

. . . . . . . I 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . .I 1. .I 260

G L S V T D L A R S I L K N L K R V H P V T T L Y K G F H G I K E E V ~ L S I P C V L G E S G I T O 270 280 290 300

F V K V N M T A E E E G L L 31 0

8 5 8 6 . . . . . J 1 + - + + + + + + . . . . .

A 11 B5-A1 la I

.............. T 33 . . J [ + + + + + + + + - + +

same as that of peptide Bl-A1 and thus peptide A1 was also derived from the blocked NH2 terminus of LDH-C. Edman degradations on peptide T1 did not yield any sequence infor- mation, presumably because of the blocked NH2 terminus. Tripeptide T3 was aligned to the COOH-terminal sequence of

320 K K S A D T L Y N M Q K N L E L

330

8 7 ........ .& " "

. . . . . . . . . . . . . . . 134 T 35

.I ~ ~ ~ t + * * " * * * + . + * * i /

T 36

MC

Leu-Ser-Arg of peptide Al . The sequence of peptide T2 was positioned at residues 5 to 15 since the sum of the compositions of peptides T1, T2, and T3 was the same as that of peptide Al . The sequence of 32 residues from peptide B2 was deter- mined and this sequence overlapped with the fiist 6 residues

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Mouse and Rat LDH-Cq Sequences 7007

of peptide A3. The composition of peptide A2 was consistent with the alignment of peptides B1 and B2. Dipeptides T4 (Cys-Lys) and T7 (Leu-Arg) corresponded to the NHP and carboxyl termini of peptide A2. The sequence of peptide T6 confumed the sequence of residues 41 to 55. The sequence of peptide T10 overlapped with the sequence of peptide A3 at residues 80 to 81. The sequence of peptide T11 containing arginine was aligned to the carboxyl terminus of peptide A3 at residues 89 to 97. The complete sequence of peptide B3 provided the linear ordering of peptides A4, A5, and A6. The sequence of 49 residues from peptide B4 overlapped the sequences of peptides A6 and A7. The sequence of 40 residues from peptide B4-A9a was determined, and this peptide must be derived from the carboxyl terminus of peptide B4 because of the presence of homoserine and homoserine lactone as well as the absence of arginine. Dipeptide B4-A8 (Phe-Arg) was placed at residues 168 to 169, since the two other arginine- containing peptides, B4-A6a and B4-A7, have already been positioned within CNBr peptide B4. Furthermore, the com- position of peptide B4-C8 was consistent with residues 169 to 171. Peptides T25 and B4-T25a possessed the same composi- tion except for an additional lysine residue in peptide T25. Thus, the sequence of peptide T25 can be aligned to the carboxyl terminus of peptide B4. The relative ordering of peptides T22 + T23 and T24 was based on the composition of peptide B4-Cl0. Furthermore, peptides B4-Tl5, B4-Tl6, B4- T17, B4-Tl8, B4-Tl9, B4-T20, B4-T22 + T23, B4-T24, and B4-T25a were derived from tryptic digestion of CNBr peptide B4. The sequences of peptides B5, B6, B7, All , T33, T35, and T36 established the sequence of residues 243 to 330. Tryptic peptide T36 contained neither lysine nor arginine, and corre- sponded to the COOH terminus of peptide B7. CNBr peptide B7 must be derived from the carboxyl terminus of LDH-C subunit because of the absence of homoserine and homoserine lactone. Therefore, a structure for the 330 residues could be established on the basis of single-residue overlapping at posi- tions 98 and 243. The proposed amino acid sequence of mouse LDH-C (Fig. 3) is in agreement with the composition of mouse LDH-C, (Table I) determined in this study as well as that previously reported (14) except that the underestimated val- ues of valine and isoleucine may be due to the presence of many Val-Val, Val-Ile, and Ile-Ile bonds in this protein. The exact molecular weight of mouse LDH-C subunit is 35,688.

It should be noted that a tentative amino acid sequence of mouse LDH-C based on the x-ray diffraction data at 2.9 8, has recently been proposed by Musick and Rossmann (10). A comparison of the x-ray sequence of mouse LDH-C subunit with the chemical sequence determined in this study indicates that the tentative sequence based on the x-ray data contains two extra amino acids between residues 16 and 17, three missing amino acids at residues 208, 249, and 330, and only 56% of residues with correct side chain assignment.

Rat LDH-C4 Sequence-Fig. 2 summarizes the partial se- quences and/or compositions of the peptides purified from rat LDH-C4. Automatic Edman degradations on peptides Bl and A1 did not yield any sequence information, and these two peptides were presumably derived from the blocked NH2 terminus of rat LDH-C,. The sequence of peptide T2 was tentatively assigned to residues 5 to 15, since the sum of compositions of peptides T1, T2, and T3 was the same as that of peptide Al. The sequence of peptide A2 corresponded to residues 19 to 33 and it overlapped the sequence of peptide B2. The sequence of residues 32 to 98 was derived from the sequences of peptides B2, T6, T9, T10, H7, B2-C4, B2-C7, B2- P1, B2-P3, and B2-P4 as well as the compositions of other peptides as indicated. The sequence of residues 98 to 183 was established from those of peptides B3, B4, B5, A5, A7, T12,

TABLE I Amino acid composition of mouse and rat LDH-C subunits

Mouse LDH-C Rat LDH-C

Sequence Analysis Sequence Analysis

Cysteineb 6 5.22 7 7.33 Aspartic acid 34 34.68 33 34.14 Threonine' 18 17.74 16 14.86 Serine' 24 23.94 29 28.73 Glutamic acid 22 24.98 25 26.76 Proline 11 10.21 10 9.69 Glycine 31 32.27 29 28.63 Alanine 18 19.46 19 21.84 Valined 39 35.37 37 34.60 Methionine 6 6.39 5 4.62 Isoleucined 23 21.47 25 24.10 Leucine 38 37.71 40 39.55 Tyrosine 5 6.17 6 5.84 Phenylalanine 7 8.41 5 5.31 Histidine 7 6.65 5 4.92 Lysine 25 24.40 26 25.87 Arginine 10 10.10 7 7.18 Tryptophan' 6 4.79 6 6.02

Total 330 329.96 330 329.99

Amino acid"

a The numbers of each amino acid were calculated on the basis of each subunit of 36,000. The values are averages of 24-, 48-, and 72-h hydrolyses except where indicated.

Cysteine was determined as carboxymethylated cysteine. The values for threonine and serine were extrapolated to zero

The values for valine and isoleucine were those of 72-h hydroly-

e Tryptophan was estimated after hydrolysis with 6 N HC1 contain-

time hydrolysis.

sate.

ing 4% thioglycolic acid.

T14, T15, T17 + 18, and T20. The sequence and composition of peptide H22 was tentatively aligned to residues 187 to 197. The sequences of peptides T22 + 23, T24, T25, and H25 were consistent with the proposed sequence of residues 211 to 240. The sequences of peptides A8, T28, T31, T32, and B5-T28 + 29 provided the overlapping sequence of residues 264 to 297. The sequence of peptides B6, T33, T35, T36, and B6-Cl4 established the COOH-terminal sequence of residues 304 to 330, because CNBr peptide B6 did not contain homoserine or homoserine lactone. In short, the amino acid sequence of 85% of rat LDH-C4 has been unambiguously determined. The sequences of residues 1-3, 16-17, 184-186, 195-210, 241-244, 248-263, and 298-302 as well as acid/amide assignments of residues 102, 136, 139, 179, 190, 203, 213, 219, 221, 223, 234, 238, and 241 were based on peptide compositions and sequence homologies with the established lactate dehydrogenase se- quences including mouse LDH-C4, and remain to be positively identified. The proposed sequence is in agreement with the amino acid composition of rat LDH-C4 (Table I) determined in this study as well as that previously reported (15). The exact molecular weight of rat LDH-C subunit is 35,534.

A comparison (Fig. 3) of the proposed rat LDH-C sequence with the complete covalent structure of mouse LDH-C indi- cates that 27 differences are located in the established rat LDH-C sequence of 280 residues and that 5 additional differ- ences are in the tentative sequence of the remaining 50 amino acids. The locations of these 32 differences in the lactate dehydrogenase molecule as well as the correlation of these differences with the immunological properties of LDH-C4 isozymes will be described in the second paper (12). The evolutionary relationship of vertebrate lactate dehydrogenase isozymes Aq (muscle), B4 (heart), and C4 (testis) will be dis- cussed in the third of these reports (13).

Sequencing Methodology-The methodology used to es- tablish the amino acid sequences of LDH-C4 isozymes war-

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7008 Mouse and Rat LDH-Cq Sequences

S T V K E E L I ~ N L A P D K ~ S R C K I l V V G V G O V G M A C A I S l L L K G L A O E L A L V O 10 20 30 40 50

6 1 6 2

A 1 A 2 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + " + * " * " + * " + * * *

I . . . . . . . . . . . . . . . . . . ~ + * " * * * * + + * * + + . . . . . . . . . . . . . . . . . 1 . . , + * + + * * * + + * c * * * + + * * + +

. . . . .I 1. . .

1 1 1 2 1 6

H 6

. . . . . . 82 - P1 L+""++

60 A D E O K L K G E A L D L L H G S L F L S l P K I V F G K D Y S V S A N S K L V I I l A G A R M V S

70 BO 90

6 2

A 2 I 1 A 3 + + + * * + * . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ]I+ +

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T 9 1 10

T 10a TlOb

H 11 . . . . .

H 6 H 7 I . . ( I . j

. . . . . . . I . . . . . . I . . . * . . . . . . . . . . i

G ~ S R L A L L ~ R N V l I H K A l V P G V I Q N S P O C K I H I V l N P V D I L l Y V V U K I S G 120 130 140 150

8 3 6 4 8 5

A 3 . I A 5

160 L P V S S ~ I G S G C N L ~ S A R F R ~ L ~ G E K L G V N P T S C H G W V L G ~ N P T ~ ~ H G ~ ~ L G E H G ~ ~ ~ ~ P ~ ~ ~

170 180 190 200

8 5

A 5 A 7

1 17+18, 65-T17+18

FIG. 2, Amino acid sequence ofthe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . peptides purified from rat LDH-C4. Thermolysin and V8 protease peptides are designated by H and P, respectively. '* * * + * * * * * + * * * * .' . ~I~ ' ! l - ' * * +

The other notations are the same as in Fig. 1.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

85-119 85-T20 H 19 HZ0 .II. .I 1. II 21 II 22

. . . . . . . . . . . . . . . . . . . . . . BS-CB

1. 1 G V N V A G V T L K S L N P A I G S D S N K q E ~ K T V I I K q V V D G G Y E V L D L K G V T S U A I

210 220 230 240 250

6 5

A 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T 22tT23 I 85-122+123 . . . . . . . ti23 H 25 I . . .

. . . 260 270 280 290

G L S V T D L A E S I L K N L K R V H A V T T L V K G L Y G I K E E I F L S I P C V L G E S G I T O 6 5

A 7 A 8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I + * * * + * * + . * * + * * + * . * * + . . . . . . . . . . . . .

~~**+*i~***++**,++-+,,. 131 T32 . . . . . . . .

I1 27 . . . . . . . . B5-CI0

. . . . . . . . . . .

L V K V N M N T E E E A L F K K S C O I L U N I Q K N L E L 310 320 330

8 5 6 6

A8 . . . . . _ ( ~ * + + + + * * * . . * * + * + * * + * + . . . , * . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65-1 33a , 133 T34 1 35 1 36 .I , . ~ ~ * * * * * + + * * * * - I H P + + + * . . . . AI++* .I

H 34 I *+ . H 35 ,

66-CI4 . . . . . . . .

8 5 - C I l 8 6 4 1 2 'I 8 6 4 1 3 1. .(

I. . . . . I [ . . . . . . . . II . . . .( 1. + * * + * . .(

RC

rants some comments. The hydrophobic nature of the LDH- improved the solubility of the modified protein for tryptic C4 isozymes presented difficulties in peptide purification (14, cleavage at arginine residues, and these limited tryptic (argi- 15). During this investigation this problem was overcome by nine) peptides were immediately separated on Sephadex G-75 the following approaches: the citraconylation of native enzyme columns (five 2.5 X 100 cm in 0.1 M NH4HCOs) without

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Mouse and Rat LDH-Cq Sequences 7009

In 20 Ser-Thr-Val-Lys-Glu-Glu-Leu-Ile-Gln-Asn-Leu-Val-Pro-Asp-Lys-Leu-Ser-Arg-Cys-Lys-

.- ~~

Ala Gln 30 40

Ile-Thr-Val-Val -Gly-Val -Gly-Asp-Val-Gly-Met-Ala-Cys-Ala-Ile-Ser-~le-Leu-Leu-Lys-

50 60

G1 u LYS 70 a0

Gly-Leu-Ala-Asp-G1u-Leu-Al~-Leu-Val-Asp-Ala-Asp-Thr-Asp-Lys-Leu-Arg-Gly-Glu-Ala-

Leu-Asp-Leu-Leu-His-Gly-Ser-Leu-Phe-Leu-Ser-Thr-Pro-Lys-Ile-Val-Phe-Gly-Lys-Asp-

90 100 Tyr-Asn-Val-Ser-Ald-Asn-Ser-Lys-Leu-Val-Ile-Ile-Thr-Ala-Gly-Ala-Arg-Met-Val-Ser-

Ser 110 120

Gly-Gln-Thr-Arg-Leu-As~-Leu-Leu-Gln-Arg-~sn-Val-Ala-Ile-Met-Lys-Ala-I~e-Val-Pro- Ser A1 a Thr

130 140 Gly-Val-Ile-Gln-Asn-Ser-Pro-Asp-Cys-Lys-Ile-Ile-Val-Val-Thr-Asn-Pro-Val-Asp-Ile-

150 Met Ile

160

FIG. 3. Sequence comparison of Leu-Thr-Tyr-Val-Val-Trp-Lys-Ile-Ser-Gly-Phe-Pro-Val-Gly-Arg-Val-Ile-Gly-Ser-Gly-

Leu Ser Ser rat and mouse LDH-C4 isozymes. 170 1 a0 Only residue differences are indicated. Cys-Asn-Leu-Asp-Ser-Ala-Arg-Phe-Arg-Tyr-Leu-Ile-Gly-Glu-Lys-Leu-Gly-Val-Asn-Pro- RC, rat LDH-C<; MC, mouse LDH-C,.

1 90 200 Thr-Ser-Cys-His-Gly-Trp-Val-Leu-Gly-Glu-His-Gly-Asp-Ser-Ser-Val-Pro-Ile-Trp-Ser-

Val 21 0 220

Gly-Val-Asn-Val-Ala-Gly-Val-Thr-Leu-Lys-Ser-Leu-Asn-Pro-Ala-Ile-Gly-Thr-Asp-Ser- Ser

230 240 Asn-Lys-Gln-His-Trp-Lys-Asn-Val-His-Lys-Gln-Val-Val-G1u-Gly-Gly-Tyr-Glu-Va1-Leu-

Glu Thr ASP 250 260

Asp-IYet-Lys-Gly-Tyr-Thr-Ser-Trp-Ala-Ile-Gly-Leu-Ser-Val-Thr-Asp-Leu-Ala-Arg-Ser- Leu

270

Ala Leu Tyr

Glu 280

Ile-Leu-Lys-Asn-Leu-Lys-Arg-Val-His-Pro-Val-Thr-Thr-Leu-Val-Lys-Gly-Phe-His-Gly-

290 300 Ile-Lys-Glu-Glu-Val-Phe-Leu-Ser-Ile-Pro-Cys-Val-Leu-Gly-Glu-Ser-Gly-Ile-Thr-Asp-

Ile

Ile

freezing and lyophilizing to avoid aggregation of hydrophobic peptides. For total tryptic digestion at lysine and arginine residues, the carboxymethylated protein was first denatured a t 37 "C for 30 min in 0.3 M NH4HC03 containing 6 M urea, and then diluted with 2 volumes of distilled water before 2% of trypsin was added (16). After 4 h at 37 "C, the tryptic digest was initially separated on a Sephadex G-75 column in 0.1 M NH4HC03, and the peptides present in the separated fractions were further purified by two-dimensional paper chromatog- raphy and electrophoresis at pH 4.4. Edman degradations on all peptides were performed on a liquid-phase sequenator (Beckman 890C) using Polybrene as a carrier (17), and most of the peptide sequences were determined by quantitative analysis of PTH derivatives after HI hydrolysis (18). The acid/amide side chains of Asx and Glx were assigned from direct identification of PTH derivatives by high performance liquid chromatograph as well as from the electrophoretic mobilities of peptides at pH 4.4 and/or pH 6.5 (19).

Acknowledgments-We thank Dr. C.-Y. Lee for providing the proteins; Drs. J . P. Marciniszyn and S.-M. T. Chang for their contri- bution to the early work of this investigation; Dr. H. H. Kiltz for informing us of the pig lactate dehydrogenase sequences before pub- lication; Drs. C. Aquadro, F. B. Armstrong, J. H. Harrison, J . L. Irvin, G. B. Kitto, A. F. Riggs, and S. Slaughter for critical reading of the

310 320 Phe-Val-Lys-Val-Asn-Met-Thr-Ala-G1u-G1u-G1u-Gly-Leu-Leu-Lys-Lys-Ser-Ala-Asp-Thr- Leu Asn Thr Ala Phe Cys Ile

Leu-Trp-Asn-Met-Gln-Lys-Asn-Leu-Glu-Leu MC RC

330

manuscript; Drs. B. H. Judd and H. V. Malling for their interest and support in this project; and Leslie Gardner for her excellent help in preparing the manuscript.

1.

2. 3.

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Y C Pan, F S Sharief, M Okabe, S Huang and S S Liand rat testes.

Amino acid sequence studies on lactate dehydrogenase C4 isozymes from mouse

1983, 258:7005-7016.J. Biol. Chem. 

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