Spleen Enzyme Research Articles

Brain-type prostaglandin D synthetase occurs in the rat cochlea.

Prostaglandin D synthetase [(5Z, 13E)-(15S)-9 alpha, 11 alpha-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase, EC 5.3.99.2] activity was found in the high-speed (100,000 x g, 1 hr) supernatant of the homogenate of the cochlea of adult rats. The specific activity (14.0 nmol/min per mg of protein) was 6- to 7-fold higher than that in the central nervous system. The enzyme showed full activity with 1 mM glutathione, 1 mM 2-mercaptoethanol, or 0.5 mM dithiothreitol and was almost completely inhibited by 1 mM 1-chloro-2,4-dinitrobenzene. The Km value for prostaglandin H2 was about 20 microM. These catalytic properties are the same as those of rat brain prostaglandin D synthetase but different from those of rat spleen prostaglandin D synthetase. The activity decreased to less than 20% of its initial level after incubation with excess amounts of a polyclonal or a monoclonal antibody against the brain enzyme, but the activity remained unchanged with a polyclonal antibody against the spleen enzyme, indicating that the brain-type enzyme synthesizes prostaglandin D2 in the cochlea. When cryosections of 5-week-old (adult) rat cochleas were stained by an immunoperoxidase method with antibodies against the brain enzyme, the immunoreactivity was found in inner and outer hair cells, Claudius' cells, Deiters' cells, marginal cells, basal cells, and cells of Reissner's membrane. In 8-day-old rats, the immunoreactivity was found in all of these cell types except hair cells. The immunoreactivity in hair cells was found in only one specimen from 9-day-old animals, and no immunoreactivity was found in spiral ganglion cells at any of the ages examined. These findings indicate that prostaglandin D2 is produced by the brain-type synthetase in the indicated types of cochlear cells.

Biochemical and immunological characterization of rat spleen prostaglandin D synthetase.

Rat spleen prostaglandin D synthetase (Christ-Hazelhof, E., and Nugteren, D. H. (1979) Biochim. Biophys. Acta 572, 43-51) is very similar to rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi O. (1985) J. Biol. Chem. 260, 12410-12415) as judged by their pI (4.7-5.2), Mr (26,000-27,000), and self-inactivation during the isomerase reaction from prostaglandin H2 to prostaglandin D2. However, the amino acid compositions of these two enzymes were quite different. Furthermore, the spleen enzyme was associated with the glutathione S-transferase activity, differing from the brain enzyme. The synthetase and transferase activities of the spleen enzyme showed almost identical pH and glutathione dependencies, the optimum pH = 8.0 and Km for glutathione = 300 microM. The Km values for prostaglandin H2 and 1-chloro-2,4-dinitrobenzene (a substrate for the transferase) were about 200 microM and 5 mM, respectively. The synthetase activity was dose-dependently inhibited by 1-chloro-2,4-dinitrobenzene (IC50: approximately 5 mM) and more strongly by nonsubstrate ligands, such as bilirubin and indocyanine green (IC50: 150 and 2 microM, respectively). Both the synthetase and transferase activities of the purified enzyme dose-dependently decreased and showed identical immunotitration curves by incubation with antibody against this enzyme, but remained unchanged when treated with antibody against the brain enzyme. The antibody specific for the spleen enzyme absorbed almost all of the synthetase activity and about 10% of the transferase activity in the spleen, but not the transferase activity in the liver, heart, and testis. These results show that the two types of prostaglandin D synthetase are similar but different enzymes and that the spleen enzyme is a unique glutathione S-transferase differing from other isozymes and their subunits reported previously.

Relative deficiency of serine proteinase activities in spleen of aged mice

We examined the relation of hydrolytic enzymes in spleen to the aging process in mice over a period of 30 months. When the enzymatic activities were expressed as activities per milligrams protein, those of serine proteinases and dipeptidyl peptidase IV (Gly-Pro-AP) significantly decreased with age, whereas that of L-leucine aminopeptidase (Leu-AP) increased significantly. However, when expressed as total activities, the enzymatic activities in spleen generally tended to increase with age, except in the case of serine proteinases, because of the age-related increase in spleen weight. The results were taken to indicate that the activities of serine proteinases become relatively more deficient in the spleen as age increases. The results of a multivariate study maintained this peculiarity of serine proteinases in comparison with other enzymes. The relative deficiency of serine proteinases in spleen may be somehow related to immunodeficiency in aged animals, as judged from dimilar findings in animal models of systemic erythematodes.

Chemical evidence for the pH-dependent control of ion-pair geometry in cathepsin B. Benzofuroxan as a reactivity probe sensitive to differences in the mutual disposition of the thiolate and imidazolium components of cysteine proteinase catalytic sites.

Benzofuroxan reacts with the catalytic-site thiol group of cathepsin B (EC 3.4.22.1) to produce stoichiometric amount of the chromophoric reduction product, o-benzoquinone dioxime. In a study of the pH-dependence of the kinetics of this reaction, most data were collected for the bovine spleen enzyme, but the more limited data collected for the rat liver enzyme were closely similar both in the magnitude of the values of the second-order rate constants (k) and in the shape of the pH-k profile. In acidic and weakly alkaline media, the reaction is faster than the reactions of benzofuroxan with some other cysteine proteinases. For example, in the pH region around 5-6, the reaction of cathepsin B is about 10 times faster than that of papain, 15 times faster than that of stem bromelain and 6 times faster than that of ficin. The pH-dependence of k for the reaction of cathepsin B with benzofuroxan was determined in the pH range 2.7-8.3. In marked contrast with the analogous reactions of papain, ficin and stem bromelain [reported by Shipton & Brocklehurst (1977) Biochem. J. 167, 799-810], the pH-k profile for the cathepsin B reaction contains a sigmoidal component with pKa 5.2 in which k increases with decrease in pH. This modulation of the reactivity of the catalytic-site -S-/-ImH+ ion-pair state of cathepsin B (produced by protonic dissociation from -SH/-ImH+ with pKa approx. 3) towards a small, rigid, electrophilic reagent, in a reaction that appears to involve both components of the ion-pair for efficient reaction, suggests that the state of ionization of a group associated with a molecular pKa of approx. 5 may control ion-pair geometry. This might account for the remarkable finding [reported by Willenbrock & Brocklehurst (1984) Biochem. J. 222, 805-814] that, although the ion-pair appears to be generated in cathepsin B as the pH is increased across pKa 3.4, catalytic competence is not generated until the pH is increased across pKa 5-6.

Characterization of phosphohydrolase activity in bovine spleen extracts: Identification of a bis( p-nitrophenyl)phosphate-hydrolyzing activity (phosphodiesterase IV) which also acts on adenosine triphosphate

Several bovine spleen enzymes with acid pH optima, some of which hydrolyze bis( p-nitrophenyl)phosphate and therefore fit the definition of “phosphodiesterase IV,” were partially separated by isoelectric focusing and ion-exchange techniques. The activities were characterized by zymogram analysis with the aid of p-nitrophenyl and 4-methylumbelliferyl phosphate and phosphonate substrates. A number of these enzymes meet the criteria for phosphodiesterase I or other phosphodiesterases. However, the predominant phosphodiesterase I hydrolyzes the bis( p-nitrophenyl)- and 4-methylumbelliferyl phosphates, p-nitrophenyl and 4-methylumbelliferyl phenylphosphonate, and ATP at the β-γ bond, but not p-nitrophenyl or 4-methylumbelliferyl 5′-thymidylate (the usual PDE I substrates). These properties, as well as the pH optimum, distinguish the activity from the previously described, alkaline pH optimum PDE I. A second phosphodiesterase hydrolyzes only the phenylphosphonates. Several other activities, less well described, are apparent on zymograms. None of the phosphodiesterases IV was also a phosphodiesterase II (no hydrolysis of 4-methylumbelliferyl 3′-thymidylate).

Spectroscopic, ligand binding, and enzymatic properties of the spleen green hemeprotein. A comparison with myeloperoxidase.

The bovine spleen green hemeprotein, a peroxidase which exhibits spectrophotometric properties similar to those of granulocyte myeloperoxidase, was purified using an improved method. The ligand affinity of the ferric enzyme was spectroscopically determined using chloride and cyanide as exogenous ligands. The pH dependence of the apparent dissociation constant of the enzyme-chloride complex showed the presence of a proton dissociable group with a pKa value of 4 on the enzyme; chloride binds to the enzyme when this group is protonated with a dissociation constant of 60 microM. The cyanide affinity of the enzyme is also regulated by the group with a pKa value of 4, but in this case cyanide binds to the unprotonated enzyme with a dissociation constant of 0.6 microM; only the protonated, uncharged form of cyanide reacts with the enzyme. Cyanide binding was competitively inhibited by chloride, and chloride binding was also competitively inhibited by cyanide. The EPR spectrum of the resting enzyme exhibited a rhombic high spin signal at g = 6.65, 5.28, and 1.97 with a low spin signal at g = 2.55, 2.32, and 1.82. Upon formation of the chloride complex, the spectrum was replaced with a new high spin EPR signal with g-values of 6.81, 5.04, and 1.95. The cyanide complex showed a low spin EPR signal with g-values of 2.83, 2.25, and 1.66. Examination of the enzymatic activity of the spleen green hemeprotein by following the chlorination of monochlorodimedon has indicated that the enzyme has the same chlorinating activity as myeloperoxidase; the spleen green peroxidase can catalyze the formation of hypochlorous acid from hydrogen peroxide and chloride ion. Comparison of the present data with those of myeloperoxidase has led to the conclusion that the structure of the iron center and its vicinity in spleen green hemeprotein is very similar, if not identical, to that of myeloperoxidase. The spleen enzyme can thus be used as a model to study the active center, and its environment, in myeloperoxidase.

Human spleen dihydroorotate dehydrogenase: Properties and partial purification

Human spleen dihydroorotate dehydrogenase is associated with the mitochondrial membrane and is linked to the respiratory chain via ubiquinone. The enzyme activity was unaffected by pyridine nucleotides. The product of the reaction, orotate, was a potent inhibitor. However, a range of other naturally occurring pyrimidines or purines had no significant effect on the activity. No evidence for the involvement of a complexed metal ion or for an active sulfhydryl group was obtained. Purification of the enzyme was achieved by preparation of an acetone powder and extraction with Triton X-100, followed by preparative polyacrylamide gel electrophoresis. Activity was observed by the addition of the artificial electron acceptors, ubiquinone 50 or PMS. Purification resulted in alteration of the pH optimum and of other kinetic characteristics. Two molecular-weight species, of molecular weight 88,000 and 98,000, were consistently observed. The properties of the human spleen enzyme were similar in principle to those for the rat liver enzyme. Differences in the mode of linkage to the respiratory chain for the mitochondrially bound enzyme, and in the characteristics of the purified enzyme, were observed.

The type 5, acid phosphatase from spleen of humans with hairy cell leukemia. Purification, properties, immunological characterization, and comparison with porcine uteroferrin.

The spleens of patients with hairy cell leukemia contain high levels of a tartrate-insensitive, cationic, acid phosphatase (the human Type 5 isozyme). This phosphatase has been purified by a procedure which involves only two chromatographic steps: CM-cellulose chromatography and immunoaffinity chromatography on sheep antibodies generated against porcine uteroferrin. Uteroferrin is an abundant iron-containing acid phosphatase that can be recovered readily from porcine uterine secretions. Like uteroferrin, the purified human Type 5 phosphatase is a glycoprotein of molecular weight about 34,000. It contains two atoms of iron/molecule. The human phosphatase and uteroferrin also resemble each other closely in electrophoretic mobility, substrate specificity, and response to a variety of activators and inhibitors. Mouse monoclonal antibodies have been raised to uteroferrin and to the human Type 5 phosphatase. Three monoclonal antibodies which bind with high affinities to distinct sites on the uteroferrin molecule also recognize the human spleen enzyme, but bind to it with much lower affinity. These antibodies also recognize cationic acid phosphatases purified from bovine and rat spleens. A monoclonal antibody raised against the human enzyme, but selected for binding to uteroferrin, appears to recognize a relatively conserved site on all four phosphatases. We conclude that the human Type 5 isozyme belongs to a growing class of structurally related, iron-containing acid phosphatases which includes the iron-transport protein, uteroferrin.

Lysosomal enzymes in plasma, liver and spleen from rats with carbon tetrachloride-induced liver cirrhosis.

Plasma levels of the lysosomal enzymes, beta-hexosaminidase and beta-glucuronidase, were analyzed in rats with carbon tetrachloride induced liver cirrhosis. Out of 22 animals, 15 showed cirrhotic and 4 pre-cirrhotic livers. 4 cirrhotic animals exhibited cholestatic features with increased bilirubin, alkaline phosphatase (ALP) and aspartate aminotransferase (ASAT) in plasma. The remaining 15 pre-cirrhotic and cirrhotic rats showed clear significant changes only in ASAT levels. These 15 rats showed no consistent increase in plasma, spleen or liver lysosomal enzyme activities, whereas the 4 rats with cholestatic features exhibited considerable increases of lysosomal enzymes. The increased activities might be attributed to decreased biliary excretion and/or increased production of lysosomal enzymes by activated macrophages.

The pH dependence of breakdown of various purified brain proteins by cathepsin D preparations

In a continuing study of control processes of cerebral protein catabolism we compared the activity of cathepsin D from three sources (rat brain, bovine brain, and bovine spleen) on purified CNS proteins (tubulin, actin, calmodulin, S-100 and glial fibrillary acidic protein). The pH optimum was 5 for hydrolysis with tubulin as substrate for all three enzyme preparations, and it was pH 4 with the other substrates. The pH dependence curve was somewhat variable, with S-100 breakdown relatively more active at an acidic pH range. The formation of initial breakdown products and the further catabolism of the breakdown products was dependent on pH; hence the pattern of peptides formed from glial fibrillary acidic protein was different in incubations at different pH's. The relative activity of the enzyme preparations differed, depending on the substrate: with tubulin and S-100 as substrates, rat brain cathepsin D was the most active and the bovine spleen enzyme was the least active. With calmodulin and glial fibrillary acidic protein as substrates, rat brain and spleen cathepsin D activities were similar, and bovine brain cathepsin D showed the lowest activity. Actin breakdown fell between these two patterns. The rates of breakdown of the substrates were different; expressed as μg of substrate split per unit enzyme per h, with rat brain cathepsin D activity was 8–9 with calmodulin and S-100, 4 with glial fibrillary acidic protein, 1.8 with actin, and 0.9 with tubulin. The results show that there are differences in the properties of a protease like cathepsin D, depending on its source; furthermore, the rate of breakdown and the characteristics of breakdown are also dependent on the substrate. We recently measured the breakdown of brain tubulin by cerebral cathepsin D in a continuing study of the mechanisms and controls of cerebral protein catabolism (Bracco et al., 1982a). We found that tubulin breakdown is heterogeneous, that membrane-bound tubulin is resistant to cathepsin D but susceptible to thrombin (Bracco et al., 1982b), and that cytoplasmic tubulin was in at least two pools, one with a higher, another with a lower, rate of breakdown. The pH optimum of tubulin breakdown by cerebral cathepsin D differed significantly from the pH optimum of hemoglobin breakdown by the same enzyme. These findings showed that the properties of breakdown by a cerebral protease depend on the substrate. To further examine this dependence of properties of breakdown on the substrate, we now report measurements of pH dependence of breakdown of several purified proteins (tubulin, actin, calmodulin, S-100, glial fibrillary acidic protein [GFA]) from brain by cathepsin D preparations from three sources, rat brain, bovine brain, and bovine spleen. We also compare the rate of breakdown of the various proteins with the rate of hemoglobin breakdown.

Heme metabolism in liver and spleen of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU)-treated rats

The effect of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), an anticancer alkylating agent of the nitrosourea group, on liver and spleen enzymes involved in the control of heme metabolism was studied. A single oral dose of 50 or 100 mg/kg CCNU caused a time-dependent loss in weight of both spleen and liver. Seven days after CCNU treatment (lOO mg/kg) the weights were at 45 and 65% of controls respectively. The activity of δ-aminolevulinic acid synthetase (ALA-S), the rate-limiting enzyme in heme biosynthesis, declined in spleen and liver to 11 and 24% of control values, respectively, 7 days after CCNU treatment. Heme oxygenase activity, the rate-limiting enzyme of heme breakdown, was moderately increased in liver and spleen following CCNU administration. In liver, heme oxygenase activity was 142% of control values at 24 hr, and in spleen the activity was 180% of controls at 1 week. Pretreatment of the animals with phenobarbital (PB) (40 mg/kg/day, i.p.) for 4 days caused a reversal in the decline of liver weight with no effect on the decline in spleen weight following CCNU treatment. Similarly, PB pretreatment reversed the decline in hepatic ALA-S activity after CCNU administration but had no effect on the decline in splenic ALA-S activity. This study indicates that CCNU causes significant decreases in the activity of enzymes of heme biosynthesis in spleen and liver. The CCNU hemotoxicity in the liver was reversed by PB pretreatment whereas the splenic hemotoxicity was unchanged.

Age-related changes in glucose metabolizing enzymes in spleen, thymus, and pulmonary lavage cells from F344 rats

Healthy male Fischer 344 rats were sampled at 6, 12, 18, and 24 months of age. There was no gross pathological evidence or deviations in body weight, hermatology, or clinical chemistry that were indicative of disease. Mixed populations of thymus, spleen, and pulmonary cells were obtained for enzymatic analyses. Key enzymes from the hexose monophosphate shunt, glycolysis and the tricarboxylic acid cycle were evaluated to determine if there were tissue-specific or pathway-specific changes that occurred during aging. The enzyme responses among the tissues were not consistent during the aging process. Generally the activities of the glucose metabolizing enzymes in thymus and pulmonary lavage cells decreased with age whereas they increased in the spleen cells. Between 18 and 24 months enzymes representative of all three glucose metabolic pathways decreased in pulmonary lavage cells, whereas the decreases in thymic cells were mainly restricted to glycoltyic enzymes. By contrast there were two- to ten-fold increases during aging in all of the splenic enzymes measured except malate dehydrogenase. The alterations in tissue enzyme activities probably reflected the changing cellular populations during aging, and in the thymic and pulmonary lavage cellular environment resulted in a loss of energy production by glucose oxidation, compared to the vigorous activity maintained in spleen.

Immunological and isoelectric focusing study of β-glucocerebrosidase from normal and Gaucher disease

Comparison of normal and Gaucher disease β-glucocerebrosidase by agarose isoelectric focusing (IEF) demonstrated additional bands at the pI-6 area seen within the mutated enzyme, while both normal placenta and spleen enzyme preparations manifest only major activity at pI-5. Antiglucocerebrosidase antibodies precipitated both normal and pathological enzymes, however, more antibodies were needed to reach an equivalence with the normal enzymes than with the Gaucher's. Cross reactivity of the IEF isozymes were detected by direct immunodifusion on the prefocused gel.

An immunological study of delta-aminolevulinic acid dehydratase specificity consistent with the phylogeny of species.

Rabbit antibody directed to homogeneously purified mouse liver delta-aminolevulinic acid dehydratase cross-reacted with the enzyme in erythrocytes, spleen, kidney and brain in the mouse. The antibody also cross-reacted with the enzyme in the rat, hamster and gerbil, but not in the rabbit, guinea pig, cattle, chick embryo, and human. In contrast, rabbit antibody against the human enzyme partially recognized the monkey enzyme, but not the enzyme in the other species. The species specificity of delta-aminolevulinic acid dehydratase in this study was consistent with the phylogenetic evolution of the species examined.

Biosynthesis of Δ-aminolevulinate in greening barley leaves VI. Activation of glutamate by ligation to RNA

The components involved in the enzymic conversion of glutamate to δ-aminolevulinate have been separated into three fractions; a Blue Sepharose bound, a chlorophyllin-(or heme) Sepharose bound and an unbound fraction. Combination of these three fractions reconstituted δ-aminolevulinate synthesis from glutamate. Participation of a specific RNA in δ-aminolevulinate synthesis was established by isolating a homogeneous RNA from the chlorophyllin-Sepharose bound fraction and reconstituting δ-aminolevulinate synthesis in the presence of the unbound and Blue Sepharose bound fractions. The RNA involved in δ-aminolevulinate synthesis was purified by high-pressure liquid chromatography and preparative gel electrophoresis. In the presence of the Blue Sepharose bound fraction, ATP and Mg2+, glutamate bound covalently to this RNA. Co(III)-ATP-o-phenanthroline bound to the RNA and strongly inhibited glutamyl-RNA formation, whereas heme and Mg-protoporphyrin at 50 μM were only slightly inhibitory. The chlorophyllin-Sepharose bound fraction also contained two other glutamate acceptor RNAs. RNAase A and snake venom phosphodiesterase strongly reduced δ-aminolevulinate synthesis and glutamyl-RNA formation, whereas addition of DNAase or spleen phosphodiesterase was only slightly inhibitory. The RNA became sensitive to the spleen enzyme after phenol extraction of the chlorophyllin-Sepharose bound fraction. E. coli tRNAGlu orwheat germ tRNA did not reconstitute δ-aminolevulinate synthesis when combined with the Blue Sepharose bound and unbound fractions. The RNA involved in δ-aminolevulinate synthesis hybridised to a 3.9 kb Hind III Pst I restriction endonuclease fragment from the barley chloroplast genome located in the large single copy region 38 kb from the large subunit gene for RuBP carboxylase and 12 kb from the inverted repeats. Glutamate 1-semialdehyde aminotransferase was labelled during35S-incorporation into greening barley leaves but not during incorporation into isolated plastids. It is suggested that an NADPH-dependent dehydrogenase involved in the reduction of glutamyl-RNA to glutamate 1-semialdehyde is present in the Blue Sepharose bound fraction.

Affinity separation of an acid phosphatase from rat tissues and Gaucher spleen with immobilized Cibacron Blue.

An acid phosphatase species which was activated by Fe2+ was determined to be partially soluble but mainly particulate in rat spleen. The particulate enzyme could be extracted into 1 M KCl. This enzyme bound to Cibacron Blue-immobilized Sepharose (Blue-Sepharose) and was desorbed by 2 M KCl with a good yield, while the other acid phosphatases in rat spleen did not adsorb on Blue-Sepharose. The enzymes eluted on Blue-Sepharose chromatography of both the soluble and particulate fractions were electrophoretically identical. The enzyme hydrolyzed aryl monophosphates, phosphoproteins, and nucleoside di- and triphosphates. The activity for the three kinds of substrate was similarly activated by Fe2+, ascorbic acid and cysteine, and inhibited by molybdate, Cu2+ and F-. Cibacron Blue inhibited the enzyme competitively with respect to a substrate, p-nitrophenyl phosphate, but kinetic analysis suggested that more than one dye molecule binds to the enzyme. The Blue-Sepharose technique could be applied not only to quantitative separation of acid phosphatases similar to the spleen enzyme from bone and epidermis of rat, but also to that of a tartrate-resistant acid phosphatase from human spleen with Gaucher's disease.

Zur Substratspezifität der Phenoloxidase (EC 1.14.18.1)

Using 10 microns thick cryostat-slides of spleen and gut from white rats the following substances (in 2 different concentrations) were tested as substrates for the enzyme phenoloxidase compared with dihydroxyphenylalanine: pyrogallic acid, resorcin, 2.4-dinitrophenol, tyraminhydrochloride, o-cresol, and epinephrine. Only pyrogallic acid and resorcin resulted in positive reaction. Resorcin showed a positive reaction only when the higher concentration is employed. The investigations show clearly that the detectable enzyme in gut, spleen, and oral cavity is a polyphenoloxidase, which occurs consequently not only in granulocytes.

Endocytosis and breakdown of mitochondrial malate dehydrogenase in the rat in vivo. Effects of suramin and leupeptin.

1. The plasma clearance of intravenously injected 125I-labelled mitochondrial malate dehydrogenase (half-life 7 min) was not influenced by previous injection of suramin and/or leupeptin (inhibitors of intralysosomal proteolysis). 2. Pretreatment with both inhibitors considerably delayed degradation of endocytosed enzyme in liver, spleen, bone marrow and kidneys. 3. The tissue distribution of radioactivity was determined at 30 min after injection, when only 3% of the dose was left in plasma. All injected radioactivity was still present in the carcass. The major part of the injected dose was found in liver (49%), spleen (5%), kidneys (13%) and bone, including marrow (11%). 4. Liver cells were isolated 15 min after injection of labelled enzyme. We found that Kupffer cells and parenchymal cells had endocytosed the enzyme at rates corresponding to 9530 and 156 ml of plasma/day per g of cell protein respectively. Endothelial cells do not significantly contribute to uptake of the enzyme. 5. Uptake by Kupffer cells was saturable, whereas uptake by parenchymal cells was not. This suggests that these cell types endocytose the enzyme via different receptors. 6. Previous injection of carbon particles greatly decreased uptake of the enzyme by liver, spleen and bone marrow.

Angiotensin converting enzyme in cultured fibroblasts in Gaucher and Niemann-Pick diseases.

AbstractSimilar low levels of angiotensin converting enzyme activity were found in cultured fibroblasts from types 2 and 3 Gaucher disease, type A Niemann-Pick disease, and normal subjects. The highly elevated levels of angiotensin converting enzyme in serum and spleen in Gaucher disease is not due to a marked general cellular elevation of the enzyme, but may be due to elevated synthesis of ACE secondarily stimulated in Gaucher cells.

Subcellular distribution of phospholipid-sensitive calcium-dependent protein kinase in guinea pig heart, spleen and cerebral cortex, and inhibition of the enzyme by Triton X-100

The subcellular distribution of phospholipid-sensitive Ca 2+-dependent protein kinase in guinea pig heart was found to be: cytosol, 73%; microsome, 18%; plasma membrane, 9%; nuclei and mitochondria, < 0.1%. The enzyme in spleen and cerebral cortex was distributed nearly equally in the cytosolic and total (unfractionated) particulate fractions. The particulate enzyme in heart was released by EGTA (2.5 mM) alone but not by Triton X-100 (0.3%) alone, although a combination of the two was most effective. On the other hand, the particulate enzyme in spleen and cerebral cortex was released only by a combination of Triton X-100 and EGTA. Triton X-100 inhibited the enzyme, and this inhibition was reversed by phosphatidylserine (a phospholipid cofactor for the enzyme). The detergent, however, was without effect on cyclic AMP-dependent and cyclic GMP-dependent protein kinases.