Subcellular localization and substrate specificity of dolichol kinase from rat liver

When purified subcellular fractions were prepared from rat liver and assayed for dolichol kinase activity using pig liver dolichol as a substrate, the microsomes were found to contain the highest specific activity and greater than 75% of the total actvity. With regard to substrate specificity, the microsomal enzyme showed a marked preference for saturation of the α-isoprene: dolichol-16 and -19 were 2.5-fold more active than the corresponding polyprenols. For a given class of prenol, the 16 and 19 isoprenologs exhibited similar activity, whereas the 11 isoprenolog appeared less active. The enzyme was twice as active against the naturally occurring polyprenol-16 (α-cis-isoprene) compared to synthetic α-trans-polyprenol-16. Taken together, the data indicate that the α-isoprene specificity follows the order: saturated>cis>trans. In addition, all-trans-2,3-dihydrosolanesol was not a substrate, suggesting that at least one cis isoprene residue is required.     

Preliminary study of the physical chemistry and catalytic properties of glutathione-S-transferase from human placenta

Glutathione-S-transferase activity has been identified in the cytosol of human placenta. The specific activity measured is about 50% of that found in human liver. While some kinetic data have a close correspondence with those attributed to transferases of other sources, the molecular weight (60.000 daltons) and electric properties of this protein are unusual. The inhibitory effect of several non-substrate compounds suggests that also the placental Glutathione-S-transferase may play some role in detoxication of exogenous substances.     

Subcellular localization of intracellular forms of human chorionic gonadotropin in first trimester placenta

To determine the subcellular sites for synthesis and processing of human chorionic gonadotropin subunits in cells, first trimester placental cells were fractionated subcellularly on sucrose density gradients. Analysis of the subcellular fractions by immunobinding techniques revealed that the rough endoplasmic reticulum-rich fraction contained only intermediates having high-mannose oligosaccharides, but the Golgi-rich fraction contained not only intermediates but also mature forms which were resistant to endoglycosidase H but sensitive to neuraminidase. These results show that human chorionic gonadotropin subunits are synthesized in the rough endoplasmic reticulum as forms containing high-mannose oligosaccharides, and their maturation occurs in the Golgi apparatus by trimming with endogenous glycosidases. They are then modified by addition of complex oligosaccharides and terminal sialic acid through glycosyltransferases.     

Subcellular localization and nucleosome specificity of yeast histone acetyltransferases

We have previously reported [López-Rodas et al. (1989) J. Biol. Chem. 264, 19028-19033] that the yeast Saccharomyces cerevisiae contains four histone acetyltransferases, which can be resolved by ion-exchange chromatography, and their specificity toward yeast free histones was studied. In the present contribution we show that three of the enzymes are nuclear, type A histone acetyltransferases and they are able to acetylate nucleosome-bound histones. They differ in their histone specificity. Enzyme A1 acetylates H2A in chicken nucleosomes, although it is specific for yeast free H2B; histone acetyltransferase A2 is highly specific for H3, and histone acetyltransferase A3 preparations acetylate both H3 and H4 in nucleosomes. The fourth enzyme, which is located in the cytoplasm, does not accept nucleosomes as substrate, and it represents a canonical type B, H4-specific histone acetyltransferase. Finally, histone deacetylase activity is preferentially found in the nucleus.     

Subcellular localization and properties of lipase activities in human polymorphonuclear leukocytes

A fluorimetric assay for lipase activity has been optimized for measurement of the enzyme in human neutrophils. Activity was maximal at acid (4.5) and alkaline (9.5) pH, although there was also a neutral peak of activity at pH 6.5. Neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. The gradient fractions were assayed for acid, neutral and alkaline lipase activity and for the principal organelle marker enzymes. Neutral lipase showed a unimodal distribution with an equilibrium density of 1.19 g . cm-3, corresponding to the distribution of particulate leucine aminopeptidase. Acid and alkaline lipase activities showed very similar distribution profiles to each other with both soluble components and a broad peak of particulate activity. The broad modal density of 1.19-1.22 g . cm-3 suggests that acid and alkaline lipase activities could be localised to more than one population of cytoplasmic granule. Fractionation experiments with neutrophils homogenised in sucrose medium containing digitonin confirmed the localisation of neutral lipase and leucine aminopeptidase to the same cytoplasmic granule, and suggested that at least part of the acid lipase activity was localised to the specific granule. No lipase activity could be attributed to the alkaline phosphatase-containing granule. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and women in the third trimester of pregnancy. The specific activity of acid, neutral and alkaline lipase, and leucine aminopeptidase, in contrast to that of alkaline phosphatase, were similar in the three patient groups.     

Subcellular distribution, catalytic properties and partial purification of epoxide hydrolase in the human adrenal gland

Epoxide hydrolase in human adrenal gland was characterized with respect to catalytic properties and subcellular distribution. With human adrenal microsomes and the substrates styrene-7,8-oxide, cis-stilbene oxide, estroxide and androstene oxide the specific activities were between 1.9 and 19.0 nmol/min/mg protein. With styrene-7,8-oxide as substrate the apparent Km-value was 0.98 mM and the pH optimum was 9.2. Subcellular fractionation revealed that the bulk of the activity was confined to the endoplasmic reticulum. Different compounds known to influence rodent microsomal epoxide hydrolase activity were also tested on the human adrenal enzyme. 1,1,1-Trichloropropene-2,3-oxide (TCPO) and cyclohexene oxide (CHO) inhibited the activity while benzil and clotrimazole stimulated the activity. Partial purification of human adrenal epoxide hydrolase indicates that its molecular weight is about 51 000 and that its concentration relative total protein in the human adrenal microsomes is about 10%.     

Subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms

Nicotinamide mononucleotide adenylyltransferase (NMNAT) is the central enzyme of the NAD biosynthetic pathway. Three human NMNAT isoforms have recently been identified, but isoform-specific functions are presently unknown, although a tissue-specific role has been suggested. Analyses of the subcellular localization confirmed NMNAT1 to be a nuclear protein, whereas NMNAT2 and -3 were localized to the Golgi complex and the mitochondria, respectively. This differential subcellular localization points to an organelle-specific, nonredundant function of each of the three proteins. Comparison of the kinetic properties showed that particularly NMNAT3 exhibits a high tolerance toward substrate modifications. Moreover, as opposed to preferred NAD+ synthesis by NMNAT1, the other two isoforms could also form NADH directly from the reduced nicotinamide mononucleotide, supporting a hitherto unknown pathway of NAD generation. A variety of physiological intermediates was tested and exerted only minor influence on the catalytic activities of the NMNATs. However, gallotannin was found to be a potent inhibitor, thereby compromising its use as a specific inhibitor of poly-ADP-ribose glycohydrolase. The presence of substrate-specific and independent nuclear, mitochondrial, and Golgi-specific NAD biosynthetic pathways is opposed to the assumption of a general cellular NAD pool. Their existence appears to be consistent with important compartment-specific functions rather than to reflect simple functional redundance.     

Subcellular localization, purification, and some catalitic properties of aspartate aminotransferase from Spirodela polyrhiza

Intracellular distribution of aspartate aminotransferase (AAT) in Spirodela polyrhiza (Lemnaceae), strain SJ, has been studied by differential centrifugation. The bulk of the enzyme (73% of total cellular content) was localized in the cytoplasm and 24% activity was localized in chloroplasts. Purified cytoplasmic and chloroplastic isozymes differed by their affinity for substrates. The reaction balance was shifted towards direct and reverse transamination in the cytoplasm and chloroplast, respectively. Competitive inhibition of AAT by excessive substrates and enzyme affinity modulation by certain intermediates of the tricarboxylic acid cycle (isocitrate, succinate, and citrate) were observed. Possible involvement of AAT isozymes in the coordination of carbon and nitrogen metabolism through the regulation of 2-oxoglutarate synthesis and utilization in different cellular compartments is discussed.     

Subcellular localization and kinetic properties of arginase from the liver of Genypterus maculatus

1. From the liver of the teleost fish Genypterus maculatus, a partially purified preparation of arginase was obtained and characterized. 2. The Km value for arginine was found to be 9.1 mM at pH 7.5 and 11.5 mM at the optimum pH of 9.5. At both pH values, competitive inhibition was caused by ornithine and lysine, whereas proline, leucine, valine and isoleucine caused a non-competitive inhibitory effect. Branched chain amino acids were more inhibitory than proline. 3. The enzyme was found localized in the mitochondrial matrix of the liver of Genypterus maculatus. It is suggested that this localization would be of importance in the use of arginine as an energy source.     

Subcellular localization and some properties of lipoxygenase activity in human blood platelets.

Lipoxygenase activity was measured in human platelet subcellular fractions. From a sonicated platelet preparation, a granule fraction, mixed membranes (surface and intracellular) and cytosol fractions were separated by differential centrifugation. With respect to activities in the sonicated preparation, the lipoxygenase was slightly enriched in both the cytosol and mixed-membrane fractions and consistently de-enriched in the granule fractions. Approx. 65% and 20% of the total cell enzyme activity were found in the cytosol and mixed membranes respectively, with only 8% present in the granule fraction. Additionally we measured the lipoxygenase activity in purified surface- and intracellular-membrane subfractions prepared from the mixed membranes by free-flow electrophoresis. There was a slight enrichment in activity in the intracellular membrane fraction compared with that in the mixed membranes, and a depletion of activity in the surface membranes. Characterization of the enzyme activity, i.e. time course, pH-dependence, Ca2+-dependence, Vmax. and Km for arachidonic acid, and the carbon-position specificity for this acid, failed to reveal any significant differences between the membrane-bound and soluble forms of the lipoxygenase. These findings suggest that in human platelets the same lipoxygenase is associated with the membranes as in the cytosol and that the membrane-bound activity predominates in intracellular membrane elements.     

Subcellular localization of the human proto-oncogene protein DEK

Recent data revealed that DEK associates with splicing complexes through interactions mediated by serine/arginine-repeat proteins. However, the DEK protein has also been shown to change the topology of DNA in chromatin in vitro. This could indicate that the DEK protein resides on cellular chromatin. To investigate the in vivo localization of DEK, we performed cell fractionation studies, immunolabeling, and micrococcal nuclease digestion analysis. Most of the DEK protein was found to be released by DNase treatment of nuclei, and only a small amount by treatment with RNase. Furthermore, micrococcal nuclease digestion of nuclei followed by glycerol gradient sedimentation revealed that DEK co-sedimentates with oligonucleosomes, clearly demonstrating that DEK is associated with chromatin in vivo. Additional chromatin fractionation studies, based on the different accessibilities to micrococcal nuclease, showed that DEK is associated both with extended, genetically active and more densely organized, inactive chromatin. We found no significant change in the amount and localization of DEK in cells that synchronously traversed the cell cycle. In summary these data demonstrate that the major portion of DEK is associated with chromatin in vivo and suggest that it might play a role in chromatin architecture.     

Subcellular distribution of aromatase in human placenta and ovary.

The aromatization of androstenedione in human ovarian microsomes is inhibited by an antibody to porcine hepatic microsomal NADPH-cytochrome c reductase. Likewise, the antibody inhibits aromatization in mitochondria isolated from human ovaries and placentae. A given quantity of the antibody produces the same percent inhibition of aromatization in microsomes and mitochondria of both ovaries and placentae. These data, in addition to the low specific activity observed for the mitochondrial aromatase, indicate that aromatization in mitochondria probably results from microsomal contamination.     

Simultaneous preparation from human placenta of several enzymes of glucose metabolism

A procedure for the simultaneous purification to homogeneity of hexokinase, phosphoglucomutase 1 and 2, aldolase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase from human origin has been developed. Human placenta homogenate was first chromatographed on DE-52 column which retains hexokinase and glucose-6-phosphate dehydrogenase while the other enzymes are recovered in the unabsorbed protein fraction. The other steps in the purification involve Matrex gel and specific affinity chromatography for the DE-52 retained enzymes and phosphocellulose and Matrex gel chromatography for the other enzymes. All the enzymes mentioned were obtained in one week, with recoveries from 14 percent for glucose-6-phosphate dehydrogenase to 75 percent for hexokinase. Thus, the procedures utilized seem to be useful in obtaining large amounts of enzymes in a a homogeneous form from an easily available human tissue.     

Purification and properties of nucleotide pyrophosphatase from human placenta

Nucleotide pyrophosphatase was purified from human placenta to near homogeneity with a specific activity of about 500-fold over the Triton extract of the homogenate. Purification was achieved most effectively by successive chromatographic steps with AMP-agarose and ADP-agarose columns, based on the affinity of the enzyme towards 5'-adenylate and adenosine 3',5'-diphosphate, and a lectin-Sepharose column, based on the glycoprotein nature of the enzyme. The purified enzyme was found to be essentially homogeneous on SDS-polyacrylamide gel electrophoresis with a mobility corresponding to 130K. The purified enzyme was found to hydrolyze a wide variety of nucleotides, i.e. 3'-phosphoadenosine 5'-phosphosulfate (PAPS), adenosine 5'-phosphosulfate (APS), NADH, ATP, nucleotide sugars, oligonucleotides, and p-nitrophenyl-thymidine 5'-phosphate (PNTP). From the oligonucleotides, the enzyme produced 5'-phosphates. Mg2+ was required for full activity. Glycine and sulfhydryl compounds such as 2-mercaptoethanol and 2,3-dimercapto-1-propanol were inhibitory. Most of these properties are common to nucleotide pyrophosphatases [EC 3.6.1.9] and type I (5'-phosphate forming) phosphodiesterases [EC 3.1.4.1] from various sources. The relevance of this enzyme to a unique genetic disease, Lowe's syndrome, is discussed.     

Purification and kinetic properties of galactokinase from human placenta

Galactokinase from human placenta was purified about 350-fold using DEAE-Sephadex-A-50 chromatography followed by Sephadex-G-200 and CM-Sephadex-C-50 filtration. The final steps of purification involved electrofocusing and ammonium sulfate precipitation. In analytical disc electrophoresis the purified enzyme moved as a single protein band.     

Purification and various properties of uracil-DNA-glycosylase from human placenta

Uracil-DNA-glycosylase was isolated from human placenta and purified 2100-fold. The apparent Km value for non-methylated DNA substrate of the enzyme is 3.10(-7) M. However, Km for uracil-DNA-glycosylase was 3 times as low when methylated DNA was used as a substrate. It was shown that the initial rate of uracil excision was greater for the non-methylated than for the hypermethylated DNA. The experimental results indicate that the postreplicative methylation of DNA can interfere with uracil excision.     

Purification and properties of aldehyde reductases from human placenta

Aldehyde reductases (alcohol: NADP⁺-oxidoreductases, EC 1.1.1.2) I and II from human placenta have been purified to homogeneity. Aldehyde reductase I, molecular weight about 74 000, is a dimer of two nonidentical subunits of molecular weigths of about 32 500 and 39 000, whereas aldehyde erductase II is a monomer of about 32 500. Aldehyde reductase I can be dissociated into subunits under high ionic concentrations. The isoelectric pH for aldehyde reductases I and II are 5.76 and 5.20, respectively. Amino acid compositions of the two enzymes are significantly different. Placenta aldehyde reductase I can utilize glucose with a lower affinity, whereas aldehyde reductase II is not capable to reducing aldo-sugars. Similarly, aldehyde reductase I does not catalyse the reduction of glucuronate while aldehyde reductase II has a high affinity for glucuronate. Both enzymes, however, exhibit strong affinity towards various other aldehydes such as glyceraldehyde, propionaldehyde, and pyridine-3-aldehyde. The pH optima for aldehyde reductases I and II are 6.0 and 7.0, respectively. Aldehyde reductaase I can use both NADH and NADPH as cofactors, whereas aldehyde reductase II activity is dependent on NADPH only. Both enzymes are susceptible to inhibition by sulfhydryl group reagents, aldose reductase inhibitors, lithium sulfate, and sodium chloride to varying degrees.     

Purification and properties of arylsulfatase C from human placenta

Arylsulfatase C (ASC) was purified about 1,000-fold from human placenta. The major steps in the procedure included chromatography on Con A-Sepharose and Bio-Gel A-1.5 m. The purified enzyme was homogeneous by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The native enzyme has an apparent molecular weight of 238,000 resulting from three identical subunits of 78,000 daltons. The purified enzyme hydrolyzes the artificial substrate p-nitrophenyl sulfate (NPS), and the two natural substrates estronesulfate (ES) and dehydroepiandrosterone sulfate (DHEAS), the ratio of these three activities being constant throughout the purification. ES and DHEAS are powerful competitive inhibitors of the enzymatic hydrolysis of NPS. ASC, ESase and DHEASase activities show the same thermal stability. These results strongly suggest that a single enzyme is responsible for the hydrolysis of the two natural and the artificial substrates.