Preparation and biochemical characterisation of isolated parenchymal cells from adult sheep liver

475

Biochimica et Biophysica Acta, 496 (1977) 4 7 5 - - 4 8 3 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 28165

P R E P A R A T I O N AND BIOCHEMICAL C H A R A C T E R I S A T I O N OF ISOLATED PARENCHYMAL CELLS FROM A D U L T SHEEP LIVER

R O G E R A S H * and C H R I S T O P H E R I. P O G S O N

Biological Laboratory, University of Kent, Canterbury, CT2 7NJ, Kent (U.K) (Received July 23rd, 1976)

Summary 1. A simple procedure for the isolation of morphologically intact, metabolically viable sheep liver parenchymal cells is described in detail 2. The method is based on the initial treatment of fresh liver slices with collagenase and hyaluronidase. 3. The cell preparation was studied with respect to membrane permeability, potassium content, ATP/ADP ratio, adenylate content, and gluconeogenic capacity with respect to various substrates. 4. Data are presented with respect to the distribution of phosphoenolpyru- / vate carboxykinase in isolated cells and whole sheep liver. 5. The results are compared, where possible, with data currently available from isolated perfused sheep liver and multi-catheterised animals.

Introduction The study of ruminant liver metabolism has, in recent years, involved the use of two experimental techniques, namely, the perfusion of the excised liver [1], and the preparation of animals with chronically catheterised portal-hepatic venous systems [2--4]. These techniques require considerable surgical expertise, expensive equipment, and, in the latter case, long-term postoperative care, and thus, although both methods have much to recommend them, they are nevertheless of limited availability. It is perhaps a measure of the economic importance of the ruminant animal that such experimental techniques are being successfully employed in a limited number of laboratories. This paper describes in full a new approach to the study of ruminant liver m e t a b o l i s m which is relatively inexpensive, requires no surgical prowess, and yet allows a large number of variables to be studied under controlled experi* Present address: School of Studies in Applied Biology, Brvdford, West Yorkshire, BD7 1DP, U.K.

476 mental conditions. The method involves the preparation and utilisation of suspensions of enzymatically isolated, metabolically viable, ovine hepatocytes. Data relevant to the assessment of the integrity and metabolic viability of the isolated hepatocytes are also presented. Preliminary reports of some of these results have been published [ 5,36]. Materials and Methods

Sheep liver was obtained from a local abattoir. Control over the age, sex, previous nutrition and the physiological state of the animals was not exercised, b u t all animals used were functional ruminants varying in age from six months to approximately two years. All enzymes and fine biochemicals with the exception of hyaluronidase, glucose oxidase, peroxidase and digitonin (all from Sigma Chemical Corp., Norbiton, Surrey, U.K.) were obtained from Boehringer Corp., (London) Ltd., Lewes, Sussex, U.K. Collagenase was of grade II purity. [3H]Inulin was obtained from the Radiochemical Centre, Amersham, Bucks., U.K., and bovine serum albumin from Armour Pharmaceutical Co. Ltd., Eastbourne, Sussex, U.K. All other chemicals were of the purest grade available from standard suppliers. The methods for analysis of glucose [6], glycogen [7], malate [8], DNA [9], adenine nucleotides [10,11] have been described previously. Urea was determined using a standard Boehringer kit [12], after prior removal of NH~ from 1 ml of neutralised sample by ion-exchange chromatography (2 × 1 cm column containing 'Amberlite' resin CG-400 (C1-) Type II, 200 mesh; BDH Chemicals Ltd., Poole, U.K.). Potassium was determined by atomic absorption spectrometry (Unicam SP90A; Pye Unicam Ltd., Cambridge). Lactate and glutamate dehydrogenases were determined at 30°C by standard techniques [13]. Phosphoenolpyruvate carboxykinase was assayed at 37°C by the method of Seubert and Huth [14] as modified by Pogson and Smith [15]. Enzyme distribution data were obtained by treating the cell suspensions with digitonin as described by Zuurendonk and Tager [16], except that 0.75 ml cells was added to 2.5 ml 0.1% (w/v) digitonin buffer solution, and the incubation times was 5 min at 0 ° C. The pellets after centrifugation was dispersed in 2 ml 0.25 M sucrose/20 mM 3-(N-morpholino)propanesulphonate/3 mM EDTA, pH 7.0; containing 0.2% (w/v} Triton X-100. A further sample of the original cell suspension was also diluted 1 : 1 with the same solution. All samples were frozen and thawed 3 times {liquid N2 and water at 37°C, respectively), and centrifuged at 12 000 × g for 2 min (Eppendorf 3200 centrifuge}; the supernatants were stored in ice and assayed the same day. Portions of whole liver were homogenised in 0.25 M sucrose/20 mM 3-(Nmorpholino)propane sulphonate/3 mM-EDTA, pH 7.0 (10 m l . g wet wt. -t) and mitochondrial fractions were prepared [17]. The mitochondrial fractions and samples of the original homogenate were treated as described above. Enzyme activities were measured in the homogenates, postmitochondrial supernatants and mitochondrial pellets in each case. The distribution of phosphoenolpyruvate carboxykinase activity between the cytoplasmic and mitochondrial compartments were calculated after corrections for recovery of lactate

477 and glutamate dehydrogenase activities and for cross-contamination of fractions, assuming that these enzymes are exclusively located in the cytoplasm and mitochondria, respectively. The unit of enzyme activity is defined as the amount which converts 1 pmol of substrate into produce per min at the stated temperature. Solutions. Two buffer solutions were used in the preparation of the isolated cells. Solution A consisted of Ca2÷-free, Krebs-Henseleit buffer [18] containing 2% defatted bovine serum albumin [19] and 10 mM sodium propionate. Solution B consisted of propionate-free Krebs-Henseleit buffer containing 2% defatted bovine serum albumin and full calcium complement [18]. All buffer solutions were equilibrated at room temperature with 95% 02 : 5% CO2; the pH at ambient temperature was 7.6, and the final pH during incubation at 37°C was 7.4. All vessels used for transporting medium or incubating liver cells were flushed with 95% O2: 5% CO2 for at least 2 min prior to capping. Method of preparation of isolated hepatocytes. Cell suspensions were prepared from portions of sheep liver removed immediately after slaughter. The tissue was bathed in buffer (solution A) and slices of 0.5--1.0 mm thickness rapidly cut by hand. Approximately 5 g liver slices were transferred to a 250 ml polythene flask containing 10 ml buffer solution A. At this point the material was rapidly transported (5--8 min) to the laboratory. Enzymatic dispersion of th liver tissue was achieved by incubation for 1 h at 37°C on an orbital shaker (operating at 100 oscillations, min -~) in the presence of 10 mg hyaluronidase and 15 mg collagenase. Dispersion was found to be enhanced by the addition of 0.23 ml CaC12 (0.11 M) after 30 min of incubation. After 1 h, the contents of the flask were diluted with buffer (solution B), and filtered through one layer of nylon mesh (250 pm pore size; Henry Simon, Stockport, Cheshire, U.K.) The filtrate was collected in 50 ml plastic tubes and centrifuged at 40 × g for 2 min. The supernatant fraction was removed, and the cells washed twice in propionate-free buffer (solution B). After the final centrifugation the cells were resuspended in buffer (solution B) and used immediately. In practice it was found that sufficient cells (15 ml of 15 mg dry w t . . m1-1) could be obtained by employing an initial three flask collection regime. Preliminary experimen~ demonstrated that the yield and metabolic viability of the cells was greatest when the period between death of the animal and removal of the liver was reduced to a minimum (in practice approximately 2 min). Metabolic studies. In all studies, except where stated, 0.3 ml cell suspension (approximately 5 mg dry wt. of cells) was added to siliconised vials (20 ml capacity) containing 1.7 ml buffer (solution B) and appropriate substrate (10 mM final concentration, unless otherwise stated). The vials were capped (Subaseals, Laboratory Apparatus and Glassblowing Co. Ltd., Dewsbury, Manchester, U.K.) and gassed prior to incubation at 37°C on a reciprocating water bath operating at 100 oscillations per min (Mickle Laboratory Engineering Co., Gomshall, Surrey). Plastic wide-bore pipettes were used for all cell transfers. Incubations were terminated by addition of 0.2 ml 20% (v/v) perchloric acid (glucose and urea determinations), or 2 ml 60% (w/v) KOH (glycogen determinations). A modified incubation procedure was employed in the determination of

478 intracellular potassium and adenine nucleotide concentrations. Thus, 0.5 ml cell suspension was added to 1.5 ml solution B and the cells subsequently separated from incubation medium by the use of small separation tubes as described by Hems et al. [20]. The contents of two incubation vials for each time point were combined prior to rapid transfer to the separation tubes, and, in the case of intracellular potassium determinations approximately 1 mg [3H]inulin (0.25 pCi • mg -1 inulin) was added to facilitate subsequent allowance for contamination of the cell pellet with extracellular potassium. Dry weight determination. 1 ml of cell suspension was added to a preweighed vial and centrifuged (40 X g for 2 min). The supernatant was carefully removed and the vial dried to constant weight. The difference in weight observed was considered to represent the dry weight of cells per 1 ml of initial cell suspension. Results

Cell integrity Under examination by light microscopy (X 200) the cells appear rounded and refractile. Small groups of 4--5 cells are c o m m o n l y noted indicating that the initial digestion stage was not too severe. The quantity of erythrocytes and cell debris was negligible indicating that the washing procedure was also effective. The degree of staining with Trypan blue (0.2% for 5 min at room temperature) revealed variable quantities of stained cells. However, preparations with greater than 75% non-stained cells were routinely obtained with this method. Janus green B {0.025% for 5 min at room temperature) was used as an alternative dye to Trypan blue, but both staining techniques were abandoned in favour of a more quantitative assessment based on the ability of the cells to exclude succinate (10 mM). Thus, the integrity of the cell plasma membrane was routinely assessed by comparing the rate of succinate oxidation (malate production) by the cells under standard incubation conditions, in the presence, and absence, of 0.01% {w/v) digitonin [21]. The production of maleate in the absence of digitonin expressed as a percentage of the production in the presence of digitonin was considered to represent a quantitative assessment of cell integrity. Any preparations failing to achieve a 75% figure were discarded. The DNA content of the cells was found to range from 8--14 pg • m1-1 dry weight. Enzyme content and distribution The activities of lactate dehydrogenase glutamate dehydrogenase and phosphoenolpyruvate carboxykinase were measured in cells and in portions of liver tissue from which the cells were derived. These results demonstrate (see Table I) that there is no significant loss of lactate dehydrogenase or glutamate dehydrogenase when compared with the whole liver, a fact which further testifies to the integrity of the cell membrane(s). The digitonin-treatment technique for the separation of mitochol~.drial and cytoplasmic compartments of isolated cells [16] permits the quantitative recovery of intracellular enzyme activities with minimal cross-contamination (~ 10%). Utilising this method, and making the appropriate corrections for recoveries and cross contamination, we have

479

TABLE

I

ENZYME

ACTIVITIES

IN ISOLATED

SHEEP

LIVER

PARENCHYMAL

CELLS

A s s a y m e t h o d s a n d cell f r a c t i o n a t i o n p r o c e d u r e s w e r e as d e s c r i b e d i n t h e t e x t . Activities of phosphoenolp y r u v a t e c a r b o x y k i n a s e i n cell f r a c t i o n s w e r e c o r r e c t e d f o r c r o s s - c o n t a m i n a t i o n b y s i m u l t a n e o u s a s s a y of l a c t a t e a n d g l u t a m a t e d e h y d r o g e n a s e s . T h e n u m b e r o f o b s e r v a t i o n s is s h o w n i n p a r e n t h e s e s . Enzyme

a c t i v i t y ( u n i t s • g - w e t w t . -1 )

Whole liver Lactate dehydrogenase Glutamate

I s o l a t e d cells

6 3 . 0 -+ 9 . 1 ( 5 )

dehydrogenase

353

Phosphoenolpyruvate c a r b o x y k i n a s e

+ 77

13.5 ±

(5)

2.7 (5)

50.6 + 17.5 (3) 495

+ 66

1 3 . 0 +-

(5)

1.3 (5): 53.6 + 1.6% cytoplasmic 46.4 + 1.6% mitochondrial

confirmed the bimodal distribution of phosphoenolpyruvate carboxykinase in sheep liver and isolated hepatocytes [22], but find that approximately 46% only is associated with the mitochondrial fraction.

Intracellular potassium and adenine nucleotide concentration Although liver slices consist mainly of intact cells, their suitability for the study of liver metabolism has been criticised [23], primarily due to their apparent inability to maintain normal concentrations of adenine nucleotides. Cells prepared according to the present method exhibited a total adenine nucleotide concentration of 10.8 n m o l - m g -1 dry wt. (see Table II), which, when expressed as pmol .g-1 wet wt.), is similar to values previously reported for freeze-clamped sheep liver (3.48 pmol • g-1 wet wt.) [24]. Furthermore, no decrease in total adenine nucleotide content was observed throughout the standard incubation period in the presence of 10 mM sodium propionate. Similarly the intracellular K ÷ content was found to remain stable throughout the incubation period (169 nequiv.- mg -~ dry wt.) and was equivalent to 64% of that determined in the corresponding portions of the whole liver.

TABLE

II

INTRACELLULAR ADENINE NUCLEOTIDE LIVER PARENCHYMAL CELLS

AND

POTASSIUM

CONTENT

OF ISOLATED

SHEEP

T h e m e t h o d o f c e l l p r e p a r a t i o n , i n c u b a t i o n a n d u t i l i s a t i o n w a s as d e s c r i b e d i n t h e M a t e r i a l s a n d M e t h o d s s e c t i o n . P r o p i o n a t e ( 1 0 r a M ) w a s p r e s e n t i n all i n c u b a t i o n s . I n c u b a t i o n m e d i u m w a s s e p a r a t e d f r o m the c e l l s a s d e s c r i b e d b y H e m s e t al. [ 2 0 ] . T h e i n t r a c e l l u l a r a d e n i n e n u c l e o t i d e c o n t e n t w a s d e t e r m i n e d i n the n e u t r a l i s e d p e r c h l o r i c a c i d cell e x t r a c t . I n t r a c e i l u l a r p o t a s s i u m w a s d e t e r m i n e d a f t e r d i l u t i o n ( 1 : 5 0 ) of the p e r c h l o r i c a c i d c e l l e x t r a c t . A p p r o p r i a t e c o r r e c t i o n s w e r e m a d e f o r c o n t a m i n a t i o n o f t h e cell p e l l e t with e x t r a c e l l u l a r p o t a s s i u m ( s e e M a t e r i a l s a n d M e t h o d s ) . T h e n u m b e r o f a n i m a l s is g i v e n i n p a r e n t h e s e s ; r e s u l t s a r e m e a n s -+ S . E . M . Adenine nucleotide ( n m o l - d r y w t . -1 ) ATP

content

ADP

Potassium content ( n e q u i v • m g d r y w t . -1 ) AMP

8 . 3 7 +- 0 . 7 7

1 . 6 9 -+ 0 . 2 5

0.73 ± 0.07

(8)

(8)

(8)

Total

ATP: ADP

K+

10.79

5.15 ± 0.70

169 ± 5

(8)

(8)

(4)

480 TABLE

III

OBSERVATIONS CELLS

OF THE METABOLIC

CAPACITY

OF ISOLATED

SHEEP LIVER

PARENCHYMAL

I n c u b a t i o n a n d a s s a y t e c h n i q u e s w e r e a s d e s c r i b e d i n t h e t e x t . All s u b s t r a t e c o n c e n t r a t i o n s w e r e 1 0 r a M , w i t h t h e e x c e p t i o n o f o r n i t h i n e w h i c h w a s 2 r a M . R a t e s o f g l u c o s e p r o d u c t i o n a r e e x p r e s s e d as a p e r c e n t a g e o f t h e r a t e o b s e r v e d i n t h e p r e s e n c e o f p r o p i o n a t e . T h e n u m b e r o f a n i m a l s is g i v e n i n p a r e n t h e s e s . T o t a l g l u c o s e p r o d u c t i o n o b s e r v e d i n t h e p r e s e n c e o f p r o p i o n a t e w a s 2 0 1 + 1 8 n m o l • h -1 • m g -1 d r y w t . Glucose production Substrates

Percentage rate

Propionate (15) Fructose (5) Lactate (4) Pyruvate (3) Glycerol (3) No substrate (15)

100 104 42 41 30 28

+ 5 + 8 -+ 5 ± 3 ± 4

Alanine (3))less than, or equivalent to no Serine (3)) substratc Glycine (3)) Urea production Substrate

n m o l • h -1 • m g -1 d r y w t .

Ornithine, ammonium-chloride propionate (5)

and 1 0 9 +- 2 0

Metabolic capacity Glucose production from propionate (see Table III) was routinely used to monitor the metabolic viability of the isolated cells since its production involves the continued integrity of both cytoplasmic and mitochondrial compartments. Due to the varying nutritional condition of the slaughtered animals, the liver-cell glycogen content was found to vary (maximum 427 nmol glycogen/glucose per mg dry wt. of cells) and in those cases where high initial glycogen content was observed some interference in the linearity of glucose produc-

TABLE

IV

COMPARISON OF PERFUSED SHEEP

DATA OBTAINED FROM ISOLATED LIVER AND IN VIVO DATA

SHEEP

LIVER

PARENCHYMAL

CELLS,

V a l u e s a r e n m o l - h -1 • m g -1 d r y w t . e x c e p t f o r a d e n y l a t e s u m , w h i c h is n m o l • m g -1 d r y w t . D a t a f o r p e r fused liver and in vivo preparations were taken from the papers indicated. Data were recalculated on the basis of there being 300 mg dry wt. per 1 g of wet wt. of fresh liver, and that a 30--40 kg sheep would possess a liver of approx.

5 0 0 g. Paxenchymal

Gluconeogenesis

(from propionate)

Ureogenesis (maximum) Adenylate sum ATP : ADP ratio • Perfusion

cells

Perfused liver

I n vivo d a t a

201

160 * (30)

170 (31)

109

121 (1)

10.79 5.15 medium

nium hydroxide.

contained

propionate

plus hydrolysed

14.60 (30) 1.44 (30)

11.60 (24) 1.39 (24)

casein and was neutralised with ammo-

481 tion was noted. However, in cases of relatively low initial cell glycogen content ( < 1 0 0 nmol glycogen/glucose per mg dry wt. of cells) rates of glucose production were linear throughout the 1-h incubation (Table III). Our data indicate that the sheep liver cells possess the potential for a high rate of gluconeogenesis from propionate and fructose, which is consistent with the role of propionate in the fed animal [25]. Pyruvate and lactate were individually much less efficient as glucose precursors than either propionate or fructose, whilst the amino acids, alanine, serine and glycine, were found not to give rise to any net glucose synthesis, and in some cases actually decreased the observed endogenous glucose production. The isolated hepatocytes synthesised urea when incubated in the combined presence of ornithine, ammonium chloride and propionate(see Table III). Under these conditions urea synthesis was linear within the first 30--40 min of incubation, b u t declined to almost zero during the later 30 min of incubation (results are calculated over the linear portion of the time course)• Synthesis of urea from alanine and other individually presented amino acids was also investigated, b u t found to be negligible. Discussion Prior to the adoption of isolated cell preparations for the study of hepatic metabolism in any animal species it is first necessary to determine the level of morphological intactness and metabolic viability characteristic of the method of cell preparation employed. To date, such detailed information is only available for a limited number of species of laboratory animal [26--29]. This communication describes a simple, relatively cheap method for the preparation of isolated sheep liver hepatocytes which appear to maintain a high level of cellular and metabolic integrity. The possibility of increasing the proportion of intact cells in the final cell suspension by the application of density gradient centrifugation techniques [30], was considered• However, due to problems associated with centrifugation in an anaerobic material and the inherent variability of cell density within a normal population of liver cells [30], such a technique was rejected in favour of using the cells immediately after their final wash and resuspension. The rate of gluconeogenesis from propionate (201 nmol • h -1 • mg -I dry wt.) obtained with the ovine hepatocytes is comparable (see Table IV) to that observed with perfused liver [31] or in vivo preparations (160 and 170 nmol • h -~ • mg -~ dry wt. respectively) [32]. Similarly, Lindsay et al. [1] reported a rate of urea production b y the isolated perfused sheep liver which is comparable (121 n m o l . h -~. mg -~ dry wt.) to that achieved by the isolated cells in the presence of ornithine, NH4C1 and propionate (109 nmol • h -1 • mg -1 dry wt.). The apparent lack of utilisation of amino acids for glucose production by the isolated cells was somewhat unexpected, b u t recalculation of the data of Wolff and Bergman [33], derived from in vivo studies of gluconeogenesis from plasma amino acids in the liver of fed sheep, indicates that the maximum rate of glucose production from alanine would approximate to 40 n m o l . h -1 . m g dry wt. of cells -~. Such a value is low and would be masked by the rate of endogenous glucose production characteristic of the cells prepared by our

482 techniques. The results of the metabolic studies presented do nonetheless serve to confirm that the cytoplasmic and mitochondrial integrity of the isolated cells remain patent throughout the period of cell preparation and utilisation. It should be stressed that the results quoted in this paper have not been corrected for the percentage viability of the cell population (as determined by succinate oxidation) and since dry weight determinations include intact plus injured (leaky) cells, it is likely that all results will be an underestimation of the actual rate or concentration appropriate to the intact cells which constitute from 75--84% of the cell population. This point is particularly relevant when considering the intracellular concentration of low molecular weight constituents, and may account for the observed discrepancy between the potassium content of the whole liver and that of the isolated cells. During the preparation of this publication two reports [34,35] appeared which describe the routine preparation and utilisation of isolated hepatocytes from young lambs (10--50~lays-old). This method is based on perfusion of the excised caudate lobe with coUagenase and thus requires more sophisticated equipment and slaughter facilities than are required for the method described here. Nevertheless, it is interesting to note that the hepatocytes derived by the alternative techniques display essentially the same properties with respect to adenylate content, ATP/ADP, degree of enzyme leakage and staining with Trypan blue, In addition, despite the obvious difference in age and physiological state of the 'donor' animals in these studies, i.e. young growing lambs versus fully grown sheep, the maximum rates of gluconeogenesis from propionate are similar, and both cell systems exhibit a lack of gluconeogenic capacity with respect to the amino acids, alanine, serine and glycine.

Acknowledgements We gratefully acknowledge the help and co-operation of the staff of Canterbury Abattoir, and the technical assistance of Mrs. S.V. Gurnah. This work was supported by a grant from the Medical Research Council.

References 1 L i n d s a y , D.B., J a r r e t t , I . G . , M a n g a n , J . L . a n d Linzell, J . L . ( 1 9 7 5 ) Q u a r t . J. E x p t l . P h y s i o l . 6 0 , 1 4 1 149 2 B e r g m a n , E.N. a n d W o l f f , J . E . ( 1 9 7 1 ) A m . J. P h y s i o l . 2 2 1 , 5 8 6 - - 5 9 2 3 B a i r d , G . D . , S y m o n d s , H.W. a n d A s h , R. ( 1 9 7 5 ) J. Agric. Sci. 8 5 , 2 6 1 - - 2 9 6 4 T h o m p s o n , G . E . , G a r d n e r , J . W . a n d Bell, A.W. ( 1 9 7 5 ) Q u a r t . J, E x p t l . P h y s i o l . 6 0 , 1 0 7 - - 1 2 1 5 A s h , R., E l l i o t t , K . R . F . a n d P o g s o n , C.I. ( 1 9 7 6 ) P r o c . N u t r . Soe. 3 5 , 2 9 A 6 K r e b s , H . A . , B e n n e t t , D . A . H . , de G a s q u e t , P., G a s c o y n e , T. a n d Y o s h i d a , T. ( 1 9 6 3 ) B i o c h e m . J. 8 6 , 22--27 7 R a n d l e , P.J., N e w s h o l m e , E . A . and G~Lrland, P.B. ( 1 9 6 4 ) B i o c h e m . J. 9 3 , 6 5 2 - - 6 6 5 8 H o h o r s t , H-J. ( 1 9 6 5 ) in M e t h o d s o f E n z y m a t i c A n a l y s i s ( B e r g m e y e r H.-U., ed.), p p . 3 2 8 , A c a d e m i c Press, L o n d o n 9 B u r t o n , K. ( 1 9 5 6 ) B i o c h e m . J. 6 2 , 3 1 5 - - 3 2 1 1 0 A d a m , H. ( 1 9 6 5 ) in M e t h o d s o f E n z y m a t i c A n a l y s i s ( B e r g m e y e r , H.-U., ed.), p p . 5 3 9 , A c a d e m i c Press, London I I A d a m , H. ( 1 9 6 5 ) in M e t h o d s o f E n z y m a t i c A n a l y s i s ( B e r g m e y e r , H.-U., ed.), p p . 5 7 3 , A c a d e m i c Press, London 1 2 F a w c e t t , J . K . a n d S c o t t , J . E . ( 1 9 6 0 ) J. Clin. P a t h o l . 1 3 , 1 5 6 - - 1 5 9 13 L o n g s h a w , I.D., B o w e n , N.L. a n d P o g s o n , C.I. ( 1 9 7 2 ) E u r . J. B i o c h e m . 2 5 , 3 6 6 - - 3 7 1

483

14 15 16 17 18 19 20 21 22 23 24 25 26

27 28 29 30 31 32 33 34

35 36

Seubert, W. and Huth, W. (1965) Biochem. Z. 343, 176--191 Pogson, C.I. and Smith, S.A. (1975) Biochem. J. 152, 401 --408 Zuuren donk, P.F. and Tager, J.M. (1974) Biochim. Biophys. Aeta 3 3 3 , 3 9 3 - - 3 9 9 Chappell, J.B. and Hansford, R.G. (1969) in Subeellular C o m p o n e n t s (Birnie, G.D. and Fox, S.M., eds.), pp. 43--56, Butterworths, L o n d o n Krebs, H.A. and Henseleit, K. (1932) Z. Physiol. Chem. 210, 33--66 Chen, R.F. (1966) J. Biol. Chem. 2 4 2 , 1 7 3 - - 1 8 1 Hems, R., Lund, P. and Krebs, H.A. (1975) Bioehem. J. 150, 47--50 Mapes, J.P. and Harris, R.A. (1975) FEBS Lett. 51, 80--83 Hanson, R.W. and Garber, A.J. (1972) Am. J. Clln. Nutr. 25, 1010--1021 Krebs, H.A. (1970) Adv. Enz. Reg. 8 , 3 3 5 - - 3 5 3 Herriman, I.D., Heitzman, R.J., Priestley, I. and Sandhu, G.S. (1976) J. A ~ i c . Sci., in the Press Bergman, E.N. (1973) Comell Vet. 63, 341--382 Krebs, H.A., Cornell, N.W., Lund, P. and Hems, R. (1974) in Regulation of Hepatic Metabolism, VIth Alfred Benzon Sy mposium (Lundquist, F. and Tygstrup, N., eds.), pp. 726--750 Munksgaard, Copenhagen Seglen, P.O. (1976) in Methods in Cell Biology (Prescott, D.M., ed.), Vol. 13, pp. 30--83 Arinze, I.J. and Rowley, D.L. (1975) lliochem. J. 152, 393--399 Berry, M.N. (1974) Method Enzymol. 32, 625--632 Pretlow, T.G. and Williams, E.E. (1973) Anal. Binchem. 55, 114--122 Linzell, J.L., Setchell, B.P. and Linday, D.B. (1971) Quart. J. Exptl. Physiol. 56, 53--71 Steel, J.W. and Leng, R.A. (1973) Br. J. Nutr. 30, 475--489 Wolff, J.E. and Bergman, E.N. (1972) Am. J. Physiol. 2 2 3 , 4 5 5 - - 4 5 9 Clark, M.G.~ Filsell, O.H. and Jarrett, I.G. (1976) in Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies (Tager, J.M., SSling, H.D. and Williamson, J.R., ed.), pp. 398--401, North Holland, Amsterd am Clark, M.G., Filsell, O.H. and Jarrett, I.G. (1976) Biochem. J. 156, 671--680 Elliott, K.R.F., Ash, R., Crisp, D.M., Pogson, C.I. and Smith, S.A. (1976) in Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies, (Tager, J.M., SSling, H.D. and WiUiamson~ J.R., ed.), pp. 139--143, North Holland, Amsterdam