816
Biachimica et Biophysica Acta, 444 (1976) 816--834 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 28039
PROPERTIES AND SUgCELLULAR LOCALIZATION OF CMP-N-ACETYLNEURAMINIC ACID HYDROLASE OF CALF KIDNEY
WILLEM VAN DIJK, HANS MAIER and DIRK H. VAN DEN EIJNDEN
Department of Medical Chem/stry, Faculty of Medicine, Vr'~e Universiteit, v.d. Boeehorststmat 7, P.O. Box 7161, Amsterdam (The Ne~erlands) (Received March 19th, 1976)
Summary The properties and subcellular distribution of CMP-N-acetylneuraminic acid (CMP-NAcNeu) hydrolase were studied in the cortex of calf kidney. The pH o p t i m u m was 9.0 in both Tris. HC1 and glycine/NaOH buffer. The apparent K , was 0.47 mM and the apparent V 15.3 ~mol/h/g wet wt of calf kidney cortex. A stimulation by divalent metal ions (Ca 2÷ and Mg 2.) was demonstrated for the hydrolase. In the presence of Triton X-100 an increase in enzyme activity was observed. CMP-NAcNeu hydrolase was inhibited by EDTA, ~-mercaptoethanol, nucleoside phosphates and nucleotide-sugars. The inhibition was more pronounced when a sub-optimal CMP-NAcNeu concentration was used. The enzyme appeared t o be localized in the plasma membranes. In the plasma membrane preparation of calf kidney cortex, which was derived mainly from the proximal tubule cells, the yield of CMP-NAcNeu hydrolase (13%) and its increase in specific activity (9-fold) was as high as for the plasma membrane marker enzymes. From subceUular distribution studies it appeared that the enzyme was localized mainly at the brush border side of the plasma membrane of the proximal tubule cell. Introduction Regulation of sialoglycoprotein and sialoglycolipid biosynthesis enables the cell to control cellular processes in which these glycoconjugates are involved. Regulation can be achieved in several ways, one of which is modifying the activity of sialyltransferase by intervention in the availability of CMP-NAcNeu, the low molecular weight substrate for sialyltransferase. We have shown that CMP-NAcNeu synthetase is localized in the nuclei of calf kidney cells [1]. Since the sialylation takes place in other parts of the cell, i.e. the endoplasAbbreviation: NAcNeu, N-acetylneurL,nlnlc acid.
817 matic reticulum, the different localization of CMP.NAcNeu synthetase enables the cell to control the CMP-NAcNeu concentration at the site of incorporation of sialic acid into glycoproteins by sialyltnmsferase. Shoyab and Bachhawat [2,3] have reported the existence of a "CMP-NAcNeu degrading enz y m e " in a varieW of rat tissues and this was confirmed recently by Kean and Bighouse [4,5]. Kean and Bighouse [5] studied the subcellular localization of "CMP-NAcNeu hydrolase" in rat liver and they concluded that the enzyme is localized in the plasma membranes of rat liver cells. CMP-NAcNeu hydrolase might contribute to the regulation of the CMP-NAcNeu concentration in the cell. To study if, and to what extent CMP-NAcNeu hydrolase could function in a possible regulation mechanism of sialyltransferase activity in calf kidney, investigations on the properties and subcellular localization of CMP-NAcNeu hydrolase were performed. Materials and Methods
Materials CMP-[Ac-~4C]NAcNeu as well as non-radioactive CMP-NAcNeu was prepared as described previously [7]. All other chemicals were obtained from commercial sources and were of analytical grade. Kidneys from 3-month-old calves were obtained from a local slaughterhouse. The organs were removed as soon as possible but not longer than 30 rain after death and were transported on ice to the laboratory.
Assay for CMP-NAcNeu hydrolase activity The incubation system for the measurement of CMP-NAcNeu hydrolase activity contained the following components in a final volume of 0.2 mh 30 #mol Tris • HC1 buffer (pH 9.0), 1 pmol CaC12, 1% Triton X-100, in dependence of the assay system used 70 nmol CMP-NAcNeu (Method A) or 300 nmol CMP[~4C] NAcNeu (spec. act. 0.7--0.8 Ci/mol) (Methods B and C), and 0.03--0.50 units of enzyme. The mixture was incubated for 15--60 min at 37°C. The reaction was stopped on ice and the disappearance of CMP-NAcNeu or the appearance of NAcNeu was determined by one of the methods outlined below. When the assay according to Warren and Blacklow [8] was used (Method A) the sampies were run in triplicate, otherwise duplicates were sufficient. Correction for non-enzymatic hydrolysis of CMP-NAcNeu was obtained by running controls containing no enzyme in parallel with each incubation. Three assay methods were used to measure the hydrolysis of CMP-NAcNeu. In Method A the en, zyme activity was calculated from the difference between the amounts of CMPNAcNeu present before and after incubation of the enzyme [2], as measured by the thiobarbituric acid assay of Warren [9] after reduction of NAcNeu (present or formed) with NaBH4 according to the method of Warren and Blacklow [ 8]. For Methods B and C 0.2 m196% ethanol (--20°C) was added to the reaction mixtures to precipitate the bulk of the protein. After standing at --20°C for 15 min the tubes were centrifuged at 1000 X g in a refrigerated centrifuge for 15 rain. The supernatants were removed and the pellets were resuspended in 0.5 ml 50% ethanol and centrifuged as above. This washing step was repeated once.
818 The supernatants were combined and used for the assay of CMP-[~4C]NAcNeu and [ 14C]NAcNeu. Method B is a modification of the method of Kean [4] which involves the separation of CMP-NAcNeu and NAcNeu by ion-exchange chromatography. The pooled supernatants were placed on top of a 0.8 X 2 cm column of Dowex 1-X8 (100-200 mesh) in the HCO3-form. [~4C]NAcNeu was eluted with 25 ml 0.05 M triethylammonium bicarbonate and subsequent elution with 30 ml 1.0 M triethylammonium bicarbonate released CMP-[ ~4C]NAcNeu. The amount of radioactivity in the eluates was determined in 1-ml aliqu0ts (or in concentrates obtained by freeze-drying the eluates) using a dioxane-based scintillation fluid. In Method C separation of CMP-[14C] NAcNeu and [14C] NAcNeu is accomplished by ascending paper chromatography, as described previously [7]. 50 /~1 of the pooled supernatants were applied as a 2-cm streak on Whatman 3MM paper. Chromatography was performed overnight at 10°C with 95% ethanol/ 0.6 M NH4OH (7 : 3, v/v) as a solvent mixture. Three radioactive areas were detected with a radiochromatograph scanner (Fig. 1). From comparison with radiochromatographs with standard compounds these areas appeared to correspond to CMP-NAcNeu, NAcNeu and N-acetylmannosamine. The radioactive areas were cut out and counted either directly in a toluene-based scintillation fluid, or, after extraction of the radioactive compounds with water, in a dioxane-based scintillation fluid. The appearance of N-acetylmannosamine seems to be a consequence of the enzymatic breakdown of NAcNeu by NAcNeu aldolase (unpublished results). As a consequence of this breakdown the CMP-NAcNeu hydrolase activity has to be computed by summarizing the radioactivities in NAcNeu and N-acetylmannosamine. In Method C this is accomplished by combining the NAcNeu and the N-acetylmannosamine areas. Because N-acetylmannosamine does not bind to the anion-exchange resin used in Method B, in this method the compound is recovered in the void volume of the column, which is routinely combined with the 0.05 M bicarbonate fraction containing the radioactive NAcNeu. Finally in Method A the NAcNeu aldolase-catalyzed breakdown of NAcNeu does not interfere with t h e calculation of the CMP-NAcNeu hydrolase activity, because it is computed from the difference in the amounts of CMP-NAcNeu present before and after incubation of the enzyme preparation. In all instances the net decrease in CMP-[~4C] NAcNeu appeared to be count-
Fig. 1. R a d i o e h r o m a t o p a p h i c s c a n o f a p a p e r c h r o m a t o g r a m o f t h e r e a c t i o n p r o d u c t s resulting a f t e r inc u b a t i o n o f a c a l f k i d n e y c o r t e x h o m o g e n a t e f o r C M P - N A c N e u h y d r o l a s e activity at p H 9 . 0 . T h e i n c u b a t i o n was p e r f o r m e d and assayed as described i n M a t e r i a l s and M e t h o d s f o r M e t h o d C. A p r o l o n g e d inc u b a t i o n t i m e w a s c h o s e n t o visualize N- [ 14C ] a c e t y l m a n n o s a m i n e ( M a n N a c ) .
819
erbalanced stoichiometrically by the appearance of [~4C] NAcNeu and N-[~4C] acetylmannosamine. One unit of CMP-NAcNeu hydrolase activity is defined as the quantity of enzyme cleaving 1 ~mol of CMP-NACNeu per h a t 37°C. When a CMP-NAc-Neu hydrolase preparation was incubated under standard incubation conditions using 70 nmol CMP-[14C] NAcNeu the same activities were obtained independent whether Method A, B or C was used. The advantages of Methods B and C to Method A are (i) that the enzyme can be incubated under conditions of substrate saturation (300 nmol CMP-NAcNeu) and (ii) that the amount of NAcNeu produced as well as the residual amount of CMP-NAcNeu are measured. On the other hand the non-radioactive Method A is a more convenient (and less expensive) one because the results are obtained rapidly. Although in this method the CMP-NAcNeu hydrolase activity is 'not determined under conditions of substrate saturation (i.e., enzyme activity is lower than obtained with Methods B and C), the method proved valuable for routine determinations as is shown by the linear increasing reaction rates with respect to time and enzyme concentration (of. Fig. 5).
Assay o f other enzyme activities CMP-NAcNeu synthetase, succinate-2-(p-iodophenyl)-3~p-nitrophenyl)-5phenyltetrazolium reductase (succinate-INT reductase), ~alactosidase and glucose-6-phosphatase activities were assayed as described previously [10]. (Na÷ + K*)-dependent ATPase and Mg2÷~iependent ATPase activities were determined according to Bonting et al. [11] after treatment of the samples with 0.1% sodium deoxycholate and subsequent storage at --20°C for at least 48 h [12]. NADPH~ytochrome c reductase activity was determined according to Phillips and Langdon [13]. Alkaline phosphatase activity was assayed according to Bessey et al. [14] using p-nitrophenyl phosphate as a substrate. Phosphodiesterase I (EC 3.1.4.1) activity was assayed with p-nitrophenyl thymidine 5'phosphate according to a modification of the method of Razzell [15]. The incubation mixture contained 45/~mol Tris • acetate buffer (pH 8.9), 3.0/~mol MgC12, 1% Triton X-100, 0.3 /~mol substrate and 0.01--0.23 unit of enzyme in a total volume of 0.3 ml. Substrate was added after preincubation of the mixture at 37°C for 2 rain. After subsequent incubation for 10 rain at the same temperature the increase in absorbance at 405 nm was read immediately, emM = 12.0 Was used to calculate the amount ofp-nitrophenol liberated. Glycoprotein sialyltransferase was assayed by measuring the amount of [14C] NAcNeu incorporated into chloroform/methanol-insoluble protein using desialyzed al-acid glycoprotein as an acceptor [16]. One unit of enzyme activity is defined as the amount of enzyme producing I/~mol of product per h. Chemical assays Determination of protein and inorganic phosphate was performed as described previously [1]. Tissue homogenization and fractionation Crude homogenates were prepared by two different procedures. For kinetic studies homogenates were prepared in 0.01 M Tris • maleate buffer (pH 7.4) as described previously [17]. For subcellular distribution studies calf kidney cor-
820 tex homogenates were prepared in 0.25 M sucrose/0,01 M Tris • maleate buffer (pH 7.4) by 6 up-and-down strokes of a Teflon-glass Potter-Elvehjem type homogenizer (0.03 mm clearance) rotating at 800 rev./min. The homogenate obtained was filtered twice through two layers of surgical gauze (18 threads/ cm). The filtered homogenate was subjected to differential centrifugation. Each subcellular fraction obtained was washed twice by repeated centrifugation and the pooled supernatants were then subjected to the next centrifugation step. In this way the homogenate was separated in five particulate fractions and one soluble fraction (see first column of Table III). Preparation o f p l a s m a m e m b r a n e - r i c h fractions
For the preparation of plasma membrane-rich fractions of calf kidney cortex we used a m e t h o d which is essentially a combination of fractionation methods of Kamat and Wallach [19] and of Jacobsson [12] (Scheme I). By this SCHEME 1
kidney cortex
I I
homogenization with a Potter-glvehjem type homogenizer in 3 mM Tris buffer (pH 7.5) plus .3 vol. of 1.0 M sucrose/3 mM Tris.Hel buffer (pH 7.5) resulting in 0.25 M sucrose
I
10 rain, 10 000 X g pellet, discarded supernatant layered on 1.6 M sucrose in MSE 3)< 23 {swing-out) / 30 [min, 105 000
X
g
45 rainv g/////A ooo PM~ MSE 3× 23
MSE 3× 70
pellet resuspended in 0.25 M sucrosell mM Tris.HCI buffer (pH 8.2) and washed with 10 and 1 mM Tris. HCI buffer (pH 8.2), respectively
I I dialysis against 1 mM Tris.HC1 buffer (pH 8.2)/1 rnM MgSO4 for 2 h homogenized vigorously in 1 mM Ttis-HCI buffer (pH 8.2)
~-- ~
3.107
g. min
~
plasma membranes (PM=)
23.9 % Ficoll endoplmmaatie reticulum membranes {ER= ) M S E 3× 70
method plasma membrane fractions are obtained from a 10 000 X g supernatant of vigorously homogenized tissue. This microsomal fraction is subfractionated into plasma membrane and endoplasmatic reticulum membrane vesicles taking advantage of the differences in charge density on the inner surface of the membrane vesicles and the different behaviour towards Ficoll (see Steck and Wallach [20]). Minced kidney cortex was vigorously homogenized by 15 up-
821 and
Biachimica et Biophysica Acta, 444 (1976) 816--834 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 28039
PROPERTIES AND SUgCELLULAR LOCALIZATION OF CMP-N-ACETYLNEURAMINIC ACID HYDROLASE OF CALF KIDNEY
WILLEM VAN DIJK, HANS MAIER and DIRK H. VAN DEN EIJNDEN
Department of Medical Chem/stry, Faculty of Medicine, Vr'~e Universiteit, v.d. Boeehorststmat 7, P.O. Box 7161, Amsterdam (The Ne~erlands) (Received March 19th, 1976)
Summary The properties and subcellular distribution of CMP-N-acetylneuraminic acid (CMP-NAcNeu) hydrolase were studied in the cortex of calf kidney. The pH o p t i m u m was 9.0 in both Tris. HC1 and glycine/NaOH buffer. The apparent K , was 0.47 mM and the apparent V 15.3 ~mol/h/g wet wt of calf kidney cortex. A stimulation by divalent metal ions (Ca 2÷ and Mg 2.) was demonstrated for the hydrolase. In the presence of Triton X-100 an increase in enzyme activity was observed. CMP-NAcNeu hydrolase was inhibited by EDTA, ~-mercaptoethanol, nucleoside phosphates and nucleotide-sugars. The inhibition was more pronounced when a sub-optimal CMP-NAcNeu concentration was used. The enzyme appeared t o be localized in the plasma membranes. In the plasma membrane preparation of calf kidney cortex, which was derived mainly from the proximal tubule cells, the yield of CMP-NAcNeu hydrolase (13%) and its increase in specific activity (9-fold) was as high as for the plasma membrane marker enzymes. From subceUular distribution studies it appeared that the enzyme was localized mainly at the brush border side of the plasma membrane of the proximal tubule cell. Introduction Regulation of sialoglycoprotein and sialoglycolipid biosynthesis enables the cell to control cellular processes in which these glycoconjugates are involved. Regulation can be achieved in several ways, one of which is modifying the activity of sialyltransferase by intervention in the availability of CMP-NAcNeu, the low molecular weight substrate for sialyltransferase. We have shown that CMP-NAcNeu synthetase is localized in the nuclei of calf kidney cells [1]. Since the sialylation takes place in other parts of the cell, i.e. the endoplasAbbreviation: NAcNeu, N-acetylneurL,nlnlc acid.
817 matic reticulum, the different localization of CMP.NAcNeu synthetase enables the cell to control the CMP-NAcNeu concentration at the site of incorporation of sialic acid into glycoproteins by sialyltnmsferase. Shoyab and Bachhawat [2,3] have reported the existence of a "CMP-NAcNeu degrading enz y m e " in a varieW of rat tissues and this was confirmed recently by Kean and Bighouse [4,5]. Kean and Bighouse [5] studied the subcellular localization of "CMP-NAcNeu hydrolase" in rat liver and they concluded that the enzyme is localized in the plasma membranes of rat liver cells. CMP-NAcNeu hydrolase might contribute to the regulation of the CMP-NAcNeu concentration in the cell. To study if, and to what extent CMP-NAcNeu hydrolase could function in a possible regulation mechanism of sialyltransferase activity in calf kidney, investigations on the properties and subcellular localization of CMP-NAcNeu hydrolase were performed. Materials and Methods
Materials CMP-[Ac-~4C]NAcNeu as well as non-radioactive CMP-NAcNeu was prepared as described previously [7]. All other chemicals were obtained from commercial sources and were of analytical grade. Kidneys from 3-month-old calves were obtained from a local slaughterhouse. The organs were removed as soon as possible but not longer than 30 rain after death and were transported on ice to the laboratory.
Assay for CMP-NAcNeu hydrolase activity The incubation system for the measurement of CMP-NAcNeu hydrolase activity contained the following components in a final volume of 0.2 mh 30 #mol Tris • HC1 buffer (pH 9.0), 1 pmol CaC12, 1% Triton X-100, in dependence of the assay system used 70 nmol CMP-NAcNeu (Method A) or 300 nmol CMP[~4C] NAcNeu (spec. act. 0.7--0.8 Ci/mol) (Methods B and C), and 0.03--0.50 units of enzyme. The mixture was incubated for 15--60 min at 37°C. The reaction was stopped on ice and the disappearance of CMP-NAcNeu or the appearance of NAcNeu was determined by one of the methods outlined below. When the assay according to Warren and Blacklow [8] was used (Method A) the sampies were run in triplicate, otherwise duplicates were sufficient. Correction for non-enzymatic hydrolysis of CMP-NAcNeu was obtained by running controls containing no enzyme in parallel with each incubation. Three assay methods were used to measure the hydrolysis of CMP-NAcNeu. In Method A the en, zyme activity was calculated from the difference between the amounts of CMPNAcNeu present before and after incubation of the enzyme [2], as measured by the thiobarbituric acid assay of Warren [9] after reduction of NAcNeu (present or formed) with NaBH4 according to the method of Warren and Blacklow [ 8]. For Methods B and C 0.2 m196% ethanol (--20°C) was added to the reaction mixtures to precipitate the bulk of the protein. After standing at --20°C for 15 min the tubes were centrifuged at 1000 X g in a refrigerated centrifuge for 15 rain. The supernatants were removed and the pellets were resuspended in 0.5 ml 50% ethanol and centrifuged as above. This washing step was repeated once.
818 The supernatants were combined and used for the assay of CMP-[~4C]NAcNeu and [ 14C]NAcNeu. Method B is a modification of the method of Kean [4] which involves the separation of CMP-NAcNeu and NAcNeu by ion-exchange chromatography. The pooled supernatants were placed on top of a 0.8 X 2 cm column of Dowex 1-X8 (100-200 mesh) in the HCO3-form. [~4C]NAcNeu was eluted with 25 ml 0.05 M triethylammonium bicarbonate and subsequent elution with 30 ml 1.0 M triethylammonium bicarbonate released CMP-[ ~4C]NAcNeu. The amount of radioactivity in the eluates was determined in 1-ml aliqu0ts (or in concentrates obtained by freeze-drying the eluates) using a dioxane-based scintillation fluid. In Method C separation of CMP-[14C] NAcNeu and [14C] NAcNeu is accomplished by ascending paper chromatography, as described previously [7]. 50 /~1 of the pooled supernatants were applied as a 2-cm streak on Whatman 3MM paper. Chromatography was performed overnight at 10°C with 95% ethanol/ 0.6 M NH4OH (7 : 3, v/v) as a solvent mixture. Three radioactive areas were detected with a radiochromatograph scanner (Fig. 1). From comparison with radiochromatographs with standard compounds these areas appeared to correspond to CMP-NAcNeu, NAcNeu and N-acetylmannosamine. The radioactive areas were cut out and counted either directly in a toluene-based scintillation fluid, or, after extraction of the radioactive compounds with water, in a dioxane-based scintillation fluid. The appearance of N-acetylmannosamine seems to be a consequence of the enzymatic breakdown of NAcNeu by NAcNeu aldolase (unpublished results). As a consequence of this breakdown the CMP-NAcNeu hydrolase activity has to be computed by summarizing the radioactivities in NAcNeu and N-acetylmannosamine. In Method C this is accomplished by combining the NAcNeu and the N-acetylmannosamine areas. Because N-acetylmannosamine does not bind to the anion-exchange resin used in Method B, in this method the compound is recovered in the void volume of the column, which is routinely combined with the 0.05 M bicarbonate fraction containing the radioactive NAcNeu. Finally in Method A the NAcNeu aldolase-catalyzed breakdown of NAcNeu does not interfere with t h e calculation of the CMP-NAcNeu hydrolase activity, because it is computed from the difference in the amounts of CMP-NAcNeu present before and after incubation of the enzyme preparation. In all instances the net decrease in CMP-[~4C] NAcNeu appeared to be count-
Fig. 1. R a d i o e h r o m a t o p a p h i c s c a n o f a p a p e r c h r o m a t o g r a m o f t h e r e a c t i o n p r o d u c t s resulting a f t e r inc u b a t i o n o f a c a l f k i d n e y c o r t e x h o m o g e n a t e f o r C M P - N A c N e u h y d r o l a s e activity at p H 9 . 0 . T h e i n c u b a t i o n was p e r f o r m e d and assayed as described i n M a t e r i a l s and M e t h o d s f o r M e t h o d C. A p r o l o n g e d inc u b a t i o n t i m e w a s c h o s e n t o visualize N- [ 14C ] a c e t y l m a n n o s a m i n e ( M a n N a c ) .
819
erbalanced stoichiometrically by the appearance of [~4C] NAcNeu and N-[~4C] acetylmannosamine. One unit of CMP-NAcNeu hydrolase activity is defined as the quantity of enzyme cleaving 1 ~mol of CMP-NACNeu per h a t 37°C. When a CMP-NAc-Neu hydrolase preparation was incubated under standard incubation conditions using 70 nmol CMP-[14C] NAcNeu the same activities were obtained independent whether Method A, B or C was used. The advantages of Methods B and C to Method A are (i) that the enzyme can be incubated under conditions of substrate saturation (300 nmol CMP-NAcNeu) and (ii) that the amount of NAcNeu produced as well as the residual amount of CMP-NAcNeu are measured. On the other hand the non-radioactive Method A is a more convenient (and less expensive) one because the results are obtained rapidly. Although in this method the CMP-NAcNeu hydrolase activity is 'not determined under conditions of substrate saturation (i.e., enzyme activity is lower than obtained with Methods B and C), the method proved valuable for routine determinations as is shown by the linear increasing reaction rates with respect to time and enzyme concentration (of. Fig. 5).
Assay o f other enzyme activities CMP-NAcNeu synthetase, succinate-2-(p-iodophenyl)-3~p-nitrophenyl)-5phenyltetrazolium reductase (succinate-INT reductase), ~alactosidase and glucose-6-phosphatase activities were assayed as described previously [10]. (Na÷ + K*)-dependent ATPase and Mg2÷~iependent ATPase activities were determined according to Bonting et al. [11] after treatment of the samples with 0.1% sodium deoxycholate and subsequent storage at --20°C for at least 48 h [12]. NADPH~ytochrome c reductase activity was determined according to Phillips and Langdon [13]. Alkaline phosphatase activity was assayed according to Bessey et al. [14] using p-nitrophenyl phosphate as a substrate. Phosphodiesterase I (EC 3.1.4.1) activity was assayed with p-nitrophenyl thymidine 5'phosphate according to a modification of the method of Razzell [15]. The incubation mixture contained 45/~mol Tris • acetate buffer (pH 8.9), 3.0/~mol MgC12, 1% Triton X-100, 0.3 /~mol substrate and 0.01--0.23 unit of enzyme in a total volume of 0.3 ml. Substrate was added after preincubation of the mixture at 37°C for 2 rain. After subsequent incubation for 10 rain at the same temperature the increase in absorbance at 405 nm was read immediately, emM = 12.0 Was used to calculate the amount ofp-nitrophenol liberated. Glycoprotein sialyltransferase was assayed by measuring the amount of [14C] NAcNeu incorporated into chloroform/methanol-insoluble protein using desialyzed al-acid glycoprotein as an acceptor [16]. One unit of enzyme activity is defined as the amount of enzyme producing I/~mol of product per h. Chemical assays Determination of protein and inorganic phosphate was performed as described previously [1]. Tissue homogenization and fractionation Crude homogenates were prepared by two different procedures. For kinetic studies homogenates were prepared in 0.01 M Tris • maleate buffer (pH 7.4) as described previously [17]. For subcellular distribution studies calf kidney cor-
820 tex homogenates were prepared in 0.25 M sucrose/0,01 M Tris • maleate buffer (pH 7.4) by 6 up-and-down strokes of a Teflon-glass Potter-Elvehjem type homogenizer (0.03 mm clearance) rotating at 800 rev./min. The homogenate obtained was filtered twice through two layers of surgical gauze (18 threads/ cm). The filtered homogenate was subjected to differential centrifugation. Each subcellular fraction obtained was washed twice by repeated centrifugation and the pooled supernatants were then subjected to the next centrifugation step. In this way the homogenate was separated in five particulate fractions and one soluble fraction (see first column of Table III). Preparation o f p l a s m a m e m b r a n e - r i c h fractions
For the preparation of plasma membrane-rich fractions of calf kidney cortex we used a m e t h o d which is essentially a combination of fractionation methods of Kamat and Wallach [19] and of Jacobsson [12] (Scheme I). By this SCHEME 1
kidney cortex
I I
homogenization with a Potter-glvehjem type homogenizer in 3 mM Tris buffer (pH 7.5) plus .3 vol. of 1.0 M sucrose/3 mM Tris.Hel buffer (pH 7.5) resulting in 0.25 M sucrose
I
10 rain, 10 000 X g pellet, discarded supernatant layered on 1.6 M sucrose in MSE 3)< 23 {swing-out) / 30 [min, 105 000
X
g
45 rainv g/////A ooo PM~ MSE 3× 23
MSE 3× 70
pellet resuspended in 0.25 M sucrosell mM Tris.HCI buffer (pH 8.2) and washed with 10 and 1 mM Tris. HCI buffer (pH 8.2), respectively
I I dialysis against 1 mM Tris.HC1 buffer (pH 8.2)/1 rnM MgSO4 for 2 h homogenized vigorously in 1 mM Ttis-HCI buffer (pH 8.2)
~-- ~
3.107
g. min
~
plasma membranes (PM=)
23.9 % Ficoll endoplmmaatie reticulum membranes {ER= ) M S E 3× 70
method plasma membrane fractions are obtained from a 10 000 X g supernatant of vigorously homogenized tissue. This microsomal fraction is subfractionated into plasma membrane and endoplasmatic reticulum membrane vesicles taking advantage of the differences in charge density on the inner surface of the membrane vesicles and the different behaviour towards Ficoll (see Steck and Wallach [20]). Minced kidney cortex was vigorously homogenized by 15 up-
821 and
