Increase In Km Value Research Articles

Effects of coordination of diammineplatinum(II) with DNA on the activities of Escherichia coli DNA polymerase I.

The effects of the reaction of cis- and trans-diamminedichloroplatinum(II) with DNA have been measured with regard to DNA synthesis, 3'-5' exonuclease (proofreading), and 5'-3' exonuclease (repair) activities of Escherichia coli DNA polymerase I. Both isomers inhibit DNA synthetic activity of the polymerase through an increase in Km values and a decrease in Vmax values for platinated DNA but not for the nucleoside 5'-triphosphates as the varied substrates. The inhibition is a consequence of lowered binding affinity between platinated DNA and DNA polymerase, and of a platination-induced separation of template and primer strands. Strand separation enhances initial rates of 3'-5' excision of [3H]dCMP from platinated DNA (proofreading), while total excision levels of nucleotides are decreased. In contrast to proofreading activity, the 5'-3' exonuclease activity (repair) discriminates between DNA which had reacted with cis- and with trans-diamminedichloroplatinum(II). While both initial rates and total excision are inhibited for the cis isomer, they are almost not affected for the trans isomer. This differential effect could explain why bacterial growth inhibition requires much higher concentrations of trans- than cis-diamminedichloroplatinum(II).

Kinetics of alkaline phosphatase activity and phosphorus availability for phytoplankton and bacterioplankton in lake plu\see (North German Eutrophic Lake)

Annual studies of kinetics of alkaline phosphatase (APA) activity and phosphorus availability for microplankton in the photic zone of an eutrophic lake are reported. The total APA activity of microplankton varied strongly. Vmax was highest during summer P depletion, and in autumn and winter total APA activity was low. The total APA specific activity of the microplankton was also highest (average 3.55 pmole PO4 (3-) ng ATP(-1) min(-1)) when ambient orthophosphate concentrations were very low. Both Vmax and specific APA activity were not dependent on the biomass of microplankton; they were strongly affected by P available for microplankton. A differential filtration technique was used for separation of microplankton into two size classes, i.e., algal, larger than 3μm, and bacterial fraction with size 0.2-3.0μm. The algal size fraction had lower specific APA activity (average 1.224 pmole PO4 (3-) ng ATP(-1) min(-1)) and higher KM values (38.8μmole × liter(-1)) than microorganisms which were smaller than 3μm (2.011 pmole PO4 (3-) ng ATP(-1) min(-1) and 25.4μmole liter(-1), respectively). The KM values of free, dissolved APA (36.8μmole liter(-1)) indicated that free APA was probably released by algae. Phytoplankton were major APA activity producers in the photic zone of the lake from March to November, and their activity constituted, on the average, 48.6% of the total APA activity in the water. Bacteria were the dominant APA activity producers in winter (41.3-44.9%); however, during other periods they contributed significantly (average 21.7%) to total APA activity. When surplus constituted less than 10% of particulate P in seston, phytoplankton produced high specific APA activity, and when surplus P was higher than 15%, the specific APA activity of phytoplankton size fraction rapidly decreased. APA of the bacterial size fraction of the seston was not affected by P concentrations. Orthophosphate was a competitive inhibitor of APA produced by microorganisms of the size fraction larger than 3.0μm, and increasing concentrations of inorganic phosphate caused an increase in KM values. The hypothetical metabolic-coupling between phytoplankton and bacterioplankton in the phosphorus cycle in conjunction with carbon metabolism in the lake is discussed.

Substrate specificity and stereospecificity of calf spleen phosphodiesterase towards deoxyribonucleosidyl 3'-(4-nitrophenyl phosphates) and phosphorothioates.

Phosphodiesterase from calf spleen exhibits nucleotidyltransferase activity when incubated with either the (PR) or the (PS) diastereomer of thymidyl 3'-(4-nitrophenyl phosphorothioate). Thymidylyl(3'-5')thymidyl phosphorothioate 3'-(4-nitrophenyl phosphorothioate) was identified as the main product of the enzyme-catalyzed reaction and the absolute configuration at the internucleotide phosphorus atom of the product was determined. The nucleotidyltransferase reaction is shown to proceed with retention of configuration at phosphorus, implying involvement of a double displacement mechanism with the formation of a nucleotidylated enzyme intermediate. To study the substrate specificity of spleen phosphodiesterase a series of deoxyribonucleosidyl 3'-(4-nitrophenyl phosphates) and phosphorothioates were synthesized, and Km and V parameters for each substrate were measured. The results obtained show virtually no specificity for substrates with different nucleosidyl moieties, while about a 20 - 30-fold drop in V and a slight increase in Km values is observed for phosphorothioate analogues as compared with corresponding phosphates. The enzyme showed no significant stereoselectivity towards phosphorothioates of opposite configurations at phosphorus.

Pressure-adaptive differences in NAD-dependent dehydrogenases of congeneric marine fishes living at different depths

The pressure sensitivities of the apparent Michaelis constant of coenzyme were compared at 5°C for three NAD-dependent dehydrogenases purified from the white muscle of two congeneric fishes living at different depths.Sebastolobus altivelis adults are common between 550 and 1,300 m;S. alascanus adults between 180 and 440 m. Two isozymes of cytoplasmic malate dehydrogenase (MDH, EC 1.1.1.37, NAD+:l-malate oxidoreductase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12, NAD+:d-glyceraldehyde 3-phosphate oxidoreductase [phosphorylating]) were compared. For these enzymes, the homologues fromS. alascanus were markedly sensitive to moderate hydrostatic pressures (Fig. 1). TheK m(NADH) ofS. alascanus MDH-1 and theK m(NAD+) ofS. alascanus GAPDG double between 1 and 68 atm and continue to increase at a slower rate up to 476 atm, the highest pressure tested. For MDH-2 ofS. alascanus, theK m(NADH) triples between 1 and 68 atm and increases at a slower rate to 340 atm; between 340 and 476 atm, theK m decreases slightly from the value at 340 atm. TheK m of coenzyme values are pressure-independent for the MDH-1 and GAPDH homologues ofS. altivelis up to 476 atm (Fig. 1). TheK m(NADH) of theS. altivelis MDH-2 is sensitive to pressure, but much less so than the homologue ofS. alascanus (Fig. 1). TheK m increases 63% between 1 and 68 atm and remains constant at this higher value at higher pressures up to 476 atm. The relative increases inK m values for theS. alascanus enzymes between 1 and 68 atm are large (Table 1). Higher pressures are not as effective in perturbing theK m of coenzyme values. Perturbation ofK m of coenzyme by moderate hydrostatic pressures (50–100 atm) may seriously impair the function of dehydrogenases ofS. alascanus at pressures experienced by the deeper-living congener in its habitat. The reduction of the pressure-sensitivity of theK m of coenzyme in NAD-dependent dehydrogenases may be an important and ubiquitous feature of adaptation to the deep sea.

Interaction of Mg2+ with human liver aldehyde dehydrogenase. I. Species difference in the mitochondrial isozyme.

The dehydrogenase activity of the mitochondrial isozyme (E2) of human liver aldehyde dehydrogenase was stimulated about 2-fold by the presence of low concentrations (about 120-140 microM) of Mg2+ in the assay at pH 7.0 using propionaldehyde as substrate. The stimulation was totally reversible by treatment with EDTA. Maximum stimulation was dependent on the concentration of NAD+ used in the assay; an increase in Km value of NAD+ was observed to parallel the increase in maximal velocity with increasing Mg2+ concentration, indicating that alterations in the catalytic properties of the E2 isozyme occur in the presence of Mg2+. The presteady state burst of NADH product was observed to decrease in the presence of Mg2+, suggesting that the rate-limiting step of the dehydrogenase reaction is altered by Mg2+. No evidence for Mg2+-induced alterations in the molecular weight properties of the E2 isozyme was observed using gel filtration column chromatography and fluorescence polarization techniques. In addition, no alterations in the inactivating properties of iodoacetamide or disulfiram were produced by Mg2+. These results suggest that the mechanism by which human mitochondrial aldehyde dehydrogenase (E2) is stimulated by Mg2+ is different from that of the horse enzyme, representing a significant species difference.

Protein phosphotransferases from liver of ageing rats.

Cyclic AMP-dependent and -independent protein kinase have been isolated from livers of rats at 3, 9, 18 and 30 months of age. A preliminary characterization reported here reveals that in addition to a decrease in their activities due to ageing and a likely shift to cyclic AMP-independent casein kinase activity at 30 months of age, the enzymes show modest changes in some of their kinetic properties. In the case of cyclic AMP-dependent enzymes, decreases in Vmax for ATP and Km for cyclic AMP as well as histones were observed. On the contrary, casein kinases displayed increases in Km values for casein, GTP and ATP. Molecular weights of 143,000 daltons for casein kinases from 3-, 9- and 18-month-old rats and 211,000 daltons for that from 30-month-old rats were obtained. Slight differences were also noticed in the inhibitor sensitivity (NaF and 2,3-diphosphoglyceric acid) of casein kinases isolated from rats of different ages.

Critical lysine residue at the chloride binding site of angiotensin converting enzyme.

Pulmonary angiotensin converting enzyme has been reductively methylated by using formaldehyde and sodium cyanoborohydride. This modification virtually eliminates enzyme activity toward some substrates (e.g., furanacryloyl-Phe-Gly-Gly) while less drastically affecting activity toward others (e.g., furanacryloyl-Phe-Phe-Arg). Affinity chromatography and analysis of radiolabeled reaction products reveal that this effect is due to methylation of a single critical lysine residue. Loss of activity primarily represents an increase in Km values, indicating that the critical lysine plays a role in substrate binding. This lysine can be protected by a competitive inhibitor, suggesting that it is at or near the active site. Addition of chloride at pH 6.1 specifically protects against methylation of this lysine. These findings support the idea that the critical lysine is part of the binding site for chloride and other monovalent anions which are strong activators of the enzyme.

Aging of the erythrocyte. XVIII. Changes in kinetic properties of acetylcholinesterase.

Comparison of kinetic properties of acetylcholinesterase in membrane preparations from various density (age) fractions of bovine erythrocytes revealed in increase in Km value, ascribed to decrease in membrane surface potential, with increasing cell age. Activation energy of the enzyme decreased during red cell aging.

The effect of elimination of amino acids from the carboxyl-terminal of the minor ribonuclease from Aspergillus saitoi by carboxypeptidase A on the enzymatic activity.

In order to investigate whether carboxyl-terminal amino acid is involved in the active site of a base non-specific ribonuclease from Asp. saitoi (RNase Ms), removal of carboxyl-terminal amino acid residues by digestion with carboxypeptidase A was investigated. By 24 hours digestion, about 3 serine residues and 1.0 alanine residue were removed from RNase Ms and its activity decreased to about 70% of the native enzyme so far as measured with ribonucleic acid (RNA) as a substrate. The pH optimum and Km of carboxypeptidase treated RNase Ms (CP-RNase Ms) were very similar to those of native RNase Ms so far as measured with RNA as a substrate. However, Km of CP-RNase Ms using ApC as a substrate seemed to be larger than that of native RNase Ms. The large increase in Km value was not observed in the early stage of digestion where about 3 serine residues were removed. The gross structure of CP-RNase Ms was quite similar to that of the native RNase Ms by judging from circular dichroism spectrum at wavelength between 230-205 nm. From the results described above, it was concluded that carboxyl-terminal amino acid was not involved directly in the active site of RNase Ms.

Partial purification and properties of purine nucleoside phosphorylase from rabbit erythrocytes

1. The partial purification of purine nucleoside phosphorylase from rabbit erythrocytes is described. 2. Analytical and preparative isoelectric focusing gave a pI value for the enzyme of 4.65. 3. Gel-chromatography and sucrose-density-gradient-centrifugation techniques gave estimates of the molecular weight in the range 75000-83000. 4. Lineweaver-Burk plots of kinetic data were non-linear at high inosine concentrations. Extrapolation of the linear part of such plots yielded a Km value for inosine of about 70 micrometer for the rabbit erythrocyte and liver enzymes. 5. A Hill interaction coefficient of 0.75 was obtained, suggesting negative co-operativity with respect to the binding of inosine. 6. Treatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) caused partial inactivation, and subsequent Lineweaver-Burk plots with inosine as substrate displayed complete linearity, with an increase in Km value for inosine to 200 micrometer. 7. Starch-gel electrophoresis did not reveal the presence of secondary isoenzymes; all tissue extracts examined gave electrophoretic patterns similar to those obtained with the partially purified enzyme from erythrocytes. 8. Results of hybridization studies with nucleoside phosphorylase from human foetal liver suggest that the rabbit enzyme is also a trimer.

Kinetic properties of hexokinase in resected temporal lobes of patients with drug-resistant epilepsy.

Properties of the cerebral glycolytic enzyme, hexokinase, were studied in biopsy samples of human temporal lobe, obtained during lobectomy for drug-resistant epilepsy and compared "blind" with contol biopsy samples of human cerebral cortex. No significant changes in the total activity or subcellular distribution of the enzyme were observed but the Km value for glucose was altered. The 17 control samples gave a normal mean value for Km (glucose) of 0.05 mM and the 14 epileptic samples gave a significantly higher mean value of 0.09 mM. The drugs used in previous treatment of the epilepsies were "scored" with respect to type and dose; analysis of these in relation to the kinetic results eliminated the possibility that the increase in Km value was an artifact due to the drugs. The observed change in enzyme kinetic properties is discussed in terms of potential interactions of small molecules with the isoenzymes of cerebral hexokinase.