THE CARBOHYDRATE METABOLISM OF BRAIN

II. THE EFFECT OF VARYING THE CARBOHYDRATE AND INSULIN SUPPLY ON THE GLYCOGEN, FREE SUGAR,

AND LACTIC ACID IN MAMMALIAN BRAIN

BY STANLEY E. KERR AND MUSA GHANTUS

(From the Departments of Biochemistry and Anatomy, American University of Beirut, Beirut, Lebanon)

(Received for publication, June 22, 1936)

A reexamination of the relationship of brain glycogen, free sugar, and lactic acid became necessary when it was demonstrated that autolytic changes take place in brain with such rapidity after death that “resting” levels for phosphocreatine and lactic acid can be secured only when the tissue is fixed in situ (Kerr, 1935; Avery, Kerr, and Ghantus, 1935). Moreover, the wide variation in values so far reported by various investigators for brain glycogen suggested defects in the methods used. In Paper I (Kerr, 1936) a modification of the Pfliiger procedure was presented which permits the accurate determination of glycogen in nerve tissue. Using the technique of freezing the brain in situ with liquid air, and the improved method for glycogen, we have attempted to determine whether or not the carbohydrates of brain are subject to the same fluctuations as those of liver and muscle under a variety of influences, including overfeeding, fasting, phlorhizin poisoning, pancreatectomy, and overdosage with insulin.

EXPERIMENTAL

Methods

The brains were prepared for analysis as previously described (Kerr, 1935). Glycogen was determined in duplicate or triplicate by a method (Kerr, 1936) designed to avoid errors due to the presence of cerebrosides. Lactic acid was determined in duplicate as described by Avery, Kerr, and Ghantus (1935) on zinc hy- droxide filtrates, and the free sugar in the same filtrates by the

9

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10 Carbohydrate Metabolism of Brain. II

TABLE I

Glycogen, Free Sugar, and Lactic Acid in Dog Cerebrum. E$ects of Fasting, High Carbohydrate Diet, Infusion with Glucose, or Glucose with Insulin

10 11 12 16 17 18 20

138 139 147 150

-

-

--

-

Condit,ions

Normal dog, uncontrolled diet “ “ “ “ ‘I “ “ “ “ ‘I “ “ ‘I “ “ “ ‘I I‘ “ “ “ “ “ “ ‘I ‘I I‘ I‘ I‘ “ “ “ I‘ “ “ I‘ I‘ ‘L ‘I “

Average...............................

81-E 37 38 36 39 40

Fasted 2 days “ 2 ‘I “ 3 “ ‘I 3 “ ‘I 3” “

3 “ 36 “

Average...............................

42

43 44 35

67

High carbohydrate-meat diet and glucose by mouth*

“ “ “ “

5 min. after glucose infusion of 3.3 gm. per kilo

30 min. after glucose infusion of 3 gm. per kilo

-

-

I

_-

--

-_

-

Mg. per 100 gm. cerebrum

9.7 77 45 20.1 9.5 104 51 22.:

10.1 80 52 18.: 8.8 124 86 18.:

12.2 130 56 13.1 9.1 96 62 16.: 9.8 112 48 15:

110 82 78 95

---~

9.9 98 57 17.: ----

116 49 14.c 8.6 100 55 21.t 7.7 150 33 32.: 8.4 96 71 21.~

10.4 135 48 26.1 8.5 142 47 23.1

8.7 123 51 23.: __---__

120 55 29.:

113 87 19.A 86 61 20.:

10.6 143 197 17.t

7.2 98 206 25.1

533

*High carbohydrate-meat diet for 3 days and 1.5 gm. of glucose per kilo 2 to 3 hours before the brain was frozen.

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S. E. Kerr and M. Ghantus

TABLE I-Concluded

90 min. after glucose infusion of 3 gm. per kilo

5 min. after glucose-insulin infu- sion?

60 min. after glucose-insulin infu- sion?

135 min. after glucose-insulin infu- siont

-

-

I

-

Mg. per 100 gm.

-

t Sk 8.4 4* 4 -

9.:

10.1

cerebrum

102 52 19.:

104 65 11.:

118 206 16.!

124 16 16.1

- I

95

223

28

11

-

Per cent

6.90

2.42

t 3 gm. of glucose and 3 units of insulin per kilo of body weight.

Shaffer-Somogyi (1933) procedure. Blood sugar was estimated by the Benedict (1931) procedure, with the exception of experi- ments numbered above 88, and in these the Folin (1929) procedure was used on unlaked blood filtrates. For phosphocreatine the Fiske-Subbarow (1925) method was used (see Kerr, 1935). This determination was included for the sake of ascertaining whether or not “resting” conditions were obtained during the fixation of the brain.

The experiments were conducted on dogs and rabbits, anesthe- tized intraperitoneally with amytal. The dosage was 60 to 65 mg. per kilo of body weight for dogs except the diabetic, phlor- hizinized, and fasting animals, in which 50 to 55 mg. were found less apt to cause death. In rabbits 80 mg. per kilo were used.

Results

The cerebrum of dogs, selected at random with previous history unknown, was found to contain from 77 to 130 mg. per cent of glycogen, 45 to 86 mg. per cent of free sugar, and 13 to 22 mg. of lactic acid (see Table I). After fasting periods of 2 to nearly 4 days the glycogen content in three of the six animals was found to

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12 Carbohydrate Metabolism of Brain. II

be even higher than in the non-fasting dogs, but whether this difference is significant is doubtful. A diet of rice, bread, and meat in excess for 3 days, followed by glucose (1.5 mg. per kilo) by stomach tube 2 to 3 hours before the animal was sacrificed pro- duced no evidence of storage of glycogen in the brain, or other changes.

In a further effort to demonstrate whether or not glycogen could be stored in the brain, glucose infusions of 3 gm. per kilo of body weight were given to three dogs. A 25 per cent solution was introduced slowly into the femoral vein during a period of 40 minutes. The brains were frozen 5, 30, and 90 minutes respec- tively after the completion of the glucose infusion. In one of the three (Experiment 35, Table I) the glycogen level was found to be 143 mg. per cent (somewhat above the upper limit observed in the “normal” dogs), whereas in the other two (Experiments 67 and 68) the glycogen content was near the normal average. In Experiment 35 the glucose infusion raised the free sugar of the brain to 3 to 4 times the normal value, but less than half the blood level. Within 90 minutes after the infusion the brain sugar was again within normal limits (Experiment 68).

A combined glucose and insulin infusion (3 gm. of glucose and 3 units of insulin per kilo of body weight) administered intra- venously over a 40 minute period likewise failed to increase the glycogen content of the brain above normal. In Experiment 73 the brain was frozen 5 minutes after the infusion, at which time the sugar of both blood and brain was at normal levels, the liver gly- cogen 6.9 per cent, and the brain glycogen (104 mg.) at the normal average. In Experiment 80 2$ hours elapsed between completion of the infusion and fixation of the brain, with the result that very low levels were reached for both blood sugar (28 mg.) and brain sugar (16 mg.). The glycogen content (124 mg.) was found to be within normal limits.

Depletion of the glycogen reserves of the body by removal of the pancreas failed to disturb the level of brain glycogen (Table II). In three of these experiments the glycogen of liver was low- ered to 0.15 to 0.18 per cent, while that in brain remained well within normal limits. Under these conditions the free sugar of brain was greatly increased, although remaining lower than the blood sugar, and lactic acid was not significantly altered.

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S. E. Kerr and M. Ghantus 13

Combined fasting and phlorhizin poisoning of sufficient severity to reduce the glycogen of liver to the low level of 0.049 to 0.063 per cent failed to lower the level of brain glycogen below the normal (Table II). The free sugar was, however, lowered to nearly half the normal value, as was also the blood sugar.

TABLE II

Effect of Pancreatectomy and of Phlorhinin Poisoning on Glycogen, Free Sugar, and Lactic Acid of Dog Cerebrum

s 3 fi ‘C !% r3 -

61 88 95 87 89 90 62-P 94 70 71 72

Conditions

Depancreatized (partial ?) “ “ “ “

Depancreatized “ ‘I “ “

Phlorhizinized* “ “

None ‘I “

+ +

1 +

-

w. Per Per zoo cent cc.

806.38 1176.79

5004.36 3320.15 3340.18 2860.17 400 1.98

48 0.06: 400.04’ 57 0.05’

&T. per 100 gm. cerebrum

8.6 108 7117.2 7.3 117 7725.2

104 0.7 115 22414.6 6.1 86 16638.6 9.4 107 15621.1 0.7 119 15218.9

109 306 13.6 8.7 96 3620.6 9.9 116 3517.8 8.5 103 3565.6

* These dogs were fasted for 7 days, injected subcutaneously with 1 gm. of phlorhizin emulsified in 10 cc. of olive oil on the 4th and 5th days, and with adrenalin (1 mg.) three times on the 6th day. On the 7th day the brains were frozen under amytal anesthesia.

The effect of insulin (15 to 20 units per kilo) on the brain gly- cogen of dogs is presented in Table III. In five of the eight experi- ments the glycogen was brought far below the lowest level ob- served in normal dogs (see Table I), in two others the lower normal limit was approached, and in one (Experiment 78) the

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16 Carbohydrate Metabolism of Brain. II

concentration was about the average normal. In all ten experi- ments on rabbits (Table IV) insulin caused a sharp lowering of the glycogen level in brain.

Corresponding with the fall of blood sugar after insulin, the free sugar of brain was likewise diminished to concentrations farbelow the normal. In all but three cases (Experiment 112, Table III; Experiments 104 and 105, Table IV) the brain sugar was found to be considerably less than that of blood. No consistent changes in the lactic acid content of brain were caused by the various experimental conditions, with the exception that a high level was observed in one phlorhizinized dog (Experiment 77, Table II). A relatively low content of lactic acid was found in a number of rabbits, including the control group.

None of the experimental procedures caused significant changes in the phosphocreatine content of brain.

DISCUSSION

Glycogen-Brain resembles muscle in its ability to retain gly- cogen more tenaciously than liver. Neither fasting,r pancreatec- tomy, nor phlorhizin poisoning with adrenalin caused significant changes in the glycogen of brain, whereas all these conditions result in marked depletion in the liver. After pancreatectomy the glycogen of muscle, like that of brain, remains at a high level, even if food is discontinued (Chaikoff, 1927).

It appears also that the brain cannot store a significant surplus of glycogen, since the administration of glucose orally or intra- venously with or without insulin failed to raise the glycogen level above that found in normal or fasting animals. Cori and Cori (1926, 1928, a) report a different behavior for muscle. They fed glucose to normal rats and observed a higher muscle glycogen in the animals injected with insulin than in the controls.

Whether the loss of brain glycogen caused by insulin overdosage is a direct effect of insulin or the result of a secondary output of adrenalin during the period of hypoglycemia remains to be deter-

1 The fact that three fasting dogs had higher levels of brain glycogen than any of the controls, and that the average for the fasting animals was 25 mg. higher than the control group, suggests that the fuel reserve in the brain may be slightly increased, while that in the liver is greatly dimin- ished. It is certain that the glycogen of brain is not depleted during 3 to 4 days fasting.

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S. E. Kerr and M. Ghantus 17

mined. That the lowering of muscle glycogen by insulin is ac- tually an adrenalin effect was established by Cori and Cori (1928, b).

Our experiments confirm the results of Asher and Takahashi (1924) only in part. These authors concluded that the glycogen of brain remained unaffected by severe depletion of the carbo- hydrate reserves of muscle and liver. They concluded further that insulin caused an increase of glycogen if convulsions were avoided, but, if excitation of the central nervous system occurred to such an extent that convulsions resulted, glycogen was greatly decreased. In our experiments, however, the lowest glycogen found after insulin overdosage was noted in an animal which had no convulsions (Experiment 50). In view of the fact that these authors acknowledged that their failure to separate cerebrosides resulted in values 2 to 3 times higher than they later found by a modified method, their evidence must be regarded as inconclusive. Winterstein and Hirschberg (1925), experimenting on the isolated central nervous system of the frog, concluded that insulin in high concentration caused a loss of glycogen, whereas small doses caused an increase. It has been pointed out elsewhere (Kerr, 1936) that the method they adopted for separating cerebrosides from glycogen is inadequate, and possibly explains many of the very high values they obtained for “glycogen.” The same criticism must also be made of the experiments of Kobori (1926) who used the methods of Asher and Takahashi (1924) and of Wintersteinand Hirschberg (1925).

Fermentable Sugar in Brain2-On comparing the figures we obtained for brain with those of Cori, Gloss, and Cori (1933) in rabbit muscle (non-fasting) under strictly “resting” conditions it appears that the quantity of free sugar in brain (35 to 75 mg. per cent for the rabbit) is nearly within the same range as that in

2 The nature of the free carbohydrate of brain has not been conclusively demonstrated. Holmes (1929) prepared from extracts of crab nerve ganglia an osazone with a crystal form typical of glucosazone. He states, however, that in the extraction of free sugar from the tissue with 60 per cent alcohol some glycogen also dissolved and gave rise to high values for free sugar, hence the isolation of the osazone is of doubtful significance. Within the limits of error for determining the small quantities in question we find the reducing substance in the zinc hydroxide filtrate to be entirely fermented by yeast.

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18 Carbohydrate Metabolism of Brain. II

heart muscle (46 to 59 mg.) and much higher than in skeletal muscle (13 mg.).

The concentration of free sugar in brain, as in any other tissue, must be the resultant of a number of factors, including the rate of utilization as well as the concentration in blood and the volume flow of the blood. The influence of the blood glucose level is evident from the high concentration in the brain of diabetic dogs and the low level in the phlorhizinized animals (see Table II). A rapid rate of utilization is suggested by the fact that the con- centration in brain is considerably less (half to two-thirds) than in blood, both in hypoglycemia and in hyperglycemia. After insulin the sugar concentration in brain is maintained at a consider- ably lower level than in blood in all but three of the sixteen experi- ments (Experiment 112, Table III; Experiments 104 and 105, Table IV). It should be noted that in these three experiments the blood sugar had reached the lowest points obtained. The explana- tion may be found in a similar situation noted by Cori, Gloss, and Cori (1933) in rabbit muscle. These authors found a decrease in the fermentable sugar of skeletal and heart muscle during moderate insulin hypoglycemia, but in severe hypoglycemia there occurred a marked rise of muscle sugar, so that it equaled or even surpassed the plasma sugar concentration. This secondary rise was found to be due to adrenalin secretion.

The lowering of blood sugar after insulin is undoubtedly the result rather than the cause of a lowered tissue sugar concen- tration, since the tissue is the “vacuum” into which the sugar passes and disappears. The lowering of the free sugar and gly- cogen of brain appears to be due to an increased utilization of carbohydrate in the brain itself (or to conversion into other com- pounds) rather than to transport to other tissues, since the con- centration of free sugar in brain is constantly less than in blood, and since extreme depletion of the carbohydrate reserves of the body by fasting and treatment with phlorhizin and adrenalin has no effect on brain glycogen.3 On the other hand, a secondary lowering of brain sugar could occur owing to insufficient supply from the blood, as in the hypoglycemia which follows hepatec- tomy. The disturbances of central nervous origin during insulin

* The lowering of brain glycogen by insulin occurs while there is still a good reserve of glycogen in t.he liver, but this is probably not available, owing to inhibition of glycogenolysis by insulin (Cori, 1931).

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S. E. Kerr and M. Ghantus 19

hypoglycemia are possibly due to a carbohydrate deficit caused by both factors; i.e., increased utilization by the brain and insufficient replacement from the blood. Although it is tempting to attribute the nervous disturbances and convulsions to the lowered content of free sugar or of glycogen, it should be noted that in several experiments low levels of glycogen and sugar were observed with no prostration or convulsions (see Tables III and IV). However, the symptoms of hypoglycemia are abolished by the amytal anesthesia; hence we cannot be certain of a lack of correlation between the chemical findings and the nervous disturbances.

SUMMARY

Glycogen, free sugar, and lactic acid were determined in brain frozen in situ with liquid air in amytalized dogs and rabbits under various experimental conditions.

The glycogen content of the cerebrum of normal animals was found to lie within the range 77 to 150 (average 98 mg. per 100 gm.) in well fed and fasting dogs, and 70 to 99 mg. (average 82) in rabbits. Fasting, overfeeding, glucose infusion with or without insulin, phlorhizin poisoning followed by adrenalin, and pan- createctomy all failed to cause significant changes in the brain glycogen.

Overdosage with insulin caused a marked decrease in the brain glycogen of dogs and rabbits.

The free sugar of brain in the control animals varied from 35 to 75 mg. per 100 gm. in rabbits and 45 to 86 mg. in dogs. Lowering of the blood sugar by phlorhizin poisoning or by insulin caused a corresponding decrease in brain sugar. Hyperglycemia caused by pancreatectomy or by administering glucose caused a rise of sugar in brain. The free sugar of brain was constantly lower than that of blood, except in extreme insulin hypoglycemia.

Neither lactic acid nor phosphocreatine of brain was affected significantly by any of the experimental conditions.

We are indebted to the Rockefeller Foundation for financial aid in carrying out this work.

BIBLIOGRAPHY

Asher, L., and Takahashi, K., &o&em. Z., 164,444 (1924). Avery, B. F., Kerr, S. E., and Ghantus, M., J. Biol. Chew, 110, 637 (1935). Benedict, S. R., J. Biol. Chem., 92, 141 (1931).

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20 Carbohydrate Metabolism of Brain. II

Chaikoff, I. L., J. Biol. C&m., 74, 203 (1927). Cori, C. F., Physiol. Rev., 11, 143 (1931). Cori, C. F., and Cori, G. T., J. Biol. Chem., 70,557 (1926); 76,755 (1928, a);

79,309 (1928, b). Cori, G. T., Closs, J. O., and Cori, C. F., J. Biol. Chem., 103, 13 (1933). Fiske, C. H., and Subbarow, Y., J. BioZ. Chem., 66, 375 (1925). Folin, O., J. BioZ. Chem., 82, 83 (1929). Holmes, E. G., Biochem. J., 23, 1182 (1929). Kerr, S. E., J. BioZ. Chem., 110, 625 (1935); 116, 1 (1936). Kobori, B., Biochem. Z., 173, 166 (1926). Shaffer, P. A., and Somogyi, M., J. BioZ. Chem., 100, 695 (1933). Winter-stein, H., and Hirschberg, E., Biochem. Z., 169, 351 (1925).

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Stanley E. Kerr and Musa GhantusMAMMALIAN BRAIN

FREE SUGAR, AND LACTIC ACID IN GLYCOGEN,INSULIN SUPPLY ON THE

VARYING THE CARBOHYDRATE ANDOF BRAIN: II. THE EFFECT OF

THE CARBOHYDRATE METABOLISM

1936, 116:9-20.J. Biol. Chem. 

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