Postnatal development of the actual and total activity of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues

Actual and total activities of the branched-chain 2-oxo acid dehydrogenase complex were determined in homogenates of quadriceps muscle, heart, liver, kidney and brain from rats of 0-70 days age. All rat tissues except quadriceps muscle showed a marked increase of total activity between 0 and 21 days, heart and kidney also after weaning. The actual activity rose after birth in liver, kidney and brain and after weaning in liver, kidney and heart. The activity state was always about 100% in liver and varied between 40-60% in kidney and brain, 10-23% in heart and 6-12% in quadriceps muscle. The actual activities measured indicate, that the degradation of branched-chain 2-oxo acids mainly takes place in the liver of the newborn, suckling and young-adult rat.     

The autorelease of alkaline phosphatase from the plasma membrane during the incubation of cultured liver cell homogenates

When a rat hepatoma cell (R-Y121B) homogenate was incubated at 37 degrees C, 30-70% of the total alkaline phosphatase was released into the supernatant fluid from the precipitate fractions. The release reached a plateau level after 10 h of incubation at 37 degrees C. The optimum pH value for the release was 7.4. Alkaline phosphatase activity increased during the incubation of the cell homogenates, but this increase was independent of the enzyme release. Serum increased not only alkaline phosphatase activity in the cultured cells but also enzyme release in their homogenates. In addition, we examined a rat liver homogenate and the following 11 cell lines: 3 hepatoma cell lines, including the R-Y121B cell line, 4 liver cell lines, 2 human urinary bladder carcinoma cell lines, a kidney cell line, and a mouse adrenal tumor cell line. Only in the cultured liver cell line and hepatoma cell lines, 30-60% of the total enzyme was released into the soluble fraction from the precipitate fractions; the release was not observed in the other cell lines, nor in the rat liver homogenate. The release of alkaline phosphatase took place in both heat-stable and heat-labile alkaline phosphatases. Alkaline phosphatase, extracted from cell homogenates, showed two bands during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mobilities of the two bands changed inversely with or without sodium dodecyl sulfate. In general, the alkaline phosphatase which showed slow mobility with sodium dodecyl sulfate was more readily released from the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

The metabolism of progesterone by animal tissues in vitro. Sex and species differences in conjugate formation during the metabolism of [4-14C]progesterone by liver homogenates

1. Sex and species differences during the metabolism of [4-¹⁴C]progesterone by liver homogenates from rat, rabbit, guinea pig and hamster have been investigated. 2. Liver homogenate from male rat formed `water-soluble' metabolites faster and in significantly larger amounts than did liver homogenate from female rat. About 65–70% of the added progesterone was conjugated as glucuronide by liver homogenate from male rat and about 45–50% by that from female rat. Liver homogenate from male rat also formed glucuronides faster than did liver homogenate from female rat. Sulphate formation was low (8–16%) in liver homogenates from both male and female rats. 3. Hamster-liver homogenate did not show any sex difference in the rate of formation of `water-soluble' metabolites, but a sex difference was observed in the amount of free steroids recovered at low tissue:steroid ratios. Liver homogenate from female hamster formed glucuronides faster and in significantly larger amounts than did liver homogenate from male hamster, the reverse of what was found in rat liver. 4. Liver homogenates from male and female rabbits and guinea pigs formed `water-soluble' metabolites that were almost entirely glucuronides. 5. Neither rabbit liver nor guinea-pig liver showed any significant sex difference in the rate or amount of formation of total `water-soluble' metabolites or glucuronides, but guinea-pig liver was considerably less active than rabbit liver. 6. Glucuronides were quantitatively the major type of conjugate formed by the liver homogenates from both sexes of all species except the male hamster.     

THE ISOLATION OF A CELL MEMBRANE FRACTION FROM RAT LIVER

A procedure is described for isolating cell membranes from rat liver homogenates. 20 gm. of rat liver was homogenized in a Dounce homogenizer in ice cold water buffered to pH 7.5 with NaHCO₃, rupturing all of the cells and most nuclei. The diluted homogenate was filtered through cheesecloth to remove precipitated nucleoprotein and centrifuged at 1500 g, 10 minutes, to sediment a crude membrane fraction. The membrane containing sediment was recentrifuged 3 times in conical tubes (1220 g, 10 minutes), the top layer of the 2-layered sediment being retained. Flotation in a sucrose solution d = 1.22 freed the preparation from contaminating cell fragments and nuclear membranes not previously disintegrated. The floating material ~0.4 ml. was quite homogeneous and consisted of thin amorphous membranes. Electron micrographs revealed numerous double profiles similar in shape and dimensions to apposed liver cell membranes in intact tissue.     

Co-purification of alanine-glyoxylate aminotransferase with 2-aminobutyrate aminotransferase in rat kidney

Alanine-glyoxylate aminotransferase and 2-aminobutyrate aminotransferase were co-purified from rat kidney to a single protein (about 500-fold purified from the homogenate). The activity ratios of alanine-glyoxylate aminotransferase to 2-aminobutyrate aminotransferase were constant during co-purification steps suggesting the 2-aminobutyrate aminotransferase activity was catalysed by only alanine-glyoxylate aminotransferase. The molecular weight of the enzyme was estimated to be approx. 213 000, 220 000 and 236 000 by analytical ultracentrifugation, Sephadex G-150 gel filtration and sucrose density gradient centrifugation, respectively. From the polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, the enzyme consisted of four apparently similar subunits having a molecular weight of approx. 56 000. The enzyme was almost specific to L-alanine and L-2-aminobutyrate as amino donor and to glyoxylate, pyruvate and 2-oxobutyrate as amino acceptor. The enzyme was identified with rat liver alanine-glyoxylate aminotransferase isoenzyme 2 but not with rat liver alanine-glyoxylate aminotransferase isoenzyme 1 from Ouchterlony double diffusion analysis. Absorption spectra and some kinetic properties of the enzyme were clarified.     

A Rapid Purification Procedure for Camel Kidney Glutathione S-Transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37, 000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 μ;mol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100.

The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29, 000 D and 26, 000 D to give a native molecular weight of 55, 000 D.

The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. l-chloro-2, 4-dinltrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse.

Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

Studies on isolated cell components III. A cytological study of the effects of heparin on isolated nuclei

The gel released from isolated rat liver nuclei in response to heparin treatment has been found to stain with methylene blue, azure A, and methyl green when the dyes were added to the salt-sucrose nuclear isolation medium.Azure A and methylene blue caused rapid nuclear shrinkage to as little as $\text{1}\text{4}$ the original nuclear volume. Subsequent treatment with heparin caused the nuclei to fade rapidly and swell to approximately $\text{5}\text{4}$ of the original volume. With methylene blue stained nuclei heparin caused the extrusion of deeply stained, slightly birefringent rods through apertures on the nuclear surface. Methyl green also caused nuclear shrinkage, but to a lesser degree.Studies with the Feulgen reaction demonstrated structural damage in isolated rat liver nuclei as a result of heparin action. The viscous material released by heparin was shown to be Feulgen positive by resort to hydrolysis without prior fixation, since after customary fixatives the presence of a Feulgen positive reaction outside the nucleus could not be clearly demonstrated. The possibility is suggested that the Feulgen reaction following the customary fixatives depends in part on the manner in which the DNA is bound.The nuclei of leucocytes with visually intact cell membranes included in the nuclear preparations failed to show structural damage due to heparin and it is suggested that either the cell membrane provides some protection against heparin action or that damaged cells are more susceptible to this action.Observations made provide additional basis for the conclusion that heparin replaces DNA in the nucleo-histone of the nucleus, resulting in the structural damage observed, and releasing DNA in the form of a soluble viscous protein containing complex.     

INTRACELLULAR LOCALIZATION OF ENZYMES IN SPLEEN : I. REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE CYTOCHROMEc REDUCTASE, CYTOCHROMEc OXIDASE, AND SUCCINIC DEHYDROGENASE IN THE RAT AND GUINEA PIG

1. The intracellular distribution of nitrogen, DPNH cytochrome c reductase, succinic dehydrogenase, and cytochrome c oxidase has been studied in fractions derived by differential centrifugation from rat and guinea pig spleen homogenates. 2. In the spleens of each species, the nuclear fraction accounted for 40 to 50 per cent of the total nitrogen content of the homogenate, and the mitochondrial, microsome, and supernatant fractions contained about 8, 12, and 30 per cent of the total nitrogen, respectively. 3. Per mg. of nitrogen, DPNH cytochrome c reductase was concentrated in the mitochondria and microsomes of both rat and guinea pig spleens. Seventy per cent of the total DPNH cytochrome c reductase activity was recovered in these two fractions. The reductase activity associated with the nuclear fraction was lowered markedly by isolating nuclei from rat spleens with the sucrose-CaCl₂ layering technique. The lowered activity was accompanied by the recovery of about 90 per cent of the homogenate DNA in the isolated nuclei, indicating that little, if any, of the reductase is present in spleen cell nuclei. 4. Per mg. of nitrogen, succinic dehydrogenase was concentrated about 10-fold in the mitochondria of rat spleen, and 65 per cent of the total activity was recovered in this fraction. 5. Cytochrome c oxidase was concentrated, per mg. of nitrogen, in the mitochondria of both rat and guinea pig spleens. The activity associated with the nuclear fraction was greatly diminished when this fraction was isolated from rat spleens by the sucrose-CaCl₂ layering technique. Only 50 to 70 per cent of the total cytochrome c oxidase activity of the original homogenates was recovered among the four fractions from both rat and guinea pig spleens, while the specific activities of reconstructed homogenates were only 55 to 75 per cent of those of the original whole homogenates. This was in contrast to the results with DPNH cytochrome c reductase and succinic dehydrogenase where the recovery of total enzyme activity approached 100 per cent, and the specific activities of reconstructed homogenates equalled those of the original homogenates. The recovery of cytochrome c oxidase was greatly improved when only the nuclei were separated from rat spleen homogenates. 6. Data were presented comparing the concentrations (ratio of activity per mg. of nitrogen of the fraction to activity per mg. of nitrogen of the homogenate) of DPNH cytochrome c reductase in mitochondria and microsomes derived from different organs of different animals. 7. Data were presented comparing the activities per mg. of nitrogen of DPNH cytochrome c reductase in homogenates from several organs of various animals.     

Mechanisms of lipid peroxide formation in animal tissues

1. Homogenates of rat liver, spleen, heart and kidney form lipid peroxides when incubated in vitro and actively catalyse peroxide formation in emulsions of linoleic acid or linolenic acid. 2. In liver, catalytic activity is distributed throughout the nuclear, mitochondrial and microsomal fractions and is present in the 100000g supernatant. Activity is weak in the nuclear fraction. 3. Dilute (0·5%, w/v) homogenates catalyse peroxidation over the range pH5·0–8·0 but concentrated (5%, w/v) homogenates inhibit peroxidation and destroy peroxide if the solution is more alkaline than pH7·0. 4. Ascorbic acid increases the rate of peroxidation of unsaturated fatty acids catalysed by whole homogenates of liver, heart, kidney and spleen at pH6·0 but not at pH7·4. 5. Catalysis of peroxidation of unsaturated fatty acids by the mitochondrial and microsomal fractions of liver is inhibited by ascorbic acid at pH7·4 but the activity of the supernatant fraction is enhanced. 6. Inorganic iron or ferritin are active catalysts in the presence of ascorbic acid. 7. Lipid peroxide formation in linoleic acid or linolenic acid emulsions catalysed by tissue homogenates is partially inhibited by EDTA but stimulated by o-phenanthroline. 8. Cysteine or glutathione (1mm) inhibits peroxide formation catalysed by whole homogenates, mitochondria or haemoprotein. Inhibition increases with increase of pH.     

Tissue hemolysins as lysin-inhibitor complexes

1. Lytic substances are enzymatically produced at 37°C. from tissue slices or homogenates (mouse liver, kidney, etc.) and appear in the medium in which the tissue fragments are suspended. Their concentration increases with the time during which the tissue is kept at 37°C. (preincubation), and is accompanied by pH changes, so that the lytic activity as finally measured is a function of both the time of preincubation and of the pH. The optimum pH for lysin production is above 7.0, but the lysins, once produced, hemolyze red cells more rapidly at low pH's than at high ones. The enzyme system which produces the lysins is inactivated by heating to 100°C. for 5 minutes. Sodium iodoacetate and fluoride interfere with lysin production principally by reducing the concomitant pH shift; KCN accelerates the production of lytic material in mouse liver homogenates. 2. Comparison of the lytic activity of the supernatant fluid of a preincubated homogenate with the much greater lytic activity of the substances which can be extracted from the same supernatant fluid by alcohol and ether points to these extractable substances existing in the supernatant fluid as lysin-inhibitor complexes of relatively low lytic activity. These complexes are formed enzymatically during preincubation from non-lytic complexes in the tissue. The latter may be lipoproteins, and the highly lytic ether-extractable substances may be fatty acids or their soaps. 3. The diffusibility of the lysin-inhibitor complexes is small. 4. Lytic substances which are ether-insoluble can be extracted with alcohol from tissues as well as from serum. These "lysolecithin-like" substances exist in the supernatant fluids of homogenates as lysin-inhibitor complexes. 5. Lysis of mouse red cells by substances contained in mouse tissue (liver and kidney) is often accompanied by the formation of methemoglobin and choleglobin. Mouse red cells containing choleglobin are abnormally fragile both osmotically and mechanically, and it is possible that a process involving the production of choleglobin, accompanied or followed by globin denaturation, is one which contributes towards the hemolysis which occurs in systems containing tissue slices or homogenates.     

Intracellular localization of enzymes in spleen. I. Reduced diphosphopyridine nucleotide cytochrome c reductase,cytochrome c oxidase,and succinic dehydrogenase in the rat and guinea pig

1. The intracellular distribution of nitrogen, DPNH cytochrome c reductase, succinic dehydrogenase, and cytochrome c oxidase has been studied in fractions derived by differential centrifugation from rat and guinea pig spleen homogenates. 2. In the spleens of each species, the nuclear fraction accounted for 40 to 50 per cent of the total nitrogen content of the homogenate, and the mitochondrial, microsome, and supernatant fractions contained about 8, 12, and 30 per cent of the total nitrogen, respectively. 3. Per mg. of nitrogen, DPNH cytochrome c reductase was concentrated in the mitochondria and microsomes of both rat and guinea pig spleens. Seventy per cent of the total DPNH cytochrome c reductase activity was recovered in these two fractions. The reductase activity associated with the nuclear fraction was lowered markedly by isolating nuclei from rat spleens with the sucrose-CaCl(2) layering technique. The lowered activity was accompanied by the recovery of about 90 per cent of the homogenate DNA in the isolated nuclei, indicating that little, if any, of the reductase is present in spleen cell nuclei. 4. Per mg. of nitrogen, succinic dehydrogenase was concentrated about 10-fold in the mitochondria of rat spleen, and 65 per cent of the total activity was recovered in this fraction. 5. Cytochrome c oxidase was concentrated, per mg. of nitrogen, in the mitochondria of both rat and guinea pig spleens. The activity associated with the nuclear fraction was greatly diminished when this fraction was isolated from rat spleens by the sucrose-CaCl(2) layering technique. Only 50 to 70 per cent of the total cytochrome c oxidase activity of the original homogenates was recovered among the four fractions from both rat and guinea pig spleens, while the specific activities of reconstructed homogenates were only 55 to 75 per cent of those of the original whole homogenates. This was in contrast to the results with DPNH cytochrome c reductase and succinic dehydrogenase where the recovery of total enzyme activity approached 100 per cent, and the specific activities of reconstructed homogenates equalled those of the original homogenates. The recovery of cytochrome c oxidase was greatly improved when only the nuclei were separated from rat spleen homogenates. 6. Data were presented comparing the concentrations (ratio of activity per mg. of nitrogen of the fraction to activity per mg. of nitrogen of the homogenate) of DPNH cytochrome c reductase in mitochondria and microsomes derived from different organs of different animals. 7. Data were presented comparing the activities per mg. of nitrogen of DPNH cytochrome c reductase in homogenates from several organs of various animals.     

In Vitro Metabolic Stability of Exendin-4: Pharmacokinetics and Identification of Cleavage Products

The aim of this study was to investigate the metabolic stability and cleavage sites of exendin-4 in rat tissue homogenates, as well as to identify the types of proteases involved in exendin-4 degradation. The stability of exendin-4 in kidney and liver homogenates from rats was evaluated using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) with gradient elution. Furthermore, we used a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and LC-ESI-MS/MS to identify the structures of the major degradation products of exendin-4, and peptidase inhibitors were used to characterize exendin-4 degradation in rat liver and kidney homogenates and to identify the proteases involved in exendin-4 metabolism. Exendin-4 had a half-life of 7.8 and 100.9 min in the kidney and liver homogenate, respectively. The enzymes most likely to be involved in the degradation of exendin-4 were aminopeptidases, serineproteases, and metalloproteases. Exendin-4(15-39) and exendin-4(16-39) were the predominant direct exendin-4 metabolites in the kidney, and the main product of exendin-4 metabolism in the liver was exendin-4(12-39). Our results indicated that the metabolism of exendin-4 involved an initial endoproteolytic cleavage and subsequent exoproteolytic digestion. The degradation of exendin-4 in the kidney and liver homogenates followed distinct patterns, and the primary cleavage sites of exendin-4 degradation in rat kidney homogenates were located after AA-14, and -15, whereas those in rat liver homogenates were located after AA-11.     

Sex- and species-related differences in the biotransformation of isosorbide dinitrate by various tissues of the rabbit and rat

The biotransformation of isosorbide dinitrate (ISDN) by various tissues of the rabbit and rat was examined. Incubation of 2 X 10(-7) M ISDN at 37 degrees C with tissue homogenates of liver, lung, kidney, intestine, skeletal muscle, aorta, and erythrocytes from the rabbit and rat resulted in a significant disappearance of ISDN after a 30-min incubation (also, 5-min incubation for liver). The disappearance of ISDN in each tissue homogenate was accompanied by an equimolar production of the mononitrate metabolites, isosorbide-2-mononitrate (2-ISMN) and isosorbide-5-mononitrate (5-ISMN), with the exception of liver homogenates where the loss of ISDN could not be accounted for by mononitrate formation. The relative rate of ISDN disappearance in various tissue homogenates was for the male rabbit, liver greater than lung approximately intestine greater than kidney greater than erythrocytes approximately skeletal muscle approximately aorta; for the female rabbit, liver greater than kidney approximately lung approximately intestine greater than erythrocytes approximately skeletal muscle approximately aorta; and for the male rat, liver greater than intestine greater than erythrocytes greater than skeletal muscle greater than lung approximately kidney. A sex difference in the percent disappearance of ISDN was observed in homogenates of lung and intestine from male and female rabbits. In addition, a sex difference in the ratio of metabolite (2-ISMN/5-ISMN) formed by denitration of ISDN was seen in homogenates of lung, skeletal muscle, and erythrocyte lysate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the presence of a prostaglandin E 2 -9-keto reductase in rat organs

Antibodies directed toward PGF_2α which cross react with PGE₂ only slightly were used to detect conversion of PGE₂ to PGF_2α by homogenates of several rat tissues. This conversion by rat heart homogenates was demonstrated to be reversible, lost after trypsin digestion, and inhibited by several sulfhydryl blocking agents. The activity of the rat heart homogenate was precipitable by ammonium sulphate, was not dialyzable, and was 50% destroyed when the homogenate was incubated at 50° for 5 min. In the rat, the heart had the highest activity, followed by the kidney, brain, and liver. Negligible activity was found in smooth muscle, skeletal muscle, and whole blood of rat.     

Isolation of ribonuclease-free intact polyribosomes from rat kidney.

High levels of RNAase present in rat kidney have prevented isolation of intact polyribosomes from this tissue. This problem has been circumvented by a thorough in situ arterial perfusion of rat kidney, coupled with homogenization of the perfused rat kidney in heparin and detergents-fortified high-speed supernatant prepared from rat liver. This procedure reduced RNAase activity in the homogenate by as much as 70%. Sedimentation of the polyribosomes from this homogenate through a layer of 2.0 M sucrose resulted in a 78--80% yield of polyribosomes from the rat kidney. The resulting polyribosomal pellet contained less than 8% of the RNAase activity present in polyribosomes from non-perfused rat kidney. The remaining RNAase activity was separated from the larger polyribomes by sucrose density gradient centrifugation. The majority of the polyribosomes were larger than tetramers. This procedure also incrased both the yield and size of polyribosomes from rat and mouse liver.     

Metabolic stability of long-acting luteinizing hormone-releasing hormone antagonists

Long-acting luteinizing hormone-releasing hormone (LHRH) antagonists designed to be protease resistant consisted of a series of novel decapeptides structurally similar to LHRH. The aim of this study was to evaluate the in vitro metabolic stability of the LHRH decapeptides using pancreatin and homogenates models and identify the metabolites in rat liver homogenate for the purpose of illustrating the metabolic features of the decapeptides. The major metabolites in rat liver homogenate were identified by LC-ESI-MS(n). The half-lives of the 11 LHRH decapeptides were from 44 to 330?min in the pancreatin model. The half-lives of the five decapeptides in rat liver, kidney and lung homogenates were between 8 and 462?min. The most stable decapeptides were the LY616 and LY608 peptides with half-lives of 36?min in liver homogenate. Two major cleavage sites were found by analysing the metabolites of the LY618 peptide in rat liver homogenate, between the Pal(3)-Ser(4) and the Leu(7)-Ilys(8) peptide bonds. The major metabolites were produced via cleavages of peptide bonds at these sites, and further metabolic reactions such as hydroxylation, oxidative dechlorination, alcohol dehydration and isopropyl dealkylation were also observed.     

Cholate effects on all-trans-retinyl palmitate hydrolysis in tissue homogenates: solubilization of multiple kidney membrane hydrolases

Retinyl ester hydrolysis was observed in the absence of cholate in homogenates of rat lung, liver, kidney, intestine, and testes. Eighty-four percent of the activity in kidney was membrane-associated. The kidney microsomal fraction contained 19% of the total activity and was the only subcellular fraction that had increased specific activity relative to the homogenate (about 1.5-fold). In contrast, the cytosol was the only fraction that was decreased in specific activity (about 3-fold). Cholate (18 mM), reportedly required to observe hydrolysis of all-trans-retinyl esters by rat liver preparations, was not obligatory for activity in kidney homogenates or microsomes. The microsomal activity was solubilized efficiently and with a twofold increase in specific activity by the synthetic detergent 1-S-octyl-beta-D-thioglucopyranoside. Gel-permeation chromatography of the solubilizate suggested that at least two pools of activity existed, with molecular weights in the ranges 70-95 and 30-40 kDa. Neither hydrolyzed cholesteryl oleate. Both were more active in hydrolyzing retinyl palmitate than trioleoylglycerol. The higher mass pool had decreased trioleoylglycerol hydrolase activity relative to the solubilizate. Anion-exchange chromatography separated the lower mass pool into two major peaks. A major peak, distinct from the two peaks observed with the lower mass pool, was observed upon anion-exchange chromatography of the higher mass pool. These data demonstrate that multiple retinyl ester hydrolases, more efficient at hydrolyzing retinyl esters than cholesteryl esters and triacylglycerol, occur in a retinoid target tissue.     

Sulfite oxidase from Merluccius productus

Sulfite oxidase (sulfite:oxygen oxidoreductase, EC 1.8.3.1) was purified 482-fold from liver of the Pacific hake Merluccius productus. The molecular weight of the enzyme was found to be 120 000 by gel exclusion chromatography on Sephadex G-100. Electrophoretic analysis on sodium dodecyl sulfate (SDS)-polyacrylamide gel revealed that the enzyme was composed of two subunits whose molecular weight was estimated to be 60 000. The pH optimum of the enzyme was 8.7; Ks for sulfite, 2.5 x 10(-5) M; and that for cytochrome c, 3.6 x 10(-7) M. The enzyme elicited an EPR signal at g = 1.97 characteristic of pentavalent molybdenum. Colorimetric analysis also disclosed that the enzyme contained 2 mol each of heme and molybdenum per mol of protein. This fish liver homogenate in isotonic sucrose solution was fractionated by differential centrifugation into nuclei, mitochondria, microsomes and supernatant (100 000 X g). The major portion of sulfite oxidase activity was found in mitochondria. The sulfite oxidase activity was markedly high in liver and kidney, as compared with that in heart, spleen, muscle, gill and eye.