JOURNAL OF PATHOLOGY, VOL. 158: 53-56 (1 989)

THE MICROSOMAL GLUCOSE-6-PHOSPHATASE ENZYME OF HUMAN GALL-BLADDER

ALEXANDER HILL, IAN D. WADDELL, DAVID HOPWOOD* AND ANN BURCHELL

Departments of Medicine and Pathology*, Ninewells Hospital and Medical School, University of Dundee, Dundee DDI 9SY, U.K.

Received 27 September 1988 Revised 7 November 1988

SUMMARY Microsomes isolated from adult human gall-bladders have for the first time been shown to contain specific glucose-6-

phosphatase activity. The gall-bladder glucose-6-phosphatase enzyme has the same molecular weight (36 500 daltons) and similar immunological properties and kinetic characteristics to the hepatic microsomal glucose-6-phosphatase enzyme. KEY wo~~~-GI~co~e-6-phosphatase , gall-bladder, microsomes.

INTRODUCTION

The hepatic microsomal glucose-6-phosphatase enzyme (EC 3.1.3.9) catalyses a key step in the regu- lation of blood glucose levels,' and it has recently been suggested that glucose-6-phosphatase also plays a role in regulating cytosolic Ca2+ The microsomal glucose-6-phosphatase system has been demonstrated to be present only in liver,

and cells of pancreatic islet^.^ Type 1 glycogen storage disease patients exhibit a

number of symptoms which do not appear to be related to the role of the glucose-6-phosphatase system in liver, kidney, or pancreatic islets. It is therefore important to determine whether the glucose-6-phosphatase system is also present in any other tissues.

There has been one report of the measurement of low levels of glucose-6-phosphate hydrolysis in guinea-pig gall-bladder,' but this report failed to establish whether the activity measured was due to the presence of the microsomal glucose-6-phospha- tase system or merely the non-specific hydrolysis of glucose-6-phosphate by other phosphatases in the test-tube.

Addressee for correspondence: Dr Ann Burchell, Department of Medicine, Ninewells Hospital and Medical School, University of Dundee. Dundee DD19SY, U.K.

0022-341 7/89/050053-O4 $05.00 @ 1989 by John Wiley & Sons, Ltd.

This study was therefore undertaken to establish if the specific microsomal glucose-6-phosphatase enzyme system is present in human gall-bladder.

MATERIALS AND METHODS

Eiotinylated anti-sheep antibody and streptavi- din linked peroxidase complex were purchased from Amersham International plc (Amersham, U.K.). 4-Chloro-1-naphthol, glucose-6-phosphate (monosodium salt), mannose-6-phosphate (mono- sodium salt), and prestained molecular weight markers were obtained from the Sigma Chemical Co. (Poole, Dorset, U.K.). Empigen BB was the kind gift of Albright and Wilson (Whitehaven U.K.). Nitrocellulose was obtained from Schleicher and Schuell, (Dassell, F.R.G.). CNBr-activated Sepharose was from Pharmacia (Uppsala, Sweden). All other reagents were analytical reagent grade.

Assay of glucose-6-phosphatase Microsomes were isolated from fresh (unfrozen)

human gall-bladder and liver samples as described previously.8 Glucose-6-phosphatase and mannose- 6-phosphatase activity and microsomal intactness were measured and calculated as described pre- viously9 and expressed as nmol Pi released/min/mg microsomal protein. Non-specific hydrolysis of glucose-6-phosphate was assayed and corrected for

54 A. HILL ETAL.

40

30

20

10

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as described previously.'o Protein concentrations were measured by the method of Peterson.' '

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Immunoblot analysis Microsomal samples were subjected to electro-

phoresis on a 7-16 per cent SDS polyacrylamide gel as described by LaemmliI2 or electrophoretically transferred to nitro~ellulose'~ in the presence of 1 per cent Empigen BBI4 and visualized with sheep antibodies previously shown to be monospecific for the microsomal glucose-6-phosphatase enzyme',' and a biotinylated anti-sheep second antibody and a streptavidin-linked peroxidase complex as the detection system using 4-chloronaphthol as substrate.

Human gall-bladders A total of 25 human gall-bladders were studied:

these had been obtained at the time of cholecystec- tomy undertaken for the usual medical indications. The gall-bladders were received within 10 min of ligation of the cystic artery and immediately placed in oxygenated saline 4°C. Portions of the fundus of the gall-bladder were cut after gentle washing with saline to remove surface bile and debris. The gall- bladder mucosa was carefully separated from the muscle layer by blunt dissection and then the mucosa was homogenized as described above; ulcer- ated or torn mucosa was not used. The remainder of the gall-bladder was assessed by a pathologist (D.H.) for the degree of chronic cholecystitis. The gall-bladders were assessed and graded from normal (1) to severely diseased (5 ) , based on the appearance of the epithelial cells, the muscle layer thickness, the size and site of Rokitansky-Aschoff sinuses, and the amount of chronic inflammatory infiltrate. l 5

Thecontrol adult human liver for the biochemical assays was taken at cholecystectomy for cholelith- iasis and has been described in more detail as case F in ref. 8.

Ethical approval for this study was given by the Tayside Health Board Ethical Committee.

RESULTS

Glucose-6-phosphatase activity was found to be present in microsomal fractions isolated from the mucosa of all the adult human gall-bladders studied (Fig. I). In all cases, the level of activity in the gall- bladder was much lower than the average specific

A

Fig. 1-Glucose-6-phosphatase activity in human gall-bladder. Glucose-6-phosphatase activity (expressed as nmollminjmg microsomal protein) was measured in fully disrupted micro- somes with 30 mM glucose-6-phosphate as substrate. (A-A) Gall-bladders containing gallstones; (n-n) Gall-bladders without gallstones

180- 116- 84- 58- 48- 36- 26-

Fig. 2-Immunobolt analysis of human hepatic gall-bladder microsomes. Lanes I and 4: prestained Sigma molecular weight standards (9pg); lane 2: adult human hepatic microsomes (10 p g ) ; lane 3: adult human gall-bladder microsomes ( 1 12 pg)

HUMAN GALL-BLADDER GLUCOSE-6-PHOSPHATASE 55

activity of 0.49 pmol/min/mg microsomal protein previously reported’ for the glucose-6-phosphatase enzyme in control adult human liver. The level of glucose-6-phosphatase activity found in human gall-bladder microsomes varied considerably depending on the severity of the diseased state of the gall-bladder tissue (Fig. I ) . The Km of the glucose-6- phosphatase enzyme in gall-bladder samples was found to be 1.2 f. 0.2 mM, which is very similiar to the range of K, values of 0.43-1.3 mM previously reported’ for the human adult hepatic microsomal glucose-6-phosphatase enzyme.

Microsomes isolated from human liver and mucosa gall-bladder were subjected to immunobolt analysis (see Fig. 2) using a sheep anti-hepatic microsomal glucose-6-phosphatase antiserum previously shown to be monospecific for the 36 500 dalton polypeptide which contains the active site of the hepatic glucose-6-phosphatase enzyme.5316 Lane 3 of Fig. 2 shows that the human gall-bladder contains the liver-specific glucose-6-phosphatase enzyme protein.

lmmunoblotting and enzymatic analysis both showed that all the glucose-6-phosphatase enzyme was located in the gall-bladder mucosa and that there was no glucose-6-phosphatase enzyme in the gall-bladder muscle layer (data not shown).

DISCUSSION

The hepatic microsomal glucose-6-phosphatase is a multicomponent system comprised of the glucose-6-phosphatase enzyme with its active site situated a1 the luminal surhce of the endoplasmic reticulum membrane, and a translocase T1, which mediates the entry of glucose-6-phosphate into the luminal compartment. A second translocase T2 mediates the equilibration of the product phos- phate. The mechanism of glucose permeation across the membrane has been designated T 3 . 6 , 1 6 . 1 7 3 1 8 To measure the activity of the microsomal glucose-6- phosphatase enzyme in vitro, i t is necessary to fully disrupt the microsomal vesicles to remove the rate limitations imposed on the system by the translo- cases. In this study, the intactness of all the gall- bladder microsomal samples was determined prior to fully disrupting the microsomal membranes to measure glucose-6-phosphatase activity levels. The intactness of the microsomal preparations was somewhat variable, presumably due to the contami- nation of the tissue with bile acids which have

detergent-like properties and are often used in vim to disrupt microsomal vesicle^.'^

The glucose-6-phosphatase activity in the gall- bladders assessed to be grade 1 (which are as near to normal tissue as is ethically possible to obtain) was approximately 10 per cent of the level found in human liver microsomes (Fig. 1). This was con- firmed on the immunoblot (Fig. 2), where it can be seen that 1 12 pg of human gall-bladder microsomes contain very similar levels of the 36500 dalton glucose-6-phosphatase enzyme protein to 10 pg of human hepatic microsomes.

The major role of the glucose-6-phosphatase enzyme in liver and kidney is thought to be the pro- duction of glucose, which is rapidly released into the blood stream for utilization by other tissues. It seems extremely unlikely that the low levels of glucose-6-phosphatase activity present in the gall- bladder play a significant physiological role in the regulation of blood glucose levels. The hepatic and pancreatic p cell glucose-6-phosphatase enzymes are thought to play a significant role in the regu- lation of intracellular Ca” level^,^ and in the pancreas, intracellular Ca2+ levels pla a major role in the regulation of insulin release.” It therefore seems probable that the glucose-6-phosphatase enzyme in the gall-bladder is involved in the regu- lation of the gall-bladder intracellular Ca” levels.

ACKNOWLEDGEMENTS

The authors wish to thank Miss Lesley Stewart for her excellent technical assistance. This work was supported by grants from the Scottish Hospital Endowment Research Trust and the British Diabetic Association to A.B.

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