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P B Chock

Publications and source records attributed to P B Chock.

At least 19 recordsLinked to original sources
PubMed1992

Isoforms of mammalian ubiquitin-activating enzyme.

Ubiquitin-activating enzyme, "E1," is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates and represents a potential target for regulation in the metabolic control of the conjugation reaction. Antiserum raised against human E1 recognizes two immunoreactive proteins in extracts from several human cell lines and animal tissues. We have characterized these two immunoreactive proteins in HeLa cells and present evidence that they are isoforms of E1. We have designated these isoforms as "E1(110 kDa)" and "E1(117 kDa)" to reflect their apparent molecular masses determined from SDS-polyacrylamide gel electrophoresis. These two immunoreactive proteins are immunologically similar, have nearly identical peptide maps, and comigrate with enzymatic activity characteristic of E1 in native polyacrylamide gel electrophoretic separations. Pulse-labeling experiments reveal that both isoforms are long-lived in vivo with degradation rates which are inconsistent with a proenzyme/enzyme model. Furthermore, their rates of degradation, which vary depending on the cell line studied, are kinetically distinguishable in contact-inhibited human lung fibroblasts. This work represents the first demonstration of E1 isoforms in a non-plant species and carries important implications for studies of the regulatory mechanisms controlling ubiquitin conjugation.

Animals↗
PubMed1992

Protein ubiquitination is regulated by phosphorylation. An in vitro study.

Protein ubiquitination has been implicated in ATP-dependent protein turnover and in a number of biological processes in eukaryotic cells. The ubiquitination activating enzyme, E1, and ubiquitin carrier protein, E2, are two essential enzymes in the protein ubiquitination machinery. Using purified E1 and E2 from rabbit reticulocytes and various protein kinases, which include cAMP-dependent protein kinase, protein kinase C, and protein tyrosine kinase, we demonstrated that E1 is phosphorylated by protein kinase C, with a stoichiometry of 0.65 mol of phosphate/mol of E1, and one of the E2 isoforms, E2(32kDa), is phosphorylated by protein tyrosine kinase to 2 eq of phosphate/mol of protein. Phosphorylation of E1 causes a 2-fold enhancement of its activity as monitored by ubiquitin-dependent ATP in equilibrium PPi exchange. When 1 eq of phosphate was incorporated into E2(32kDa), a 2.4-fold activation was also observed for its activity to catalyze the ubiquitination of histone H2A. The regulatory significance of this finding is discussed.

Adenosine Triphosphate↗
PubMed1992

Kinetic method for differentiating mechanisms for ligand exchange reactions: application to test for substrate channeling in glycolysis.

We have derived analytical expressions for the kinetics of the two mechanisms involved in ligand substitution reactions. These mechanisms are (i) a dissociative mechanism in which the leaving ligand is first dissociated prior to the binding of the incoming ligand and (ii) an associative mechanism where a ternary complex is formed between the incoming ligand and the complex containing the leaving ligand. The equations obtained provide the theoretical basis for differentiating these two mechanisms on the basis of their kinetic patterns of the displacement reactions. Analysis of these equations shows that an associative mechanism can only generate an increasing kinetic pattern for the observed pseudo-first-ordered rate constants as a function of increasing concentration of the incoming ligand and plateaus, in most cases, at a value higher than the off-rate constant of the leaving ligand. However, a dissociative mechanism can generate either an increasing or a decreasing (kapp decreases with increasing concentrations of the incoming ligand) kinetic pattern, depending on the magnitudes of the individual rate constants involved, and, in either case, it will plateau at kapp equal to the koff of the leaving ligand. Therefore, the decreasing kinetic pattern is a hallmark for a dissociative mechanism. This general method was used to settle the dispute of whether NADH is transferred directly via the enzyme-enzyme complex between glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27).(ABSTRACT TRUNCATED AT 250 WORDS)

Glycerolphosphate Dehydrogenase↗
PubMed1991

Association of ubiquitin-activating enzyme with HeLa cell chromosomes during mitosis.

Ubiquitin-activating enzyme (E1) is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. Antibodies raised against E1 were affinity purified and used for immunostaining HeLa cells. Condensed chromosomes in mitotic cells were found to be strongly immunoreactive. Chromosomes from metaphase-arrested HeLa cells were isolated and chromosome-associated proteins were analyzed by Western blotting. E1 was detected in fractions containing isolated chromosomes. These results suggest that E1 is associated with condensed chromosomes during mitosis.

Blotting, Western↗
PubMed1991

Electroporation by using bipolar oscillating electric field: an improved method for DNA transfection of NIH 3T3 cells.

Using the plasmid DNA pSV2-neo (which, when integrated into the cellular genome confers resistance to the antibiotic G418 for selection), we examined and compared the transfection efficiency on NIH 3T3 cells electropermeabilized by applying a sequence of high-frequency unipolar or bipolar square waves or a single square pulse. Results show that a bipolar square wave is, at least, 1.7- and 5.5-fold more efficient than the unipolar square wave and single square pulse, respectively. In the range of electric field strength used for optimum transfection, the survivability of electropermeabilized cells was comparable between the unipolar and bipolar square waves but fell considerably with the single square pulse. Qualitative comparison of cell permeabilization induced by the three types of wave forms and monitored by ethidium bromide uptake revealed that only the bipolar square wave permeabilizes the cell membrane symmetrically at the two hemispheres facing the electrodes. With unipolar square wave or single square pulse, the membrane is permeabilized either on one side or asymmetrically. Taken together, our result suggests that permeabilization of the membrane at multiple sites without affecting cell survivability may account for the improvements in transfection efficiency observed with bipolar oscillating electric fields.

Cell Line↗
PubMed1991

Distance changes at the regulatory and catalytic sites on Escherichia coli glutamine synthetase: a spin label study on the effect of substrate(s) binding.

A spin-labeled ATP analogue, 2,2,6,6-tetramethylpiperidine-1-oxyl adenosine triphosphatase (Tempo-ATP) is used to adenylate Escherichia coli glutamine synthetase (L-glutamine: ammonia ligase (ADP-forming), EC 6.3.1.2). The Tempo adenylylated glutamine synthetase (Tempo-GS) exhibits similar catalytic properties, pH profile and inhibitor susceptibility as those of glutamine synthetase adenylylated with normal ATP. Using the spin label on the enzyme as a probe and employing the spin-spin interactions between the label probe and paramagnetic Mn2+, the distances from the nitroxyl moiety of the covalently bound Tempo-AMP to the two Mn2+ binding sites, n1 and n2 were determined. The n1 site is the structural site and n2 is located at the catalytic site. The distances from Mn2+ at n1 and n2 sites to the nitroxyl radical are 19 and 16 A, respectively. Binding of the substrate, L-Glu, causes a protein conformational change which is reflected by the reduction of approximately 2 A for the n1 to Tempo-AMP distance and lengthening of approximately 2 A for the n2 to the Tempo-AMP distance. Addition of ATP to the Tempo-GS/L-Glu complex increases the distance between n1 and Tempo-AMP, and n2 and Tempo-AMP by 4 and 3 A, respectively.

Adenosine Diphosphate↗
PubMed1991

Immunocytochemical localization of ubiquitin-activating enzyme in the cell nucleus.

Ubiquitin-activating enzyme, "E1", is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. We present immunocytochemical evidence that Ubiquitin-activating enzyme is concentrated in the cell nucleus. This finding points to the nucleus as the major site of action of this enzyme. Since ubiquitin itself is not similarly compartmentalized, this result suggests a high level of ubiquitin conjugate formation in the nucleus with a rapid turnover of ubiquitin conjugates.

Animals↗
PubMed1991

Substrate channeling in glycolysis: a phantom phenomenon.

It has been proposed that glycolytic enzymes form multienzyme complexes for direct transfer of metabolites from the producing enzyme to the utilizing one. Reexamination of the supporting evidence, which involves the transfer of NADH between its complexes with glycerol-3-phosphate dehydrogenase (alpha-glycerol phosphate dehydrogenase, GPDH; EC 1.1.1.8) and with L-lactate dehydrogenase (LDH; EC 1.1.1.27), has shown that the supporting evidence is based on misinterpretation of the kinetics of ligand exchange. Srivastava et al. have responded with a revision of their own and criticism of our data. To clarify this problem, we have carried out detailed kinetic studies on NADH binding to GPHD and LDH and on the displacement of enzyme-bound NADH by LDH or GPDH. The experiments were conducted at 10 degrees C in 50 mM Hepes, pH 7.5/100 mM KCl/1 mM EDTA/1 mM 2-mercaptoethanol, using rabbit muscle GPDH and LDH. The results show that the kinetic patterns exhibited by the displacement of NADH-bound enzyme by either GPDH or LDH are consistent with a dissociative mechanism but not with a direct transfer mechanism. Theoretical analysis shows that a combined dissociative and direct transfer mechanism can explain the transient kinetic data reported by Srivastava et al. if, and only if, a majority (approximately 90%) of the enzyme present in lower concentration exists as a complex with the second enzyme. However, data from tracer and traditional sedimentation equilibrium and from gel filtration experiments show that LDH and GPDH do not form complexes in the presence of saturating NADH concentration when the enzyme concentrations are ranged between 4 and 50 microM, a concentration equal to or greater than that used by Srivastava et al. Our results demonstrate that GPDH and LDH do not form multienzyme complex and the transfer of NADH between these enzymes proceeds via a dissociative mechanism.

Animals↗
PubMed1990

Electro-permeabilization of cell membranes: effect of the resting membrane potential.

Electric field induced permeabilization of cell membranes is an important technique for gene transfection and cell hybridization. Mechanistic studies of this process revealed that the uptake of fluorescent indicator by plant protoplasts occurs predominantly on the hemisphere facing the positive electrode, while in erythrocyte ghosts the probes exit through the hemisphere facing the negative electrode. To reconcile these observations symmetrical pore formation and a mechanism of molecular exchange by electroosmosis has been proposed. In light of these controversial observations, we conducted a systematic study of electroporation of NIH3T3 cells with varying electric field strength, waveform and frequency. Our data revealed that (i) symmetrical permeabilization of the cell membrane occurs only with bipolar a.c. fields. (ii) When a critical membrane breakdown potential, Vc, is applied using either an unipolar a.c. fields or a single d.c. square pulse, the cell membrane becomes permeabilized only at the hemisphere facing the positive electrode. (iii) When the pulse-induced membrane potential, Vm, is approximately equal to or larger than the intrinsic membrane potential (i.e. using d.c. or unipolar a.c. field), asymmetric permeabilization was observed with the hemisphere facing the positive electrode being most permeable. (iv) The rate of fluorescent indicator uptake is dependent on the concentration of the indicator. These results indicate that electro-permeabilization of cell membranes is affected by its resting potential and that electroosmosis is not the dominant mechanism for the cellular uptake of foreign molecules in electroporation.

Animals↗
PubMed1990

Manganese-dependent disproportionation of hydrogen peroxide in bicarbonate buffer.

At physiological concentrations of HCO3- and CO2, Mn(II) catalyzes disproportionation of H2O2. This catalase-like activity is directly proportional to the concentrations of Mn(II) and H2O2, and it increases exponentially with increases in pH. The effect of increasing pH is almost completely attributable to the concomitant increase in HCO3- concentration. The rate is proportional to the third power of the HCO3- concentration, suggesting that 3 equivalents of HCO3- combine with 1 equivalent of Mn(II) to form the catalytic complex. It is presumed that the redox potential of the Mn(II) in equilibrium with Mn(III) couple in such a complex permits H2O2 to carry out facile reactions with Mn(II) comparable to those that occur with Fe(III) and Cu(II) chelate complexes, in which OH. and O2-. are established intermediates. The Mn-catalyzed disproportionation of H2O2 does not occur at physiological pH in the absence of HCO3-. Hepes, inorganic phosphate, and inorganic pyrophosphate inhibit the reaction catalyzed by the Mn/HCO3- system. These results are similar to those of Sychev et al. [Sychev, A.Y., Pfannmeller, U. & Isak, V.G. (1983) Russ. J. Phys. Chem. 57, 1690-1693]. The catalase-like activity of Mn(II)-bicarbonate complexes reported here, together with the superoxide dismutase activity of Mn complexes demonstrated by Archibald and Fridovich [Archibald, F.S. & Fridovich, I. (1982) Arch. Biochem. Biophys. 214, 452-463], strengthen the proposition that Mn may play an important role in the protection of cells against oxygen radical-mediated damage.

Bicarbonates↗
PubMed1990

Manganese(II) catalyzes the bicarbonate-dependent oxidation of amino acids by hydrogen peroxide and the amino acid-facilitated dismutation of hydrogen peroxide.

In bicarbonate/CO2 buffer, Mn(II) and Fe(II) catalyze the oxidation of amino acids by H2O2 and the dismutation of H2O2. As the Mn(II)/Fe(II) ratio is increased, the yield of carbonyl compounds per mole of leucine oxidized is essentially constant, but the ratio of alpha-ketoisocaproate to isovaleraldehyde formed increases, and the fraction of H2O2 converted to O2 increases. In the absence of Fe(II), the rate of Mn(II)-catalyzed leucine oxidation is directly proportional to the H2O2, Mn(II), and amino acid concentrations and is proportional to the square of the HCO3- concentration. The rate of Mn(II)-catalyzed O2 production in the presence of 50 mM alanine or leucine is about 4-fold the rate observed in the absence of amino acids and accounts for about half of the H2O2 consumed; the other half of the H2O2 is consumed in the oxidation of the amino acids. In contrast, O2 production is increased nearly 18-fold by the presence of alpha-methylalanine and accounts for about 90% of the H2O2 consumed. The data are consistent with the view that H2O2 decomposition is an inner sphere (cage-like) process catalyzed by a Mn coordination complex of the composition Mn(II), amino acid, (HCO3-)2. Oxidation of the amino acid in this complex most likely proceeds by a free radical mechanism involving hydrogen abstraction from the alpha-carbon as a critical step. The results demonstrate that at physiological concentrations of HCO3- and CO2, Mn(II) is able to facilitate Fenton-type reactions.

Alanine↗
PubMed1990

Manganese(II)-bicarbonate-mediated catalytic activity for hydrogen peroxide dismutation and amino acid oxidation: detection of free radical intermediates.

To examine the structural identities of reactive free radicals and the mechanism of the oxidative modification of proteins, we used EPR and spin-trapping methods to investigate the oxidation of amino acids by H2O2 as well as the decomposition of H2O2 itself catalyzed by Mn(II) ions. Superoxide and hydroxyl radicals (O2-. and OH.) were trapped by a spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), in a reaction mixture containing Mn(II) and H2O2 in bicarbonate/CO2 buffer. When Hepes was used in place of bicarbonate buffer, superoxide radical was not observed, indicating the importance of bicarbonate buffer. With addition of L-leucine to a similar reaction mixture, a leucine-derived radical that replaced the DMPO-superoxide adduct was detected in the absence and presence of DMPO. Using various isotope-enriched L-leucines, we successfully identified this radical as a hydronitroxide, -OOC(R)CHNHO.. The data are consistent with the formation of a transient "caged" OH. in the inner coordination sphere of Mn(II). This caged OH. is likely to undergo an intramolecular hydrogen-atom abstraction from the Mn-bound H2O2 or amino acid. Two reaction schemes are proposed to account for the experimental results shown here and in the preceding papers.

Amino Acids↗
PubMed1990

Copper, zinc superoxide dismutase catalyzes hydroxyl radical production from hydrogen peroxide.

Cu,Zn superoxide dismutase (Cu,Zn-SOD; EC 1.15.1.1) is known to be inhibited slowly by H2O2. Using EPR and the spin traps 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN), we have shown that Cu,Zn-SOD catalyzes the formation of "free" .OH radicals from H2O2 in pH 7.6 bicarbonate buffer. Supporting evidence includes the following: (i) H2O2 and active Cu,Zn-SOD are required to yield significant signals from spin-trap-OH adducts. (ii) With O2-., Cu,Zn-SOD causes the appearance of intense resonance signals due to DMPO-OH adducts. These signals were inhibited strongly by catalase. (iii) With H2O2, Cu,Zn-SOD, and DMPO, radical scavengers formate and azide, but not ethanol, decrease DMPO-OH signals while causing new intense signals due to their corresponding DMPO-radical adducts. Failure of ethanol to quench DMPO-OH signals is discussed in light of the positively charged active channel of the enzyme. (iv) With PBN as a spin trap, ethanol quenches .OH radical signals and yields PBN-trapped hydroxyethyl radical signals. (v) Mn-SOD does not catalyze "free" .OH radical formation and it also exerts no effect on the signals of DMPO-OH adducts when added together with the Cu,Zn-SOD. The capacity of Cu,Zn-SOD to generate "free" .OH radicals from H2O2 may in part explain the biological damage associated with elevated intracellular SOD activity.

Animals↗
PubMed1988

Reexamination of the kinetics of the transfer of NADH between its complexes with glycerol-3-phosphate dehydrogenase and with lactate dehydrogenase.

Srivastava and Bernhard [Srivastava, D. K. & Bernhard, S. A. (1986) Science 234, 1081-1086] have proposed that glycolytic enzymes form multienzyme complexes for the direct transfer of metabolites from the producing enzyme to the utilizing one. We have reinvestigated the evidence for direct transfer of NADH between its complexes with alpha-glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27). The results reveal the following. (i) Proper treatment of the kinetics of and equilibrium data for the transfer of NADH between GPDH and LDH indicates that NADH transfer proceeds by a free-diffusion mechanism and not by direct transfer through a ternary complex. (ii) The koff for NADH from its GPDH complex is 60 sec-1 rather than 9.4 sec-1 in Tris.HCl buffer (pH 7.4) at 25 degrees C. With this value one can explain kcat = 50 sec-1 for LDH-catalyzed hydrogenation of pyruvate with GPDH-bound NADH as coenzyme. (iii) Steady-state kinetics show that LDH inhibits the GPDH-catalyzed reaction simply by reducing the concentration of free NADH. Similarly, aldolase inhibits the GPDH-catalyzed reduction of dihydroxyacetone phosphate to glycerol-3-phosphate by binding to the substrate. The proposed direct transfer of NADH between GPDH and LDH is therefore mainly based on a misinterpretation of the experimental data.

Animals↗