Direct evidence for de novo synthesis of rat liver phenylalanine: Pyruvate transaminase after glucagon treatment

ARCHIVES OF BIOCHEMISTRY AND RIOPHYSICS Vol. 192, No. 2, February, pp. 414-420, 1979

Direct

Evidence for de Nova Synthesis of Rat Liver Phenylalanine: Pyruvate Transaminase after Glucagon Treatment’ JEAN School qf Pharmacy,

C. SHIH2 AND YUEN-LING

University

qf Southern

Cal~fomin,

Received May 15, 19’78; revised

CHAN

Los Angeles, Cdifomin

September

.YOO%‘~

13, 1978

A specific antibody to phenylalanine:pyruvatr transaminase has been used to show that the number of enzyme molecules and the rate of enzyme synthesis are increased by glucagon and N”, 0 “-dibutyryl cyclic AMP. Cycloheximide given simultaneously with glucagon or dibutyryl cyclic AMP blocked the increase in [“Hlleucine incorporation when it was injected along with glucagon, but had no cffcct when given 4 h after the glucagon. This finding suggests that the mRNA synthesis for phenylalanine:pyruvate transaminase may be completed in 4 h.

Hepatic phenylalanine:pyruvate transaminase (EC 2.6.1 aminotransferase, the exact EC number has not been assigned) catalyzes the transamination between phenylalanine and pyruvate (1). Our previous work has shown that this enzyme is specific for pyruvate as the amino group acceptor and can be separated from phenylalanine:cYketoglutarate transaminase by a Sephadex electrophoresis column (2). Heat stability and glucagon inducibility experiments showed phenylalanine and histidine to be the primary amino acid substrates for this enzyme (3). The glucagon inducibility of this enzyme has been reported by several laboratories (3-5), presumably via cyclic AMP (6). Fuller et al. (7) showed that the increase of phenylalanine:pyruvate transaminase activity in crude liver extracts after glucagon injection could be blocked by actinomycin D or cycloheximide and, based on this indirect evidence, suggested that the increase in enzyme activity after glucagon treatment was probably due to an increase in protein synthesis. I This study was supported by Grant CA 21494, awarded by the National Cancer Institute, DHEW; USC Graduate School Faculty Research Support; and BRSG Grant RR-05792 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, NIH. 2 To whom correspondence should be addressed.

0 of

1979 by Academic

reproduction in

Press, any fall;:

MATERIALS

Inc. reserved.

AND METHODS

Materials. Sprague-Dawley male rats, each weighing about 150 to 180 g, were purchased from local vendors. L-[4,5-“H(N)]Leucine, pyruvic acid, CT-‘%labeled free acid (135 mCi/mmol), and Liquiflour (2,5 - diphenyloxazole - I,4 - bisl2 - (5 phenyloxazolylllbenzene toluene concentrate) were purchased from New England Nuclear. The chemicals used for electrophoresis were all purchased as electrophoretic grade from Bio-Rad Laboratories. NCS tissue solubilizer was from AmershamJSearle; toluene was from Fisher Scientific Company. Crystalline porcine glucagon was a gift from Eli Lilly and Company. N 6, 02’-Dibutyryl adenosine 3’,5’-cyclic monophosphoric acid was purchased from Sigma Chemical Company. Cycloheximide and actinomycin D were purchased from Calbiochem. All other reagents used were of analytical grade unless otherwise specified. Enzvrrre assay. A radioassay developed in our laboratory (8) was used. Phenylalanine:pyruvate transaminase activity was determined by using [‘YJpyruvate as the amino acceptor and phenylalanine as the amino donor. The assay mixture (1 ml) contained 90 mM 414

0003-9861/79/020414-07$02.00/0 Copyright All rights

Recently, we have purified hepatic phenylalanine:pyruvate transaminase from glucagon-treated rats and prepared a specific antibody against the enzyme (8). With this antibody, we have now obtained direct unequivocal evidence that the elevation of hepatic phenylalanine:pyruvate transaminase activity by glucagon (or dibutyryl cyclic AMP) injection is via an increased synthesis of this enzyme molecule.

INDUCTION

OF PHENYLALANINE

L-phenylalanine, 46 mM [‘“C]pyruvate, 0.05 mM pyridoxal phosphate, 24 mM borate buffer, pH 8.1, and appropriate amount of enzyme. At the end of a 20.min incubation period at 37”C, the reaction mixture was passed through an anion-exchange resin, Dowex AG l-X2, 200 to 400 mesh. The eluted fraction and 4 ml water wash were collected and counted in 10 ml of Aquasolin a Beckman LS-335 liquid scintillation counter. Under this condition, the enzyme activity was linear with added enzyme extract and remained linear up to 1 h incubation. In some cases, phenylalanine:pyruvate transaminase activity was determined by the spectrophotometric method described by Linet al. (1). The reaction mixture was the same as the radioassay described above. The keto acid formed from phenylalanine and pyruvate was measured as the yellow enol-borate complex with an absorption maximum at 310 nm. The two assay systems are comparable. The radioassay has the advantages that it can be used for nonaromatic amino acid substrate. It can also be used for assaying samples containing precipitates or in a suspension which would interfere with the spectrophotometric assay. Puification o-f phenylalanine:pyrwate transaminase and antibody preparation. Two forms (A and B) of phenylalanine:pyruvate transaminase from glucagontreated rats were purified according to the method described by Shih and Chan (8). Briefly, the transaminase was purified by heating, DEAE-Sephadex A-50 column chromatography, hydroxylapatite, Sephadex G-100 chromatography, and preparative gel electrophoresis. The homogeneity of each of the enzyme was evaluated using analytical polyacrylamide gel and sodium dodecyl sulfate-gel electrophoresis. Antibody prepared against purified A form of the transaminase (8) was used in this study. Protein was determined by the method of Lowry et al. (9), in which bovine serum albumin was used as the standard protein. Electrophoresis qf phenylalanine: pyruvate transaminase. Phenylalanine: pyruvate transaminase from normal rat liver was partially purified by heating and DEAE-Sephadex A-50 column and hydroxylapatite column chromatography (8). The partially purified transaminase was subjected to electrophoresis according to the method of Davis (10). The enzyme activity was revealed by staining with p-nitrotetrazolium blue (11) utilizing phenylalanine and pyruvate as substrates. Purified phenylalanine:pyruvate transaminase from glucagon-treated rats was also subjected to electrophoresis for comparison. Isotope incorporation. Rats were treated with glucagon (1 mgikg, injected subcutaneously) or NR,OY’dibutyryl cyclic AMP (50 mgikg, intraperitoneally) for 32 h. Immediately following a glucagon or dibutyryl cyclic AMP injection, cycloheximide (2 mgikg) or actinomycine D (1 mgikg) was given intraperitoneally. A second injection of cycloheximide or actinomycin D was given 15 h later due to the short effective half-life

415

TRANSAMINASE

of the drugs (12. 13). All animals received 200 pCii100 g body wt uniformly labeled L-[4,VH(N)]leucine 31 h after glucagon (or dibutyryl cyclic AMP) and were killed 1 h after [“Hlleucine injection. Livers were removed and frozen immediately for later use. Immunochemical titration. Livers were homogenized in 10 vol (w/v) of 0.05 M Tris-HCl buffer, pH 7.6, and centrifuged at 30.0009 for 30 min. The supernatant fraction was heated at 60°C for 10 min, then reacted with the antiserum prepared against the transaminase A form. For immunochemical titrations, the antiserum was diluted with 20 mM phosphate buffer, pH 7.0 containing 1 mM EDTA, 1 mM dithiothreitol, and 5 mgiml bovine serum albumin. An appropriate dilution of antibody was incubated with various amounts of heated liver supernatant for 30 min at 37”C, then overnight at 4°C. The immunoprecipitates formed were removed by centrifugation at 1OOOg(IEC HN-S centrifuge) for 10 min. The remaining phenylalanine:pyruvate transaminase activity was then determined by radioassay. Preparation ofimmuxoprecipitate. One milliliter of antiserum against the transaminase A form was sufficient to precipitate 2368 units of phenylalanine: pyruvate transaminase activity. One unit of transaminase activity is equal to 1 nmol of product formed per minute per milligram of protein. A slight excess of antiserum was added in each experiment to ensure complete precipitation in the heated supernatant fraction. The immunoprecipitation reaction mixture conpH 7.4, 0.15 M NaCl, and taining 0.05 M Tris-HCl. 1 mM dithiothreitol was incubated at 37°C for 30 min, then overnight at 4°C. The precipitate formed was collected by centrifugation at 1OOOg(IEC HN-S centrifuge) for 10 min and washed three times with cold 0.9% NaCl in 0.05 M Tris-HCl buffer at pH 7.4. Electrophoresis of radioimmunoprecipitate on sodium dodecyl sulfate-acrylamide gels. Basically, the method of Weber and Osborn (14) was employed. About 300 pg each of washed immunoprecipitate containing [3H]leucine-labeled transaminase was dissolved in 100 yl solution containing 1% DDT,3 1% 2-mercaptoethanol, and 1% sodium dodecyl sulfate. After incubating at 37°C overnight and then at 60°C for 30 min, the sample was combined with 5~1 of bromophenol blue and 20 gl of 95% glycerol. The mixture was then layered over a 10% polyacrylamide disc gel in a 0.5 x 62.mm column. The gel contained 0.1 M sodium phosphate buffer, pH 7.0, and 0.1% sodium dodecyl sulfate. The sample was run into the gel at 4 mA/gel, then electrophoresis was continued at 6 mA/gel until the dye marker ran to the end of the gel. After electrophoresis, each gel was washed in distilled water and was frozen in ethanol containing dry ice. The gel was sliced with stacked razor blades into 1.6-mm sections. Gel slices were then placed into scintillation vials with 3 Abbreviations used: DDT, sodium dodecyl sulfate.

dithiothreitol;

SDS,

416

SHIH AND CHAK

4 ml of toluene-based scintillation tuid containing 10% NCS and 1% H,O. After incubation at 45°C for 12 h, the radioactivity was determined in a Beckman LS-335 liquid scintillation counter. Radioactivity determinations from duplicate gels agreed to within 55% and have been averaged for all data presented. When the same sample was run into more than one gel, the radioactivities from these gels were pooled. The same result was obtained when these experiments were repeated three times.

purified phenylalanine:pyruvate transaminase from normal rats is similar to the purified phenylalanine:pyruvate transaminase from glucagon-treated rats. This result suggests that the glucagon-induced enzyme is structurally similar to the basal enzyme if not identical. Immunochemical property of phenylalanine: pyruvate transaminase. As reported

by Shih and Chan (8), the antisera prepared against the A and B forms of phenylalanine: pyruvate transaminase have similar imElectrophoretic mobility of phenylalanine: munological properties. Therefore, they can pyruvate transaminase. As shown in Fig. 1, be used interchangeably. However, antibody the electrophoretic mobility of the partially prepared against A form of the enzyme was used throughout this study. At a given antiserum concentration, the immunochemical titration lines (Fig. 2) of phenylalanine: pyruvate transaminase from control and glucagon-treated rat livers are parallel and overlapping each other. Thus the equivalence points of the normal and induced enzyme are the same. This result indicates that the immunochemical properties of the basal and glucagon-induced phenylalanine: pyruvate transaminase are the same and that the increase in enzyme activity after glucagon treatment is due to an increase in enzyme concentration. Because the equivalence points for normal and glucagon-induced phenylalanine:pyruvate transaminase are the same, we may use the same antibody preparation to precipitate phenylalanine: pyruvate transaminase from both normal and glucagon-treated rats. RESULTS

AND DISCUSSION

Identification of radioactive phenylalanine: pyruvate transaminase in sodium dodecyl sulfate -acrylamide gel. An increase

G+N G

N

FIG. 1. Electrophoresis of phenylalanine:pyruvate transaminase from glucagon-treated (G) or normal (N) rat livers. Partially purified phenylalanine:pyruvate transaminase (300 pg) from normal rat liver (N), purified phenylalanine:pyruvate transaminase from glucagon (300 pg)-treated rats (G), or a mixture of the two (150 pg from each sample, G + N was applied at the top (anode) of each gel. At the end of the electrophoresis, gels were stained for transaminase activity with p-nitrotetrazolium blue to reveal the location of the enzyme. The band near the botton of the gel was from the tracking dye bromophenol blue. See text for details.

in enzyme molecules may be caused by an increase in its rate of synthesis, or by a decrease in its rate of degradation, or by changes in both rates. To test the first possibility, the rate of synthesis of phenylalanine: pyruvate transaminase was determined by pulse-labeling the enzyme with [“Hlleucine, followed by immunoprecipitation. The antibody-antigen complex formed was dissociated and subjected to SDS-gel electrophoresis as indicated under Materials and Methods. As shown in Fig. 3 the position of the radioactivity peak on SDS-gel from a glucagon-treated sample coincides with the purified phenylalanine:pyruvate transami-

INDUCTION

OF PHENYLALANINE

40

60 ENZYME

(nmol

FIG. 2. Immunochemical titration treated rats (0 0). The heated catalytic activity and reacted with at 3’i”C for 30 min. then overnight determined by radioassay. See text

product

TRANSAMINASE

80

loo

ACTIVITY

ADDED

417

120

formed ilmin / mg protein)

of phenylalanine:pyruvate transaminase from control (x. x) and glucagonsupernatant fraction from a 30,0009 centrifugation was diluted to appropriate a 1:16 dilution of anti-phenylalanine:pyruvate transaminase A antiserum at 4°C. The remaining phenylalanine:pyruvate transaminase activity Fvas for details.

nase (Fig. 3, right). Furthermore, using ovalbumin, aldolase, chymotrypsinogen A, and ribonuclease A as standard proteins, the approximate molecular weight of the radioactivity peak is 42,000, identical to that of purified phenylalanine:pyruvate transaminase. A similar result is obtained when a sample from normal liver is used (Fig. 3, left). These results indicate that the 42,000 M, radioactive peak observed on SDS-gel is phenylalanine:pyruvate transaminase. The nature of the other small radioactive peak observed on SDS-gel is not clear at the present time. In a control precipitation experiments, fivefold the amount of purified phenylalanine:pyruvate transaminase activity was added to the heated liver supernatant fraction from the [:‘H]leucine- and glucagontreated rat, and immunoprecipitation and SDS-gel electrophoresis were then followed. In this case, the radioactivity peak at a molecular weight of 42,000 decreased accordingly. This result indicates that the nonspecific binding at this radioactive peak is negligible.

Evidencefor de novo synthesis of phenylalanine:pyruvate transaminase. Radioac-

tivity profiles of the immunoprecipitates on SDS-acrylamide gels before (left) and after (right) glucagon treatment are shown in Fig. 3. When immunoprecipitates obtained from 0.3 g of glucagon-treated or 0.4 g of normal rat livers were separated on SDSgel, the [“Hlleucine incorporated into phenylalanine:pyruvate transaminase was significantly higher in the glucagon-treated animal. This result demonstrates that the increase in phenylalanine:pyruvate transaminase activity is due to an increase in newly synthesized transaminase molecules. The effects of glucagon, dibutyryl cyclic AMP, cycloheximide, and actinomycin D on [“Hlleucine incorporation and catalytic activity of phenylalanine:pyruvate transaminase are summarized in Table I. Since the half-life of the transaminase is approximately 3 days (15), the amount of radioactive transaminase lost due to degradation in 1 h of labeling time is insignificant. Glucagon was given for 32 h because Fuller et al. (16)

418

SHIH

AND CHAN

24 r 0I ! "

20

n

16

if 4 5 ,”

12

0 v Z?lI

0

4

0

FIG. 3. Radioactivity profile of rat liver phenylalanine:pyruvate transaminase on SDS-acrylamide gels before (left) and after (right) glucagon treatment. Rats weighing about 180 g each m-ere injected intraperitoneally with 200 PCiilOO g body wt of L-[4,j-:‘H(N)]leucine 1 h before death or 31 h after a single glucagon injection. The liver supernatant fraction from the 30,OOOgcentrifugation was heated at 60°C for 10 min, then reacted with antibody. The antibody-enzyme precipitate was prepared as described under Materials and Methods. Precipitates obtained from 0.4 g of normal or 0.3 g of glucagon-treated livers were separately dissolved in 1% DDT, 1% SDS, 1% 2-mercaptoethanol, and Tris-HCI buffer, pH 7.6. Each sample (350 fig immunoprecipitates) was then applied to a SDS-polyacrylamide gel and subjected to electrophoresis as described under Materials and Methods. Ovalbumin (ILf, = 45,000). aldolase (M, = 40,000), chymotrypsinogen A (M, = 25,000), ribonuclease A (M, = 13,700) were used as standard proteins in electrophoresis for molecular weight determination.

have shown that the phenylalanine:pyruvate transaminase activity reached a peak level between 24 and 48 h after glucagon injection. As indicated in Table I, 32 h after glucagon treatment the transaminase catalytic activity was increased 4.5-fold above the basal activity. Similarly, [3H]leucine incorporated into the transaminase was increased 4.5-fold. This result indicates that the glucagon induction of this transaminase is via de novo synthesis. It was also observed that a similar study with a similar conclusion has been reported for mitochondrial serine:pyruvate aminotransferase in rat liver (17). [“HJleucine incorporation into total soluble proteins was not changed in either normal of glucagontreated rats. Thus, the 4.5-fold increase in the rate of synthesis of this enzyme was

contributed neither by any increase in total protein synthesis, nor by any modifications due to differences in intrahepatic leucine specific activity in the two groups. Moreover, the increases in [“Hlleucine incorporation and catalytic activity of phenylalanine: pyruvate transaminase were both blocked if cycloheximide, an inhibitor of protein synthesis, was given simultaneously with glucagon (Table I). Cycloheximide probably blocked the formation of mRNA for the transaminase, or destabilized it. When actinomycin D was given at the same time as glucagon, the catalytic activity was decreased to the basal level, 12.5 units (Table I). The percentage relative synthesis rate for phenylalanine:pyruvate transaminase was 0.06, identical to that in normal

419

INDUCTIONOFPHENYLALANINETRANSAMINASE TABLE1 THE

EFFECT

OF GLUCAGON, DIBUTYRYL CYCLIC AMP, ACTINOMYCIN D, AND CYCLOHEXIMIDE ON THE INDUCTION OF PHENYLALANINE:~YRUVATE TRANSAMINASE” Phenylalanine:pyruvate transaminase catalytic activity

Treatment Normal rats Glucagon-treated rats Cyclohexirmde and glucagon given simultaneously Actmomycin D and glucagon given simultaneously Actmomycin D given at -1 h after glucagon Dibutyryl cyclic AMPtreated rats Cyclohexinude and dibutryl cyclic AMP aven simultaneously

nmollminlmg protein

Ratio compared to normal

I’HILeuane Phenylalanine: pyruvate transaminase, A (cpmimg protein)

incorporated

Total soluble protein, B (cpmlmg protein)

Relative synthetic rate, A/B (‘0

Ratio compared to nornlal

12.3 54.9

1.0 4.5

1.12 6.73

2.m 252x

0.06 0.27

1.11 1.5

17.1

1.4

2.29

5207

0.07

1.2

12.5

1.0

1.26

2528

0.06

1.0

5.5

1.5

7.02

2436

0 29

1.x

61.3

5.0

6.03

1X.U

0.33

5.5

15.1

1.2

1.26

1x44

0.07

1.2

” Cycloheximide (2 mgikg) or actinomycm D (1 mg/kg) was given intraperitoneally at either 0 01‘ 1 h after glucagon (or cychc AMP) injection, A second dose of these two drugs was given at 15 h aPer glucagon (or cyclic AMP) injectIon due to their short effective half-lives (12. 1:3) I’HILeueine (200 &ii100 g body wt) was injected intraperitoneally 31 h after a single glucagon or dlbutyryi cyclic .4MP Injection. The same dosage of I’Hlleucine was injected mto control animals. The rats were decapitated 1 h later. One and one-half grams OS each hver WBP homoyenized and centrifuged. The supernatant fraction from the 30,OOOy centrifugation was heated at 60°C for 11~rmn. The precipitates were removed by centrifugatlon. The clear supernatant from 15 ml homogenate (about 1‘2 mg protein/ml) was reacted with antiserum prepared against A form of phenylalanine:pyruvate transaminase. The antigen-antibody precipitates formed were dissocmted and run on two to three SDS-gels. [ ‘Hll,eucme incorporated into the transammase was determmed by counting the corresponding gel slices. [~‘HILeucine incorporated mto the total soluble proterns was determined by 10’4 trichloroaeetic aad precipitation of the 30,OOOg supernatant fraction. The phenylalanine:pyru\~te transaminase catalytic activity was determined by spectrophotometric method. See text for details. This experiment was repeated three times. The data reported herr are from one of the experiments. Each value reported here 1s the average for two to three animals. The -tandard error was less than lo’;.

rats (0.06). Blockage of the glucagon effect by cycloheximide and actinomycin D reinforces the view that this induction is via de no~o synthesis. However, actinomycin D did not inhibit glucagon induction when it was given 4 h after glucagon injection. Under this condition, the percentage relative synthesis rate of the transaminase was 0.29; the rate of synthesis of the transaminase was increased 4.8-fold over the controls, and the catalytic activity of phenylalanine: pyruvate transaminase was 4.5-fold higher than the normal. The failure of actinomycin D given 4 h after glucagon to block enzyme synthesis suggests that the transcription of phenylalanine:pyruvate transaminase may be completed within 4 h after glucagon injection. This result is consistent with Fuller’s report on phenylalanine:pyruvate transaminase activity in crude liver extracts (7). The involvement of RNA metabolism in enzyme regulation in animal tissues has also been reported in other systems, such as the effect of glucocorticoid on rat tryptophan pyrrolase

and tyrosine transaminase (18) an? ’ %rogen on chick oviduct ovalbumin synthesis (19). As indicated in Table I, when dibutyryl cyclic AMP instead of glucagon was given to rats, phenylalanine:pyruvate transaminase activity was 5.0-fold higher than the normal. Similar finding was reported by Fuller et al. (6). Interestingly, [“Hlleucine incorporation was also increased, by 5.5-fold. Furthermore, the increase in catalytic activity and [“Hlleucine incorporation can be blocked by cycloheximide. These results suggest that the mechanism of glucagon induction of phenylalanine: pyruvate transaminase may be mediated by cyclic AMP. This suggestion is supported by the recent finding that the induction of tyrosine aminotransferase (20,21) or phosphoenolpyruvate carboxykinase (22) by dibutyryl cyclic AMP is accompanied by an increase in the translational mRNA coding for the enzyme. The induction of phenylalanine:pyruvate transarninase appears to be directly the result of glucagon (or cyclic AMP), since Fuller et al.

420

SHIH

AND CHAN

(6) have demonstrated the lack of effect of insulin on elevation of this transaminase by glucagon and dibutyryl cyclic AMP. In summary, the present study indicates that the basal and glucagon-induced phenylalanine:pyruvate transaminase are similar in their electrophoretic mobilities, equivalence points, and molecular weights. Furthermore the glucagon induction of this enzyme is shown to be via de novo synthesis, possibly mediated by cyclic AMP. REFERENCES 1. LIN. E. C. C., PITT, B. M., CIVEN, M., ANDKNOX. W. E. (1958) J. Biol. Chen!. 233, 668-673. 2. SHIH, J. C. (1975) Life Sci. 17, 627-632. 3. SHIH, J. C., CHIU, R. H., AND CHAN, Y. I,. (1976) Biochewt. Biophy.s. Res. Cornmurr. 68, 13481355. 4. GIVEN, M., TRIMMER, B. M., AND BROWN. C. B. (1967) Lfe Sci. 6, 1331-1338. 5. BROWN, C. B.. AND CIVEN, M. (1969)Endoo-ino/ogy 84, 381-385. 6. FULLER, R. W., SNODDY. H. D., AND BROMER. W. W. (1972) &‘o!. Pharrrmcol. 8, 343-352. 7. FULLER, R. W., BAKER, J. C., AND BROMER, W. W. (1973) E&oc?i~oloyy 93, 238-240. 8. SHIH, J. C., AND CHAN, Y. L. (1978) Arch. Biochen~. Biophys. 189, 343-338.

9. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.. AND RANDALL, R. J. (1951) J. Riol. Che?~. 193, 265-275. 10. DAVIS, B. J. (1964) AWL 1%‘.Y. Acad. Sci. 121, 402-427. 11. VALERIOTE, F. A., AURICCHIO, F., TOMKINS, G. M., AND RILEY, D. (1969) J. Biol. Chew. 244, 3618-3624. 12. ROTHBLUM, L. I., DEVLIN, T. M., AND CH’IH, J. .J. (1976) Biochew,. JI. 156, 151-157. 13. SCHWARTZ, H. S.. SODERGREN. J. E., AND STERNBERG, S. S. (1965) Ca>lcer Res. 25, 307-317. 14. WEBER, K., AND OSBORN, M. (1969)J. Biol. Che),r. 244, 4406-4412. 1.5. CHAN. Y. L., AND SHIH, J. C. (1978) Bitching. Biophys. Acta 526, 100-106. 16. FULLER, R. VI;., BAKER, J. C., AND SNODDY, H. D. (1974) Biochern. Med. 9, 301-308. 17. FUKUSHIMA, M., AIHARA, Y., AND ICHIYAMA, A. (1978) J. Biol. Chem. 253, 1187-1194. 18. GARREN, L. D., HOWELL, R. R., TOMKINS, G. M., AND CROCCO, R. M. (1964), Proc. ‘Vat. Acad. Sci. USA 52, 1121-1129. 19. PALMITER, R. I)., AND SCHMIKE, R. T. (1973) d. Biol. Chem. 248, 1502-1312. 20. ERNEST, M. J., AND FEIGELSON, P. (1978)6. Biol. Chew?. 253, 319-322. 21. NOGUCHI, T.. DIESTERHAFT, M., AND GRANNER, D. (1978) J. Biol. Chem. 253, 1332-1335. 22. IYP~EDJIAN, P. B., AXD HANSON, R. W. (1977) d. Riol. Chrrn. 252, 655-662.