Comp. Biochem. Ph.rsiol.. Vol. 62B, pp. 85-87.

0305-0491/79/0115-0085502.00/0

© Pergamon Press Ltd 1979. Printed in Great Britain

SUBCELLULAR LOCALIZATION OF GLUCOSE-6-PHOSPHATASE IN ANIMAL TISSUES JEAN E. VORHABEN and JAMES W. CAMPBELL Department of Biology, William Marsh Rice University, Houston, TX 77001, U.S.A. (Received 24 April 1978) Abstract--1. Glucose-6-phosphatase (EC 3.1.3.9 D-glucose-6-phosphate phosphohydrolase) was found to be localized mainly in the endoplasmic reticulum (microsomal fraction) of all species of vertebrate liver tissue examined. 2. Hepatopancreas tissue from gastropod molluscs was found to be unique in showing the localization of glucose-6-phosphatase in the cytosol (soluble fraction). INTRODUCTION

MATERIALS AND METHODS

Glucose-6-phosphatase is a multifunctional enzyme catalyzing the hydrolysis of glucose-6-phosphate and inorganic pyrophosphate and the transfer of a phosp h o r y l function from pyrophosphate and carbamoylphosphate to glucose (Nordlie, 1971; Lueck et al., 1972; Hefferen & Howell, 1977). The enzyme is widely distributed among organisms and among mammalian tissues (Nordlie, 1971; Colilla et al., 1975). Since its first association with the endoplasmic reticulum (Hers et al., 1951), glucose-6-phosphatase has routinely been used as the marker enzyme for this structure (operationally, the microsomal fraction) in studies on the subceilular distribution of enzymes and other substances (Allfrey, 1959). In this report, we show that hepatopancreas from terrestrial snails is unique in showing the localization of this enzyme in the soluble fraction.

Tissue was fractionated by differential centrifugation in either 0.25M sucrose or in Greenawalt's medium (Greenawalt, 1974) as previously described (Vorhaben & Campbell, 1972, 1977) except that hepatopancreas homogenates were initially subjected to a low speed centrifugation (30g for 10rain). Cytochrome oxidase was used as the marker enzyme for the mitochondrial fraction and lactate dehydrogenase, pyruvate kinase or glucose-6-phosphate dehydrogenase for the soluble fraction. References for the specific assays used for these activities as well as for glucose-6-phosphatase activity are given by Vorhaben & Campbell (1972, 1977). RESULTS AND DISCUSSION

The subcellular distribution of glucose-6-phosphatase activity in liver tissue from various vertebrate species is shown in Table 1. In general, 60~ or more

Table 1. Summary subcellular distribution of glucose-6-phosphatase in vertebrate liver percent of total r~cover~ activity Fraction Species BIRDS C,uZZ~e domeetp[,c.~at CoZumba Zivlcc~ REPTILES Th~0nnophSa eZegana~ AMPHIBIANS l ~ e o ~ maeuZosua %#nopue Lmrois BUrG vaZZioeps Ran~ ea~asbeia~a Adult Tadpole F~S~ FWIo~aZumu~ Gamma TaZapia moesm.Z~ou

Soluble

Percent of homogenate un/ts r~cover~ in fmactions

63.6 (23.7)

3.3 (34.1) 0 (45.4)

92.1 83.3

2.9 (12.8)

93.4 (33.9)

3.6 (49.2)

121.7

8.4 (7.4) 6,0 (7.8) 8.6 (7.7)

78.3 (26.6) 81.3 (21.5) 88.8 (25.4)

13.0 (50.4) 5.6 (52.5) 0.4 (58.5)

iii.0 62.2 88.6

3.7 (7.8) 25.8 (8.2)

20.5 (13.1) 21.4 (5.3)

63.0 (22.2) 32.9 (18.4)

12.8 (57.0) 19.9 (76.1)

92.4 102.9

1.5 (4.5) 2.1 (8.0)

19.i (21.1) 30.2 (24.1)

77.9 (21.2) 61.4 (16.8)

i.~ (53.2) 6.3 (51.1)

91.3 83.9

25.0 34.5 16.4 17.7 27.8

86.5 43.3 71,3 72.0 62.1

Unlts~/g tissue Nuclear

Mitochond~ialf

223.2 (128.1) 678.7 (153.9)

3.8 (4.3) 9 8.6 (12.4)

28.1 (28.2) 27.8 (18.5)

54.9 (33.4)

201.6 (109.0)

0.i (4.1)

80.4 (103.7) 34.3 (89.5) 70.1 (105.9)

0.2 (5.8) 7,1 (18.2) 2,1 (8.4)

11.1 (89.8) 18.4 (49.0)

945.1 (121.7) 83.0 (53.6)

Microsc~al

SS-

D~a'12~o omer4,oana t4i~on u~d~Z4~8 //op/,t~utim ~ GuZei~hCh~a f e Z i s Chust, odipt, ez.=s fol~r ~Zua kete~oeZi~ FW-adapted SW-adapte~

20.6 104.0 77.8 162.3 385.0

~36.6) (92.0) (91.2) (121.5) (133.7)

252.1 (92.7) 484,2 (113.7)

4.8 10.2 8.5 6.1 5.0

(12.8) (4.6) (9.1) (8.1) (3.9)

10.2 (13.3) 6.7 (10.5)

(13.7) (19.2) (9.5) (27.2) (23.1)

13.0 (17.8) 8.4 (13,9)

(21.3) (21.1) (20.3) (25.1) (23.6)

72.4 (32.3) 79.9 (34.7)

4.0 12.0 3.8 4.~ 5.0

(52.2) (55.1) (61,1) (39,6) (49.4)

108.8 155.6 102.8 164.2 93.8

4.4 (38,7) 5.0 (40.8)

70.8 71.7

* #moles/hr; sum of activity in separate fractions. The sum of the protein in the separate fractions is given in parentheses in this column. The recovery of homogenate protein in different fractions was quantitative, i.e. the mean recovery for all species was 99.8yo. i" Values for "light" and "heavy" mitochondrial fractions combined. :~From Vorhaben & Campbell (1972). Percentage age of total recovered protein in fraction given in parentheses. 85

86

JEAN E. VORHABENand JAMES W. CAMPBELL Table 2. Subceilular distribution of glucose-6-phosphatase in gastropod hepatopancreas Glucose-6-phosphaTe l~action

Frotein (mg/g

~ (units/s

tissue)

tissue)

HeZ£Z aepml,sa: Homogenate 30 x g pellet Nuclea~ Heavy mitochort(h',ial L i g h t mitochond~ial Mica~m~a~al Soluble"

113.3 13.5 22.1 13.7 5,5 11.5 59.7

117.2 27.7 12.1 i0.i 4.6

Nuclear Heavy mitochund~ial .~M~tochondr4al

ll.l Solkd~le

58.1

I~' I p ~ t ee l l

-~ 6.6 21.0 61.3 8.4 2.8 0

glh~ose-6-phosphatase (u~tslg

tlgsue)

(% of total r~oovered )

66.6 7.0 5.4 3.2 1.8 7.i 45.9

9.9 7.6 4.5 2.6 i0.i 66.3

94.5 208.8 69.5 122.4 170.2 16.1 9.9 0

% of h:mogenate value z'~coweg~d i n f'msct ,Jpns 105.5

mmol,~z~

r ~ )

671.4 41.3 133.1 388.8 53.3 18.1 0

of hmmg~ate vaZue r ~ m a ~ e d in fractloms 111.3 OtaZa Za~l~:~a: i ' ~ t e 30 X g pelle~c

cgidase (% of total

P3a~vate kinase (unitslg (% of total tissue) r~oovered

478.8 15.0 17.7 12.0 5.4 32.7 '+00.7

105.6 -17.9 31.5 43.9 4.1 2.6 0

13. i 8,3 2.8 8.0 67,9

(% of total

recovered)

187.8 0 9.3 14.~ ~.8 9.3 177.2

i01.0

39.0 -7.0 4.4 1.6 4.3 36.2

186.9

-3.1 3.7 2.5 i.i 6.8 82.9

dehydrogepase (units/g tissue)

--0 4.3 6.7 2.2

4.3 82.4 114.5

296.1

---

114.2

---

5.1 5.1 7.1 20.2 345.7

i.-~

;-

~--

136.8

4.8 5.1 4.3 67.6

1.3 1.9 5.3 90.2 129.q

5.9 6.2 5.3 62.8 71,6

obZo,w ~ : ,t

P~avy ,K1x~hongh~d 1.~ n t i ~ Soluble

% of hcm~v~na~ value in n ~ t l o m

139.3 25.7 I0.0 10.6 7.2 12.2 67.7

96.s

186.7

--

14.4 25.9 97.2 28.3 20.0 6.4

7.5 13,5 50,6 14.7 10.4 3.3

93.0 ~ 9.6 5.9 6.6 10.4 46.6

97.1

---12.3 7.5 7.2 13.3 59.7

63.6

153.9

---

5.7 3.7 6.3 3.7 173,3

~T~

125.8

1.9 3.3 1.9 89.9 125.2

--

~--

~'-

Ii.i 5.1

17.2 7.3

0

0

48.4

74.9 51.~

A unit of activity equals 1 #mole/hr.

of the recovered activity was present in the microsomal fraction. The two exceptions were Rana catesbeiana tadpoles and the Atlantic croaker Micropogon undulatus. A high percentage of the phosphatase activity in tadpole liver was present in the nuclear and mitochondrial fractions. Glucose-6-phosphatase is a known activity of the nuclear membrane (Gunderson & Nordlie, 1975) and is present but to a lesser extent in the aduR bullfrog nuclear fraction. N o more than 10°/O of the phosphatase activity was present in the nuclear fraction in the other species examined. Neither the nuclear nor the mitochondrial fraction of tadpole liver was contaminated with the postmitochondrial fraction containing the microsomes. The nuclear fraction, for example, contained 1.3% of the recovered lactate dehydrogenase activity and the mitochondrial fraction (combined heavy and fight mitochondrial fractions) contained only 0.9%. In the croaker, the nuclear fraction contained 0.4% of the recovered lactate dehydrogenase and the mitochondrial fraction, 6.4%. In none of the species that, showed relatively high mitochondrial glucose-6-phosphatase activity was this due to contamination by the postmitochondrial fraction since more than 90% of their lactate dehydrogenase activity was recovered in the soluble fraction. The subcellular distribution of glucose-6-phosphatase in hepatopancreas tissue from three species of terrestrial snails is shown in Table 2. In contrast with vertebrate liver, 60% or more of the activity in this tissue is localized in the soluble fraction with no more than 13% occurring in the microsomal fraction. In both Helix and Otala, about half of that present in the microsomal fraction could be due to contamination by the postmicrosomal fraction since the microsomal fraction contained approx 5% of the pyruvate kinase and glucose-6-phosphate dehydrogenase

activities. In Strophocheilus, the microsomal fraction contains a slightly higher percentage of phosphatase activity. The localization of glucose-6-phosphatase in the soluble fraction of hepatopancreas indicates that either the enzyme is a cytosolic one in these species or else is easily solubilized from the endoplasmic reticulum during fractionation. Electron microscopy of the tissue has shown the presence in several cell types of the hepatopancreas of what appears to be typical smooth and rough endoplasmic reticulum (B. D. Boyan-Salyers, unpublished; Aboling-Krogis, 1970). Acknowledgements--This work was supported by grants from the National Science Foundation (PCM 75-13161) and the USPHS (AI 05006). REFERENCES ABOLINg-KRoGIs A. (1970) Alterations in the fine structure of cytoplasmic organelles in the hepatopancreatic cells of shell-regenerating snail, Helix pomatia L. Z. Ze//forsch. 108, 516--529. ALLFREY V. (1959) The isolation of subceUular components. In The Cell, Vol. I (Edited by BRACHET J. & MmSKY A. E.), pp. 193-290. Academic Press, New York. COULLA W., JORGENSON R. A. & NOgDLIE R. C. (1975) Mammalian carbamyl phosphate:glucose phosphotransterase and glucose-6-phosphate phosphohydrolase:' extended tissue distribution. Biochem. biophys. Acta 377,' 117-125. GgEENAW^LT J. W. (1974) The isolation of outer and inner mitochondrial membranes. Meth. Enzym. 31, 310-323. GUNDERSON H. M. & NORDL1E R. C. (1975) Carbamyl

phosphate:glucose phosphohydrolase of nuclear membrane. J. biol. Chem. 2JO, 3552-3559. ~ g ~ L N P. M. & HOWELLR. R. (1977) Genetic evidence for the common identity of glucose-6-phosphatase, pyrophosphate-glucose phosphotransferase, carbamylphosphate-glueose phosphotransferase and inorganic pyrophosphatase. Biochim. biophys, Acta 496, 431--435.

Subcellular localization of glucose-6-phosphatase in animal tissues HERS H. G., BERTHETJ., BERTHETL. & DE DUVE C. (1951) Le syst/~me hexose-phosphatasique. III. Localisation intra-ceilulaire des ferments centrifugation fractionn6e. Bull. Soc. Chim. biol. 33, 21-41. LUECK J. D., HEgaMANJ. L. & NORDLIER. C. (1972) General kinetic mechanism of microsomal carbamyl phosphate: glucose phosphotransferase, glucose-6-phosphatase, and other associated activities. Biochemistry 11, 2792-2799. NORDLIE R. C. (1971) Glucose-6-phosphatase, hydrolytic

87

and synthetic activities. In The Enzymes, Vol. 4. (Edited by BOYER P. D.), 3rd edition, pp. 543-610. Academic Press, New York. VOgHABENJ. E. & CAMPBELLI. W. (1972) Glutamine synthetase. A mitochondrial enzyme in uricotelic species. J. biol. Chem. 247, 2763-2767. VOgHABEN J. E. ~ CAMPBELL J. W. (1977) Submitochondrial localization and function of enzymes of glutamine metabolism in avian liver. J. Cell. Biol. 73, 300-310.

Subcellular localization of glucose-6-phosphatase in animal tissues.

Comp. Biochem. Ph.rsiol.. Vol. 62B, pp. 85-87. 0305-0491/79/0115-0085502.00/0 © Pergamon Press Ltd 1979. Printed in Great Britain SUBCELLULAR LOCAL...
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