Localization of oxalacetate keto–enol-tautomerase

1976 ◽  
Vol 54 (3) ◽  
pp. 233-237 ◽  
Author(s):  
James C. Wesenberg ◽  
Aravind Chaudhari ◽  
Robert G. Annett
Keyword(s):  
Enzymic Activity ◽  
The Other ◽  
Nuclear Fraction ◽  
Marker Enzymes ◽  
Animal Cells ◽  
Intracellular Location ◽  
Pig Kidney ◽  
Fractionation Procedure ◽  
Mitochondrial Fractions ◽  
Soluble Phase

The intracellular location of oxalacetate keto–enol-tautomerase (oxaloacetate keto–enol-isomerase) (EC 5.3.2.2) has been determined in two types of animal cells, rat liver and pig kidney. Two fractionation procedures were adopted and modified to suit each type of tissue. One fractionation procedure gave the soluble phase, microsomal and mitochondrial fractions, while the other isolated the nuclear fraction. The tautomerase is distributed among the soluble phase, microsomes and mitochondria in both tissues. Fractionation efficiency was checked by determining percentage recoveries of enzymic activity and total protein after each step, by microscopy studies and by determining the distribution of several marker enzymes.

scholarly journals Localization of fumarylacetoacetate fumarylhydrolase in rat liver

1978 ◽  
Vol 56 (9) ◽  
pp. 866-868 ◽  
Author(s):  
Kirit S. Doshi ◽  
Donald E. Schmidt Jr.
Keyword(s):  
Rat Liver ◽  
Liver Tissue ◽  
Specific Activity ◽  
Nuclear Fraction ◽  
Marker Enzymes ◽  
Intracellular Location ◽  
Relative Specific Activity ◽  
Fractionation Procedure ◽  
Soluble Phase ◽  
Microscopic Studies

The intracellular location of fumarylacetoacetate fumarylhydrolase (EC 3.7.1.2) has been demonstrated in rat liver tissue. Two fractionation procedures involving homogenization and differential centrifugation were adopted. The first fractionation procedure isolated the nuclear fraction while the second gave the mitochondrial, microsomal, and soluble phase fractions. The hydrolase is localized in the soluble phase of the rat liver tissue. The enzyme also showed a high relative specific activity in the soluble phase fraction. Fractionation efficiency was checked by microscopic studies and by determining the distribution of a number of marker enzymes.

scholarly journals Studies on the sterols and sterol esters of the intracellular organelles of maize shoots

1968 ◽  
Vol 110 (1) ◽  
pp. 119-125 ◽  
Author(s):  
R. J. Kemp ◽  
E. I. Mercer
Keyword(s):  
Linolenic Acid ◽  
Microsomal Fraction ◽  
The Other ◽  
Nuclear Fraction ◽  
Unesterified Cholesterol ◽  
Sterol Esters ◽  
Mitochondrial Fractions ◽  
Intracellular Organelles

1. The composition of the esterified and unesterified sterols of the nuclear, chloroplastidic, mitochondrial and microsomal fractions of 21-day-old maize shoots was examined. 2. The microsomal and mitochondrial fractions contain the bulk of the sterols of the tissue. 3. Only 1% of the sterol isolated from all the organelles is esterified. 4. The nuclear fraction has the greatest proportion of esterified sterol and the microsomal fraction the least. 5. 4-Demethyl sterols constitute the bulk of both esterified and unesterified sterols in all organelle fractions. 6. Cholesterol is the major esterified 4-demethyl sterol of the nuclear and chloroplastidic fractions, but only the nuclear fraction has an appreciable proportion of unesterified cholesterol. 7. Sterol esters of linolenic acid are more abundant in the mitochondrial and microsomal fractions than in the other two fractions.

Characterization of Different Deoxyribonucleases in Human Lymphocytes

1975 ◽  
Vol 30 (11-12) ◽  
pp. 781-784 ◽  
Author(s):  
E. Jürgen Zöllner ◽  
Hans Störger ◽  
Hans-Joachim Breter ◽  
Rudolf Zahn
Keyword(s):  
Human Lymphocytes ◽  
Disc Electrophoresis ◽  
The Other ◽  
Lower Activity ◽  
Nuclear Fraction ◽  
Polyacrylamide Gels ◽  
Cytoplasmic Fraction ◽  
Acid Deoxyribonuclease ◽  
Deoxyribonuclease Activity

Abstract Deoxyribonucleases, Disc Electrophoresis, Lymphocytes Four groups of deoxyribonuclease activities from human lymphocytes have been characterized by deoxyribonuclease assay in DNA-containing polyacrylamide gels following their separation by disc-electrophoresis. All activities hydrolyse DNA endonucleolytically. One neutral deoxyribo­ nuclease found in the cytoplasmic fraction prefers native or UV-irradiated DNA over denatured DNA as substrate and is a 5′-monoester former. Two groups of acid deoxyribonuclease activities are detectable in the nuclear fraction. Both are 3′-monoester formers. One is as well active with denatured DNA as with native DNA, the other one shows the same activity with native and UV-irradiated DNA but lower activity with denatured DNA. An alkaline deoxyribonuclease activity, also localized in the nucleus, is a 5′ -monoester DNA as substrate.

scholarly journals Purification, properties and comparative specificities of the enzyme prolyl-transfer ribonucleic acid synthetase from Phaseolus aureus and Polygonatum multiflorum

1965 ◽  
Vol 97 (1) ◽  
pp. 112-124 ◽  
Author(s):  
PJ Peterson ◽  
L Fowden
Keyword(s):  
Carboxylic Acid ◽  
Enzyme Preparation ◽  
Enzymic Activity ◽  
The Other ◽  
Asparagus Officinalis ◽  
Acid Activation ◽  
Phaseolus Aureus ◽  
Substrate Specificities ◽  
Imino Acids ◽  
Ammonium Sulphate Fractionation

1. A prolyl-s-RNA synthetase (prolyl-transfer RNA synthetase) has been purified about 250-fold from seed of Phaseolus aureus (mung bean), a species not producing azetidine-2-carboxylic acid, and more than 10-fold from rhizome apices of Polygonatum multiflorum, a liliaceous species containing azetidine-2-carboxylic acid. The latter enzyme was unstable during ammonium sulphate fractionation. 2. The enzymes exhibited different substrate specificities towards the analogue. That from Phaseolus, when assayed by the ATP-PP(i) exchange, showed azetidine-2-carboxylic acid activation at about one-third the rate with proline. Both labelled imino acids gave rise to a labelled aminoacyl-s-RNA. The enzyme from Polygonatum, however, activated only proline. 3. The enzyme from Polygonatum also formed a labelled prolyl-s-RNA with Phaseolus s-RNA but at a lower rate than when the Phaseolus enzyme was used. No reaction occurred when the Phaseolus enzyme was coupled with Polygonatum s-RNA, and only a very slight one was observed when both enzyme and s-RNA came from Polygonatum. 4. Protein preparations from seeds of Pisum sativum, another species not producing azetidine-2-carboxylic acid, also activated the analogue in addition to proline, whereas those from rhizome and seeds of Convallaria, the species from which the analogue was originally isolated, failed to activate it. However, a liliaceous species not producing the analogue, Asparagus officinalis, activated it. 5. Of the other proline analogues investigated, only 3,4-dehydro-dl-proline and l-thiazolidine-4-carboxylic acid were active with the enzyme preparation from Phaseolus. 6. pH optima of 7.9 and 8.4 were established for the enzymes from Phaseolus and Polygonatum respectively. 7. The Phaseolus enzyme was specific for ATP and PP(i). Mn(2+) partially replaced the requirement for Mg(2+) as cofactor. Preincubation with p-chloromercuribenzoate at a concentration of 0.5mm or higher produced over 99% inhibition of the Phaseolus enzyme. One-half the enzymic activity was destroyed by preheating for 5min. at 62 degrees in tris-hydrochloric acid buffer, pH7.9. 8. All experimental evidence supports the hypothesis that azetidine-2-carboxylic acid and proline are activated by the same enzyme in Phaseolus preparations, whereas the analogue was inactive in all Polygonatum preparations. The possible nature of this different substrate behaviour is discussed.

Lactate dehydrogenase isoenzyme 1 with the centrifugal analyzer after immunochemical removal of other isoenzymes.

1981 ◽  
Vol 27 (6) ◽  
pp. 922-923 ◽  
Author(s):  
D E Bruns ◽  
J C Emerson ◽  
R L Bertholf ◽  
K E Hill ◽  
J Savory
Keyword(s):  
Lactate Dehydrogenase ◽  
Reference Interval ◽  
Enzymic Activity ◽  
The Other ◽  
Dehydrogenase Isoenzyme

Abstract We describe a centrifugal analyzer method for measuring the LD-1 isoenzyme of lactate dehydrogenase (EC 1.1.1.27) in serum, after immunochemical precipitation of the other four isoenzymes. Enzymic activity was measured kinetically at 30 degrees C with the pyruvate-to-lactate assay. The method for LD-1 was linear to 1000 U/L. The precision (CV) of the assay was 1.0--2.2% within-run and 3.1--4.5% day-to-day. The reference interval was 26--73 U/L (n = 51), corresponding to 21--35% of total LD activity.

scholarly journals Rat liver cytosol contains NADPH- and GSH-dependent factors able to restore ornithine decarboxylase inactivated by removal of thiol reducing agents

1988 ◽  
Vol 250 (1) ◽  
pp. 53-58 ◽  
Author(s):  
F Flamigni ◽  
C Guarnieri ◽  
C M Caldarera
Keyword(s):  
Ornithine Decarboxylase ◽  
Rat Liver ◽  
Gel Filtration ◽  
Enzymic Activity ◽  
Effective Concentration ◽  
The Other ◽  
Limited Effect ◽  
Liver Cytosol ◽  
Rat Liver Cytosol ◽  
Dependent Mechanism

Removal of dithiothreitol (DTT) from partially purified ornithine decarboxylase (ODC) led to an almost complete inhibition of enzymic activity. The inactivation was reversed by addition of millimolar concentrations of DTT, whereas natural reductants such as NADPH or NADH were ineffective, and GSH had only a limited effect. Addition of rat liver cytosol to the incubation mixture resulted in a noticeable re-activation of ODC; however, dialysed cytosol had little effect unless NADPH or GSH was present. Fractionation of rat liver cytosol by gel filtration on Sephadex G-75 yielded two fractions involved in the NADPH- and GSH-dependent re-activation of ODC: one designated ‘A’, eluted near the void volume (Mr greater than or equal to 60,000), and the other designated ‘B’, eluted later (Mr approx. 12,000). The NADPH-dependent mechanism required both fractions A and B for maximal ODC re-activation; the most effective concentration of NADPH was 0.15 mM, although a significant effect was observed at a concentration more than 10-fold lower. The GSH-dependent mechanism involved the mediation of Fraction B only, and operated at millimolar concentrations of GSH. These results suggest the existence of reducing systems in the cytosol, which may play a role in maintaining, and potentially in regulating, ODC activity by modulation of its thiol status.

scholarly journals Distribution of secretory component in hepatocytes and its mode of transfer into bile

1980 ◽  
Vol 190 (3) ◽  
pp. 819-826 ◽  
Author(s):  
Barbara M. Mullock ◽  
Richard H. Hinton ◽  
Miloslav Dobrota ◽  
Jane Peppard ◽  
Eva Orlans
Keyword(s):  
Particle Size ◽  
Plasma Membrane ◽  
Electron Microscope ◽  
Immunoglobulin A ◽  
Secretory Component ◽  
The Other ◽  
Marker Enzymes ◽  
Liver Homogenates ◽  
Fraction Bound

Immunoglobin A in bile and other external secretions is mostly bound to a glycoprotein known as secretory component. This glycoprotein is not synthesized by the same cells as immunoglobulin A and is not found in blood. We now report the mechanism by which secretory component reaches the bile and describe its function in immunoglobulin A transport across the hepatocyte. Fractionation of rat liver homogenates by zonal centrifugation was followed by measurement of the amounts of secretory component in the various fractions by rocket immunoelectrophoresis. Secretory component was found in two fractions. One of these was identified as containing Golgi vesicles from its isopycnic density and appearance in the electron microscope; the other contained principally fragments of the plasma membrane of the sinusoidal face of the hepatocyte, as shown by its particle size and content of marker enzymes. Only the latter fraction bound 125I-labelled immunoglobulin A added in vitro. At 5min after intravenous injection of [14C]fucose, the secretory component in the Golgi fraction was labelled, but not that in the plasma membrane. The secretory component in the sinusoidal plasma membrane did, however, become labelled before the first labelled secretory component appeared in bile, about 30min after injection. We suggest that fucose is added to the newly synthesized secretory component in the Golgi apparatus. The secretory component then passes, with the other newly secreted glycoproteins, to the sinusoidal plasma membrane. There it remains bound but exposed to the blood and able to bind any polymeric immunoglobulin A present in serum. The secretory component then moves across the hepatocyte to the bile-canalicular face in association with the endocytic-shuttle vesicles which carry immunoglobulin A. Hence there is a lag before newly synthesized secretory component appears in bile.

scholarly journals The biosynthesis of serine and glycine in Pseudomonas AM1 with special reference to growth on carbon sources other than C1 compounds

1971 ◽  
Vol 121 (5) ◽  
pp. 753-762 ◽  
Author(s):  
W. Harder ◽  
J. R. Quayle
Keyword(s):  
Essential Role ◽  
Carbon Sources ◽  
Enzymic Activity ◽  
Serine Hydroxymethyltransferase ◽  
The Other ◽  
Wild Type ◽  
Phosphoserine Phosphatase ◽  
Wild Type Phenotype ◽  
Phosphoglycerate Dehydrogenase ◽  
Serine Biosynthesis

1. A mutant, 20S, of Pseudomonas AM1 was obtained that requires a supplement of serine to grow on succinate, lactate or ethanol. This mutant lacks phosphoserine phosphatase and revertants to wild-type phenotype regained this enzymic activity showing that the phosphorylated pathway of serine biosynthesis is necessary for growth on these three substrates. 2. The requirement for supplemental serine by mutant 20S could be met by glycine, suggesting that Pseudomonas AM1 can obtain C1 units from glycine. 3. Mutant 20S grows on C1 compounds at a lower rate compared with the wild type. Supplementation with serine stimulated the growth rate of the mutant suggesting that the phosphorylated pathway of serine biosynthesis plays some role, but not an essential role, during growth on C1 compounds. 4. A mutant, 82G, was obtained that requires a supplement of glycine to grow on succinate, lactate or ethanol. When grown in such supplemented media, the mutant lacks serine hydroxymethyltransferase and revertants to wild-type phenotype regained enzymic activity showing that during growth on succinate, lactate or ethanol, glycine is made from serine via serine hydroxymethyltransferase, and that the organism can obtain C1 units from glycine. 5. Mutant 82G grew on methanol and then contained serine hydroxymethyltransferase suggesting that this enzyme is necessary for growth on C1 compounds and that Pseudomonas AM1 may synthesize two such enzymes, one used in growth on C1 compounds, the other used in growth on other substrates. Mutant 82G might lack the latter enzyme. 6. Phosphoglycerate dehydrogenase is specifically inhibited by l-serine and the regulatory implications of this are discussed.

scholarly journals Extrachromosomal DNA in the Apicomplexa

1997 ◽  
Vol 61 (1) ◽  
pp. 1-16
Author(s):  
R J Wilson ◽  
D H Williamson
Keyword(s):  
Cytochrome B ◽  
The Other ◽  
Intracellular Location ◽  
Human Malaria Parasite ◽  
Apicomplexan Parasites ◽  
Organellar Dna ◽  
Mtdna Sequence ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum ◽  
Mt Genome

Malaria and related apicomplexan parasites have two highly conserved organellar genomes: one is of plastid (pl) origin, and the other is mitochondrial (mt). The organization of both organellar DNA molecules from the human malaria parasite Plasmodium falciparum has been determined, and they have been shown to be tightly packed with genes. The 35-kb circular DNA is the smallest known vestigial plastid genome and is presumed to be functional. All but two of its recognized genes are involved with genetic expression: one of the two encodes a member of the clp family of molecular chaperones, and the other encodes a conserved protein of unknown function found both in algal plastids and in eubacterial genomes. The possible evolutionary source and intracellular location of the plDNA are discussed. The 6-kb tandemly repeated mt genome is the smallest known and codes for only three proteins (cytochrome b and two subunits of cytochrome oxidase) as well as two bizarrely fragmented rRNAs. The organization of the mt genome differs somewhat among genera. The mtDNA sequence provides information not otherwise available about the structure of apicomplexan cytochrome b as well as the unusually fragmented rRNAs. The malarial mtDNA has a phage-like replication mechanism and undergoes extensive recombination like the mtDNA of some other lower eukaryotes.

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