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At least 19 recordsLinked to original sources
PubMed1977

RNase H and RNA-directed DNA polymerase: associated enzymatic activities of murine mammary tumor virus.

The RNA-directed DNA polymerase of murine mammary tumor virus, a type B RNA tumor virus, was purified sequentially through DEAE-cellulose, phosphocellulose (step gradient), and phosphocellulose (linear salt gradient) chromatography followed by glycerol sedimentation centrifugation. During all stages of purification, coincident peaks of RNA-directed DNA polymerase activity, templated by polyribocytidylate-oligodeoxyguanidylate, and RNase H digestion of [3H]polyriboadenylate-polydeoxythymidylate were observed, and both enzymatic activities displayed a cation preference for magnesium. Under conditions that removed adventitiously associated nucleases, RNase H activity was found to co-purify with polymerase. The specificity of this nuclease was assayed with various prepared substrates, which indicated that the polymerase-associated RNase H activity was directed only against the RNA strand of an RNA-DNA hybrid. It is highly probable that RNase H (RNA-DNA hybrid: ribonucleotide-hydrolase, EC 3.1.4..34) and RNA-directed DNA polymerase of type B viruses are associated enzymatic activities analogous to those observed for avian and mammalian type C RNA tumor viruses.

Mammary Tumor Virus, Mouse↗
PubMed1979

Enzymatic activities associated with a purified simian virus 40 T antigen-related protein.

A protein antigenically related to the simian virus (SV 40) A gene product has been purified to near homogeneity from cells infected with the adenovirus-SV 40 hybrid virus Ad2(+)D2 and shown to contain ATPase (ATP phosphohydrolase, EC 3.6.1.3) and protein kinase (ATP:phosphotransferase, EC 2.7.1.37) activity. Both enzymatic activities copurify with the protein through six stages including one gel filtration column, two ion exchange columns, and a heparin affinity column. Analogous fractions from extracts of cells uninfected or infected with adenovirus 2 alone do not contain these enzymatic activities. The D2 hybrid protein resolves into two forms (I and II) during ion exchange chromatography. Form I, the major species (85%) of the D2 hybrid protein, elutes from DEAE-Sephadex in 0.37 M NaCl and is able to catalyze the hydrolysis of ATP to ADP + P(i) at a rate of 3 mumol/hr per mg. The remaining 10-15% of the D2 hybrid protein consists of form II which elutes from DEAE-Sephadex in 0.29 M NaCl and is able to hydrolyze ATP as well as to incorporate phosphorus from ATP into either the D2 hybrid protein itself or other protein acceptors such as phosvitin. Although both forms are able to bind DNA, the ATPase activity of form I cosediments with SV 40 DNA more efficiently than does the protein kinase activity of form II during glycerol gradient centrifugation. The ATPase activity of form I is efficiently inhibited by addition of anti-T gamma globulin to the reaction mixture whereas control gamma globulin has no effect. Similarly, the phosphorylation of the D2 hybrid protein by form II is inhibited by anti-T gamma globulin. By contrast, phosphorylation of phosvitin is specifically inhibited by antibody only when the immune complex is removed from the reaction mixture. Thus, it appears likely that one and possibly two enzymatic activities are carried out by the D2 hybrid protein. These findings are discussed in terms of mechanisms of SV 40 DNA replication and virally induced transformation.

Adenosine Triphosphatases↗
PubMed1976

The relationship of structure and function in human Hageman factor. The association of enzymatic and binding activities with separate regions of the molecule.

Three regions of the human Hageman factor molecule termed the c, d, and e regions have been defined. Division of the molecule into these three regions is based on the analysis of fragments obtained by enzymatic cleavage during fluid-phase activation. The three regions have the following properties: (a) the c region has a mol wt of 40,000, has the capacity to bind to negatively charged surfaces, and does not have detectable enzymatic activity; (b) the e region possess a mol wt of 28,000 has enzymatic activity, and does not bind to negatively charged surfaces; (c) the d region has a mol wt of 12,000, is located between the c and e fragments but has not been detected as a freely existing polypeptide, and can bind firmly to negatively charged surfaces. The preparation of antibodies specific for the c and e regions is described as well as their use in defining the electrophoretic characteristics of the cde, cd, de, c, and e polypeptide fragments of Hageman factor. Evidence is given showing that the e region, but not the c or d, is released from a negatively charged surface when bound Hageman factor is exposed to proteolytic enzymes or whole plasma and that when this occurs in the presence of normal plasma, the e fragment becomes bound to C1 esterase inhibitor.

Electrophoresis↗
PubMed1977

Protein modification enzymes associated with the protein-synthesizing complex from rabbit reticulocytes. Protein kinase, phosphoprotein phosphatase, and acetyltransferase.

A number of protein modification activities are present in the protein-synthesizing complex isolated from rabbit reticulocytes. These enzymes are solubilized by sedimentation of the ribosomes through buffered sucrose containing 0.5 M KCl, and have been partially purified from the high salt wash fraction by chromatography on DEAE-cellulose and phosphocellulose. The ribosomal-associated enzymatic activities include cyclic AMP-regulated and cyclic nucloetide-independent protein kinase, phosphoprotein phosphatase, and acetyltransferase activities. These enzymatic activities have been shown to modify specific ribosomal and ribosomal-associated proteins. The cycli c AMP-regulated protein kinase phosphorylate the 40 S ribosomal subunit from rabbit reticulocytes. One of the cyclic nucleotide-independent protein kinase catalyzes the phosphorylation of two different factors involved in the initiation of hemoglobin synthesis. A single phosphoprotein phosphatase activity is shown to remove phosphate from 40 S ribosomal subunits. The major acetyltransferase activity associated with ribosomes acetylates a 60 S ribosomal protein.

Acetyl-CoA C-Acetyltransferase↗
PubMed1978

Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa.

Neurospora crassa wild type STA4 NADPH-nitrate reductase (NADPH : nitrate oxidoreductase, EC 1.6.6.3) has been purified 5000-fold with an overall yield of 25--50%. The final purified enzyme contained 4 associated enzymatic activities: NADPH-nitrate reductase, FADH2-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase. Polyacrylamide gel electrophoresis yielded 1 major and 1 minor protein band and both bands exhibited NADPH-nitrate and reduced methyl viologen-nitrate reductase activities. SDS gel electrophoresis yielded 2 protein bands corresponding to molecular weights of 115 000 and 130 000. A single N-terminal amino acid (glutamic acid) was found and proteolytic mapping for the two separated subunits appeared similar. Purified NADPH-nitrate reductase contained 1 mol of molybdenum and 2 mol of cytochrome b557 per mol protein. Non-heme iron, zinc and copper were not detectable. It is proposed that the Neurospora assimilatory NADPH-nitrate reductase consists of 2 similar cytochrome b557-containing 4.5-S subunits linked together by one molybdenum cofactor. A revised electron flow scheme is presented. p-Hydroxymercuribenzoate inhibition was reversed by sulfhydryl reagents. Inhibitory pattern of p-hydroxymercuribenzoate and phenylglyoxal revealed accessible sulfhydryl and arginyl residue(s) as functional group(s) in the earlier part of electron transport chain as possibly the binding site of NADPH or FAD.

Copper↗
PubMed1978

Use of trypsin for rapid and efficient purification of murine sarcoma and leukemia virus.

A method is described for purification of MSV-MuLV from culture supernatant of chronically infected 78A,1 rat embryo cell line. This method involves direct polyethylene glycol-NaCl precipitation of the low speed supernatant of culture fluid followed by digestion of the pellet with trypsin. This procedure efficiently disrupts large aggregates which normally entrap most of the virus. Highly purified virus can be obtained in very good yield by a combination of sedimentation velocity and isopycnic centrifugation : yields up to 100 A280 units (17 mg of protein) of purified virus per liter of culture fluid can be observed. This procedure appears well suited for large scale isolation of virion associated enzymatic activities.

Animals↗
PubMed1977

Myocardial injury following myocardial revascularization. Detection by isoenzyme analysis.

To clarify the value of isoenzymes in the detection of acute myocardial injury (AMI) associated with coronary artery bypass grafting, 350 consecutive patients (804 grafts) were evaluated with serial electrocardiograms (ECG) and serum levels of serum glutamic oxaloacetic transaminase (SGOT), total creatinine phosphokinase (CPK), and lactic dehydrogenase (LDH) on Days 0, 1,2,3,4,5, and 7. Graft patency 1 to 6 months postoperatively was 92%. Sixty patients (18%) had ECG evidence of AMI. Four patients died (1.1%) Of the 185 patients with serial LDH isoenzyme analysis, 46 had elevated cardiac fraction (LDH-1). Only 35 of these had AMI by ECG. The others had nonspecific ECG changes, but associated enzymatic andhemodynamic evidence of AMI. The CPK-MB band was elevated in all patients in the immediate postoperative period, but did not correlate with ECG, hemodynamic, or enzymatic evidence of AMI. The diagnosis of AMI following coronary artery bypass could not be substantiated by evaluation of total LDH, CPK, or CPK-MB. LDH-1 is specific in detecting AMI after a coronary artery bypass graft.

Adult↗
PubMed1977

Biochemistry and immunochemistry of membrane-bound enzymes.

Membrane-bound enzymes have certain specific differences compared with soluble enzymes. Membrane-binding often enables greater catalytic activity of associated enzymatic reactions, their regulation by low molecular weight substances (substrates and allosteric effectors, hormones) and compartmentation, etc. On the other hand, the binding of enzymes to membranes causes considerable difficulties as regards their isolation and the determination of their homogeneity and substrate specificity. Membrane enzymes provide a unique opportunity for studying the biogenesis of membranes and their physiological properties, however. These problems are discussed in relation to two types of membranes--the inner mitochondrial membrane and the membrane of the brush border of the small intestine. An example of the utilization of immunochemical methods is given in the results of a study of biosynthesis of the cytochrome oxidase complex in yeast cells. In the case of the brush border of the mammalian small intestine, the fact that certain enzymes, which are also of clinical significance from the aspect of congenital genetic defects, can be isolated only as complexes, constitutes a very real problem. This applies particularly to the sucrase-isomaltase complex and the lactase-beta-glucosidase complex. Solving questions of substrate specificity is of significance for the choice of a suitable analytical or histochemical method. The common regulation of these complexes gives an insight into the problems of membrane biogenesis, however. Immunochemical methods can be employed as sensitive criteria to support biochemical and morphological studies. Collaboration between the biochemist and histochemist proved especially valuable when determining the substrate specificity of enzymes (glycosidases) in relation to histochemical substrates, when applying histochemical methods for detecting enzymatic activity in immunoprecipitates and acrylamide gels and in immunohistochemical studies of the localization and developmental differentiation of the enzymes of the brush border of the small intestine.

Animals↗
PubMed1979

Five different enzymatic activities are associated with the multienzyme complex of fatty acid oxidation from Escherichia coli.

The purified multienzyme complex of fatty acid oxidation from Escherichia coli was found to possess 3-hydroxyacyl-coenzyme A (CoA) epimerase and cis-delta3-trans-delta2-enoyl-CoA isomerase activities in addition to the previously identified enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoactyl-CoA thiolase activities. Evidence is presented in support of the proposed association of all five enzyme activities with one protein which apparently is composed of two types of subunits and which can exist in several aggregated forms. The five component enzymes of the complex were rapidly inactivated by tris(hydroxymethyl)aminomethane, whereas they remained active in the presence of potassium phosphate.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1976

A deoxyadenylate kinase activity associated with polynucleotide phosphorylase from Micrococcus luteus.

We report here the presence of two enzymatic activities associated with highly purified preparations of polynucleotide phosphorylase from Micrococcus luteus. The first, a nuclease activity, which is not separated from the phosphorylase on hydroxylapatite, may be due to substitution of H2O for phosphate in the phosphorolysis reaction. The second activity, a deoxyadenylate kinase, the bulk of which is not resolved from the phosphorylase using gel filtration, sucrose density gradient centrifugation, DEAE-Sephadex, or hydroxylapatite chromatography, may represent a new activity of polynucleotide phosphorylase or be due to an enzyme which is tightly bound to the phosphorylase. Several properties of the kinase are described and its possible significance with respect to the overall enzyme mechanism is discussed.

Adenosine Monophosphate↗
PubMed1977

Synthesis of the Escherichia coli K12 isoenzymes of ornithine transcarbamylase, performed in vitro.

The in vitro synthesis of enzymatically-active ornithine transcarbamylase (OTCase) directed by each of the E. coli K-12 OTCase genes (argF and argI) is described. The E. coli OTCase isoenzyme subunits are not identical, whether synthesized in vivo or in vitro, the argF-coded product being about 5% smaller. The OTCase protomers are enzymatically inactive but associate in vitro to an enzymatically active multimer. The rates of subunit association of argF and argI isoenzymes are considerably different. Utilizing the facile assay protocol presented, the regulation of in vitro OTCase synthesis by the specific holorepressor of the arginine regulon is demonstrated. Calculations based upon data presented indicate that there are about 65 molecules of argR gene product per bacterium, a substantially lower estimate than previously reported.

Arginine↗
PubMed1976

Intracellular distribution of phosphate in the underfed rat developing weakness and coma following total parenteral nutrition.

Underfed rats infused intravenously with a glucose-amino acid solution at the rate of 390 kcal/kg/day developed a syndrome of muscular weakness, neuropathy, lethargy and precoma or coma associated with severe hypophosphatemia. The movement of phosphate into the cells was studied to determine where it went and into which organic compounds it was incorporated. All but 8% of the labeled phosphate was found in liver, muscle, bone, and carcass residue. Liver cells took up as much phosphate as bone and twice as much as muscle, on weight basis. About 90% of the labeled phosphate entering liver was found in the acid-soluble fraction. The specific activity of liver phosphate increased in the infused underfed rats compared to uninfused underfed rats. Infusion of the underfed rat until signs of the syndrome appeared was associated with a 2.7- to 5-fold increase over the correspondingly infused normal rat in the labeling of glucose-6-phosphate, glucose-1-phosphate, and 6-phosphogluconate. No increase over the infused normal rat was observed in most of the other sugar phosphate compounds nor in the non-sugar phosphate compounds such as phospholipids, nucleic acids or proteins. he changes in sugar phosphates observed in the underfed rats probably reflect the enzymatic atrophy associated with underfeeding and the consequent inability to respond to the huge glucose load.

Animals↗
PubMed1978

Protein kinase activity associated with the avian sarcoma virus src gene product.

Incorporation of phosphorus from [gamma-32P]ATP into protein was catalyzed by specific immunoprecipitates from avian sarcoma virus (ASV)-transformed avian and mammalian cells. This incorporation was observed only when antiserum from tumor-bearing rabbits able to specifically precipitate the ASV sarcoma gene product, p60src, was used to immunoprecipitate antigens from transformed cell lysates. Immunoprecipitates of extracts from normal cells or cells infected with a transformation-defective ASV mutant showed no activity in this assay, nor did any immune complexes formed with normal rabbit serum and any of the cell extracts tested. The expression of the protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) was growth temperature-dependent in cells infected with an ASV mutant temperature-sensitive for the transformation. These results on an enzymatic activity associated with the ASV transforming protein are discussed in terms of protein phosphorylation as a mechanism for viral transformation.

Alpharetrovirus↗
PubMed1979

[Studies for proteolysis in malignant tumours (author's transl)].

It's well known that increased proteolytic activity in plasma as well as in interstitial fluids is often associated with malignant tumours. There are numerous reports that cell cultures of malignant origin contain plasminogen activators responsible for an increased proteolysis. Our studies showed that patients with a carcinoma excrete in their urine a significant higher amount of an acid-stable inhibitor than healthy patients. This urine inhibitor is a split product (MW: 30,000) of the Inter-alpha-trypsin inhibitor (MW: 180,000). We conclude that the increased amount of acid-stable split products is the result of increased proteolysis in patients with malignant tumours. In contrast to other authors we do not think that the activation of plasmin by plasminogen is responsible for the proteolytic cleavage of ITI. Otherwise proteolysis by Cathepsin G and granulocytic Elastase would be reasonable. So enhanced proteolysis seems to be a sign of general enzymatic change associated with transformation.

Carcinoma, Squamous Cell↗
PubMed1976

Studies on the interaction of propranolol with erythrocyte membranes.

The interaction of propranolol with erythrocyte membranes at concentrations stabilizing intact erythrocytes against hypotonic hemolysis produced corresponding perturbations in membrane protein and particularly membrane phospholipid components as monitored by increases in the reactivity of membrane amino and sulfhydryl groups towards trinitrobenzenesulfonic acid and 5,5'-dithio-bis(2-nitro-benzoic acid), respectively. Membrane-propranolol interactions were also analyzed in terms of alterations produced in the kinetic properties of membrane enzymes. These experiments provided evidence that propranolol-induced perturbations were sufficiently generalized as to influence the activity of enzymatic processes associated with both inner and outer membrane surfaces. Configurational changes in membrane phospholipids were implicated in these effects of propranolol, which included alterations in functionally significant membrane-cation interactions, It is suggested that the findings described here may provide a basis for understanding molecular aspects of membrane stabilization in other systems.

4-Nitrophenylphosphatase↗