Molecular weight of microsomal glucose-6-phosphatase of rat and human liver

BIOCHEMICAL

Molecular

MEDlCINE

Weight

10,

:31%-319

( 1974)

of Microsomal of Rat and

P. J. COLLIPP, Research Laboratory, State University

Glucose-&Phosphatase

Human

Liver

A. CARSTEN,’ S. Y. CHEN, AND V. T. MADDAIAH

J. THOMAS,

Department of Pediatrics, Nassau County Medical Center, of New York, Stony Brook Health Sciences Center, East Meadow, New York 11554 Received

September

21,

1973

Hepatic glucose-6-P is a microsomal bound enzyme possessing pyrophosphatase and phosphotransferase activities in rats and human (l-3). Treatment of microsomes with detergents (4) or NH,OH (5) or A&O, (6) increases glucose-6-P and other associated activities. The enzyme has not yet been purified as the activity is either lost or becomes unstable when microsomes are treated with lipases or detergents for solubilization (7, 8). When Triton X-100 solubilized microsomal preparation was chromatographed on a column of Sephadex G-100 the enzymatic activity appeared in a protein peak corresponding to a molecular weight of 100,006 (3). Recently, Hinman and Phillips (9) have observed that 30+#5% of the protein of hepatic microsomal membranes on acrylamide gel electrophoresis migrated as a single band corresponding to a molecular weight of 52,000. We have now used acrylamide gel electrophoretic (10) as well as radiation inactivation technique (11, 12) to estimate the molecular weight of microsomal glucose-6-P activity from normal and diabetic rat liver and human liver obtained at autopsy. MATERIALS

AND

METHODS

Microsomes from 3 autopsied human livers and from liver of rats (Charles River, USA, weighing 150-260 g ) fasted for 24 hr were prepared and lyophihzed as described (6, 13). Diabetes was induced by an intraperitoneal injection of a single dose of alloxan monohydrate (250 mg/ kg of body weight). Induction of diabetes was checked by measuring blood and urinary sugar and the rats were used 4-6 weeks after alloxan administration. Experimental conditions for the separation of microsomal preparations into supernatant and residue fractions after treat1 Medical York. Copyright All rights

Research

Department,

Brookhaven

312 ($3 1974 by Academic Press, Inc. of reproduction in any form reserved.

National

Laboratory,

Upton,

New

MOLECULAR

WEIGHT

OF MICROSOMAL

GLUCOSE-6-PHOSPHATASE

313

ment with Al,O, and for measurement of glucose-6-P activity at 30” have been described ( 6, 13). Enzyme activity was expressed as nmoles of inorganic phosphate formed per minute per mg protein. Salt-washed microsomes were prepared as described by Hinman and Phillips (9). Polyacrylamide

Gel Electrophoresis

Proteins were electrophoresced by a modification of the method of Maize1 (14). Samples were dissolved in 0.01 M Tris-acetate buffer, pH 9.0, containing 1.0% sodium dodecyl sulfate (SDS), 0.001% EDTA, 2.0 bt urea and 0.1% betamercaptoethanol and the solution was made 10% with sucrose before applying to acrylamide gel columns. The polymerizing solution contained 5.0% acrylamide, 0.133 percent bis acrylamide, 0.95% ammonium persulfate, 0.05% N,N,N’,NI-tetramethylene diamine, 1.0% SDS, 2.0 M urea, 0.001% EDTA and 0.1 M Tris-acetate buffer, pH 9.0. Electrophoresis buffer was 0.1 M Tris-acetate, pH 9.0, 1.0% SDS, 0.1% beta-mercaptoethanol and 0.01% EDTA. Each microsomal preparation was electrophoresed in duplicate with bromophenol blue, providing a reference dye, for three hours in an ice cold water bath at 2.4 mA per gel. For protein detection the gels were stained for 3 hr in a solution of 1.0% amido black, 7.0% acetic acid and lO!% ethanol and destained overnight in a solution of 7.0% acetic acid and 10% ethanol. Method for molecular weight determination by SDS-polyacrylamide gel electrophoresis was as described by Osborn and Weber ( 10). The method used for staining glucose-6-P activity in acrylamide gels is a modification ( 15) of the Gomori ( 16) histochemical technique. Following electrophoresis, the gels were incubated at 37” for 30 min in a substrate solution at pH 6.5 containing 0.2 M glucose-6-P, 0.1 mg/ml asolectin (a mixture of soybean phosphatide from Associated concentrates) 5% partially hydrolyzed starch, 0.05 M sodium maleate. The gels were washed briefly with distilled water at room temperature and were then transferred to a solution containing 0.08 M Tris-maleate and 0.003 M Pb (NO,)., at pH 7.0. After 1 hr at 25”, the gels were again washed with several changes of water over a period of 24 hr and were then placed for 10-20 min in a solution containing 5% ammonium sulfide, and washed with water repeatedly. After the staining the gels were always longer than those that were stained for proteins by amido black. Irradiation Different microsomal preparations containing 30 mg protein in 10 ml capacity tubes were lyophilized. After lyophilization, the tubes were fitted with rubber stoppers, evacuated and stored at - 15”. Samples were gamma irradiated in a 60r, source array at a dose rate of approximately

314

COLLIPP

ET

AL.

10: rad/hr and a temperature of 30”. Dosimetry measurements 11ere made using a Perspex XH dosimeter ( l’i). L~nirradiated control samples were maintained at 30” in an oven for the same length of time as the irradiated samples. After irradiation, samples were stored at -15” until enzyme activities were assayed. Samples were resuspended in 1 ml ice-cold water just before assay. Enzyme activities of irradiated samples were expressed as percent activity of the corresponding unirradiated samples. RESULTS

Figure 1 shows the electrophoretic pattern of whole microsomes, saltwashed microsomes and Al,O,, treated supernatant preparation from human liver. Whole microsomes as well as the A&O, treated supernatant showed several protein bands but showed only one band for glucose-6-P activity after the gels were stained with ammonium sulfide. Salt-washed microsomes had one minor protein band and again showed only one glucose-62 activity band. The major protein band bearing the enzymatic activity appears to be common to all the three preparations. Glucose-62 activities of whole microsomes, salt-washed and Al,O, treated supernatant preparations were 40, 55 and 80 nmoles of P/mg protein/min, respectively. Treatment of microsomes with Al,O,, has

FIG. 1. Polyacrylamide gel electrophoresis of human liver on microsomal preparations. 1, Whole microsomes; 2, salt-washed microsomes; 3, AljO treated microsomal supematant. A. Gels stained for proteins with amido black; B. gels stained for glucose6-P activity.

MOLECULAR

WEIGHT

EFFECT

Whole microsomes Washed microsomes

OF MICROSOMAL

OF W.ISHING

TABLE 1 ON CLI-cosd-P

GLUCOSE-6-PHOSPHATASE

315

ACTIVITY~~~

Normally fed rat, liver

1 l)ay fasted rat, liver

110 176

190 540

Alloxan diabetic rat liver 440 67.5

1 Each value is an average of from three rats. ? A&vity is expressed as nmoles P/mg proteiri/min.

already been shown (6) to activate glucose-6-P and other associated activities. Glucose-6-P activities of unwashed and salt-washed microsomes of normally fed, 1 day fasted and alloxan diabetic rat liver are shown in Table 1. Enzymatic activity showed a similar increase after salt-washing in all the three preparations. Electrophoretic pattern of the three saltwashed preparations are shown in Fig. 2. In all three preparations the pattern and rate of migration were similar. There was one major protein band with l-2 minor bands. There was only one glucose-6-P activity band

FK:. 2. Polyacrylamide gel electrophoresis of salt-washed liver microsomes from rats under different treatment. 1, Normally fed; 2, 1 day fasted; 3, aloxan diabetic. A. Gels stained for proteins with amido black; B. gels stained for glucose-&phosphatase activity. The arrow indicates gel origin.

316

COLLIPP

ET

AL.

whose migration rate was identical with that obtained with human liver preparations. In Fig. 3 a semilog plot of the relative migration rate against molecular weight of marker proteins, serum albumin ( 68,000)) pepsin (35,000), trypsin (23,300) and cytochrome C (11,700) is shown. A linear relationship between migration rate and log molecular weight becomes apparent. The migration distance of glucose-6-P activity band obtained after staining with ammonium sulfide corresponded to a molecular weight of 63,000 +: 6,800. Whole microsomes, Al,O, treated supernatant, and residue fractions of human and rat liver were irradiated and glucose-6-P and pyrophosphatase activities were expressed as percent activity of the corresponding sample. There was no statistically significant difference in the percent survival of enzymatic activities of whole microsomes and the supernantant and residue fractions, obtained after the treatment of microsomes with Al,O,?, at a given radiation dose. Therefore, the percent surviving activities of whole microsomes, Al,O, treated supernatant and residue fractions were pooled and were subjected to computer analysis for both a linear and quadratic fit. The analysis indicated that these data were best represented by a single exponential equation and yielded 9.8, 8.1, 10.3 and 8.16 Mrad for 37% survival of glucose-6-P and pyrophosphatase activities of human and rat liver, respectively. As there was no significant difference

FIG. 3. Electrophoretic on polyacrylamide gels.

migration

rate

of marker

proteins

and

glucose-6-P

activity

MOLECULAR

WEIGHT

OF MICROSOMAL

317

GLUCOSE-6-PHOSPHATASE

between slope and intercept terms in the linear regression equations of glucose-6-P and pyrophosphatase activities of human and rat Iiver the two enzymatic activities were combined. Computer generated semilogarithmic plot of percent surviving activity from human liver against radiation dose is shown in Fig. 4 from which a value of 10.16 Mrad was obtained for 37% survival of enzymatic activity (D:<;). A radiation dose of 8.12 Mrad was similarly obtained from a semiIog plot of data from rat liver (not shown). Considering the nature of the experiment the two D3? values appear to be within experimenta error. Radiation “target size” may be calculated from D,; value which represents an average of one inactivating event per active unit. According to the classical “‘target” theory of Lea (B), the average value of 9.12 Mrad for 37% survival of glucose-64 and pyrophosphatase activities of microsomes from human and rat liver would correspond to a molecular weight of 77,000. A similar value of 70,000 could be obtained from the cahbration curve constructed by Keyner and Macy (11) for radiation inactivation of proteins of known molecuIar weight. The two metho& use a slightly different vahre for the average energy IOSS per inactivating event. As a result, the estimate of moIecular weight from Lea’s CaIcuIations (18) will be slightly higher (12).

0

I 2.0

4.0I

I 6.0

I 6.0 Rodiatm

I 1 r 10.0 12.0 14.0 Dose (M fads)

I 16.0

I 16.0

I 20.0

FIG. 4. Radiation inactivation of human liver microsomal glucose-6-P bols) and pyrophosphatase (closed symbols) activities. 0, 0, whole A, A, Al,03 treated supernatant preparation; PA, +, Al&& treated residue

(open symmicrosomes; preparation.

31s

COLIA’P

ET

AL.

IXSCUSSIOiX

The protein electrophoretic pattern reported herein for whok microsomes from human liver appears to be similar to that reported for rat liver by Hinman and Phillips. Successive washing of rat liver microsomes with salt solutions removed 5%70% of the proteins and the remainder migrated as a single protein band on polyacrylamide gel electrophoresis. Hinman and Phillips (9) concluded that washing removed the contaminant cytoplasmic and ribosomal proteins and that the remaining protein represented the microsomal membrane. Successive washing of microsomes of human liver also resulted in one major protein band with an electrophoretic mobility identical to that from rat liver. Treatment of whole microsomes with Al,O,, to raise the pH to 9.0 did not reduce the number of protein bands, This is in agreement with our previous conclusion reached by ultracentrifugation and electron microscopic studies that Al,O, treatment caused fragmentation of microsomes ( 6). The electrophoretic mobility of the protein band that showed glucose6-P activity on incubation with the substrate and subsequent staining with ammonium sulfide was identical in all preparations including washed microsomes from liver of rats on different nutritional and hormonal status. These results suggest that there are no significant change or size differences between glucose-62 activity of human and rat liver and among normally fed, 1 day fasted and alloxan diabetic rats. Teleologically it would make sensethat glucose-6-P is an integral part of microsomal membrane and significantly contribute towards structure and function of the membranes. The conclusion that glucose-62 protein of human and rat liver may be similar to one another is further confirmed by their similarity in the rate of their inactivation on irradiation. Radiation values for 37%survival of enzymatic activity, i.e., D,;, which is related to “target volume” of the sample being irradiated, are similar within experimental error to one another. However, whole microsomes of human liver were antigenically different from rat microsomes (6). Molecular weight deduced from electrophoretic mobility measurements (63,000 ?I 6,800) is in fair agreement with that estimated from radiation inactivation data (70,000). That the two methods based 011 entirely different assumptions and procedures gave a similar estimate is encouraging. SUMMARY

Salt-washed and unwashed and aluminum oxide treated microsomes of human liver, and washed microsomes of liver of normally fed, 1 day fasted and alloxan diabetic rats were subjected to polyacrylamide gel electrophoresis in presence of sodium dodecylsulfate and urea. Unwashed and aluminum oxide treated microsomes showed several protein bands,

MOLECULAR

WEIGHT

OF

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319

GLUCOSE-6-PHOSPHATASE

but salt-washed microsomes gave only one major protein band. This major protein band which appeared in all the preparations showed gincase-6-P activity on incubation of the electrophoresed gel with the substrate and subsequent staining with ammonium sulfide. Molecular weight of this protein was estimated to be 63,000 1 6,800. Glucose-6-P and pyrophosphatase activities were measured after subjecting lyophilized microsomes, aluminum oxide treated supernatant and residue fractions of human and rat liver to ionizing radiation in vacua at different doses (Mrads). There was no significant difference between either the surviving fraction of glucose-6-P and pyrophosphatase activities or among the different preparations of human and rat liver at a given radiation dose. Computer analysis of the pooled data gave a value of 9.16 Mrads for 37% survival of the activity which corresponds to a molecular weight of 70,000. ACKNOWLEDGMENTS

We thank

Dr. Keith Thompson Francis Rizzo for assistance with Meadowbrook Medical Education Atomic Energy Commission.

for assistance the irradiation. and Research

with computer analysis and Mr. Research was supported by the Foundation, Inc., and the U. S.

REFERENCES 1. 2. 3.

NORDLIE, R. C., AND ARION, W. J., J. Biol. Chem. 239, 1680 (1964). STETTEN, M., J. Biol. Chem. 239, 3576 ( 1964). COLLIPP, P. J., CHEN, S. Y., AND HALLE, M., Biochim. Biophys. Acta

(1968). 4. NoRDLIE,R.C., 5. STETTEN, M.

167,

141

AND kON, W.J.,J. Biol.Chem.240,2155 (1965). BURNETT, V. V., Biochim. Biophys. Acta 139, 136 (1967). 6. CHEN,S. Y., P. J., MADDAIAH, V. T., REZVAXX, I., AND DUFFY, J. L., Biochem. Med. 5, 237 ( 1971). 7. DUTTERA, S. M., BYRNE, W. L., AND GANOZA, M. C., J. Biol. Chem. 243, 2216 R., AND CO~IPP,

(1968). ZAICIM, D., J. Biol. Chem. 245, 4953 (1970). HINMAN, N. D., AND PHILLIPS, A. H., Science 170, 1222 (1970). WE==, K., AND O~BORN, M., J. Biol. Chem. 244, 4406 (1569). KEPNER, G. R., AND MACEY, R. I., Biochim. Biophys. Acra 163, 188 (1968). BI=M, E., AND ALPER, T., Biochem. J. 122, 677 ( 1971). 13. MADDAIAH, V. T., CHEN, S. Y., REZVANI, I., SHARMA, R., AND COLLIPP. P. J., Biochem. Biophys. Res. Commun. 43, 114 ( 1971). 14. MAIZEL, J. S., Science 151, 988 (1966). 15. DAVIS, C. H., SCHLISELFELD, L. H., WOLF, D. P., LEAVITT, C. A., AND KREBS, E. G., J. Biol. Chem. 242, 4824 (1967). 16. C(~MORr, G., Microscopic Biochemistry, The University of Chicago Press, Chicago, 1952. 17. R~zzo, F. J., CUNNINGHAM, G., AND GALANTA, L., Trans. A~~. iv&. Sot. 10, 53 (1967). 18. LEA, D., Actions of Radiation on Living Cells, Cambridge University press, London, 1965, p. 69.

8. 9. 10. 11. 12.