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At least 37 records · Page 2Linked to original sources
PubMed1977

Enzyme inactivation by a cellular neutral protease: enzyme specificity, effects of ligands on inactivation, and implications for the regulation of enzyme degradation.

A protease from Tetrahymena pyriformis inactivated eight of nine commercially available enzymes tested, including lactate deyhdrogenase, isocitrate dehydrogenase (TPN-specific), glucose-6 phosphate dehydrogenase, D-amino acid oxidase, fumarase, pyruvate kinase, hexokinase, and citrate synthase. Urate oxidase was not inactivated. Inactivation occurred at neutral pH, was prevented by inhibitors of the protease, and followed first order kinetics. In those cases tested, inactivation was enhanced by mercaptoethanol. Most of the enzyme-inactivating activity was due to a protease of molecular weight 25,000 that eluted from DEAE-Sephadex at 0.3 M KCl. A second protease of this molecular weight, which was not retained by the gel, inactivated only isocitrate dehydrogenase and D-amino acid oxidase. These two proteases could also be distinguished by temperature and inhibitor sensitivity. Two other protease peaks obtained by DEAE-Sephadex chromatography had little or no no enzyme inactivating activity, while another attacked only D-amino acid oxidase. At least six of the enzymes could be protected from proteolytic inactivation by various ligands. Isocitrates dehydrogenase was protected by isocitrate, TPN, or TPNH, glucose-6-dehydrogenase by glucose-6-P or TPN, pyruvate kinase by phosphoenolypyruvate or ADP, hexokinase by glucose, and fumarase by a mixture of fumarate and malate. Lactate dehdrogenase was not protected by either of its substrates of coenzymes. Citrate synthase was probably protected by oxalacetate. Our data suggest that the protease or proteases discussed here may participate in the inactivation or degradation of a least some enzymes in Tetrahymena. Since the inactivation occurs at neutral pH, this process could be regulated by variations in the cellular levels of substrates, coenzymes, or allosteric regulators resulting form changes in growth conditions or growth state. Such a mechanism would permit the selective retention of enzymes of metabolically active pathways.

Animals↗
PubMed1999

Design of immuno-enzymosomes with maximum enzyme targeting capability: effect of the enzyme density on the enzyme targeting capability and cell binding properties.

Immuno-enzymosomes have been proposed for the targeting of enzymes to cancer cells to achieve site specific activation of anticancer prodrugs. Previously, we reported that the enzyme beta-glucuronidase (GUS), capable of activating anthracycline-glucuronide prodrugs, can be coupled to the surface of inmunoliposomes directed against human ovarian cancer cells (OVCAR-3). This study aimed at the design of an immuno-enzymosome formulation with maximum enzyme targeting capability. By purification of the commercially available enzyme beta-glucuronidase (GUS), a 2-fold increase in the enzyme specific activity and a 4-fold increase in the enzymatic activity of immuno-enzymosomes was achieved. As a result, upon incubation with human ovarian cancer cells (OVCAR-3), cell-associated enzymatic activity increased correspondingly. The optimized immuno-enzymosomes were shown to bind to the target cells in a specific fashion. Above a GUS/Fab' molar ratio of 0.5, impairment of the target cell binding ability of the immuno-enzymosomes was observed. This was likely due to a steric hindrance effect mediated by the presence of large amounts of bulky GUS molecules on the liposome surface. Nevertheless, increasing the GUS density on the surface of the immuno-enzymosomes to levels by far exceeding the GUS/Fab' molar ratio of 0.5, yielded a considerably improved enzyme targeting capability.

Antibodies, Monoclonal↗
PubMed2003

GSTM1 enzyme concentration and enzyme activity in correlation to the genotype of detoxification enzymes in squamous cell carcinoma of the oral cavity.

BACKGROUND: Differences in genotype and phenotype of detoxification genes could be one reason for conflicting results in studies dealing with gene polymorphisms as susceptibility factors for tobacco associated cancer. OBJECTIVES: The objective of this study was to investigate gene polymorphisms of detoxification enzymes and to determine whether the enzyme concentration and activity of glutathione S transferase microliter 1 correlates with the genotype in patients with cancer of the oral cavity. MATERIAL AND METHODS: In 73 cancer patients and 136 matched healthy controls, the polymorphisms of glutathione S-transferase mu1 and theta (GSTM/GSTT), cytochrome p450 1A1 and CYP2D6 were detected. Simultaneously, GSTM1 protein concentration and total GSTM1-activity were determined. RESULTS: Only the coincidence of GSTM1 and GSTT null genotype was associated with oral cavity cancer. GSTM1 protein concentration and enzyme activity in null-genotype patients was significantly lower than in GSTM1-allele-carrier. But the enzyme concentration did not correlate with the activity. CONCLUSION: We assume that detoxification enzymes are functionally redundant and that only the simultaneous deficiency of several detoxification enzymes increases the risk for oral cancer.

Adolescent↗
PubMed1975

Malate dehydrogenase, circular dichroism difference spectra of porcine heart mitochondrial and supernatant enzymes, binary enzyme-coenzyme, and ternary enzyme-coenzyme-substrate analog complexes.

Circular dichroism spectra and circular dichroism difference spectra, generated when porcine heart mitochondrial and supernatant malate dehydrogenase bind coenzymes or when enzyme dihydroincotinamide nucleotide binary complexes bind substrate analogs, are presented. No significant changes are observed in protein chromophores in the 200- to 240-nm spectral range indicating that there is apparently little or no perturbation of the alpha helix or peptide backbone when binary or ternary complexes are formed. Quite different spectral perturbances occur in the two enzymes with reduced coenzyme binding as well as with substrate-analog binding by enzyme-reduced coenzyme binding. Comparison of spectral perturbations in both enzymes with oxidized or reduced coenzyme binding suggests that the dihydronicotinamide moiety of the coenzyme interacts with or perturbs indirectly the environment of aromatic amino acid residues. Reduced coenzyme binding apparently perturbs tyrosine residues in both mitochondrial malate dehydrogenase and lactic dehydrogenase. Reduced coenzyme binding perturbs tyrosine and tryptophan residues in supernatant malate dehydrogenase. The number of reduced coenzyme binding sites was determined to be two per 70,000 daltons in the mitochondrial enzyme, and the reduced coenzyme dissociation constants, determined through the change in ellipticity at 260 nm, with dihydronicotinamide adenine dinucleotide binding, were found to be good agreement with published values (Holbrook, J. J., and Wolfe, R. G. (1972) Biochemistry 11, 2499-2502) obtained through fluorescence-binding studies and indicate no apparent extra coenzyme binding sites. When D-malate forms a ternary complex with malate dehydrogenase-reduced coenzyme complexes, perturbation of both adenine and dihydronicotinamide chromophores is evident. L-Malate binding, however, apparently produces only a perturbation of the adenine chromophore in such complexes. Since the coenzyme has been found to bind in an open conformation on the surface of the enzyme and the substrate analogs bind at or very near the dihydronicotinamide moiety binding site, protein conformational changes are implicated during ternary complex formation with D-malate which can effect the adenine chromophore at some distance from the substrate binding site.

Animals↗
PubMed1993

Enzyme immunoassay of beta-hexosaminidase A and B in serum: carrier detection of GM2-gangliosidoses, and equivalence of enzyme activity and enzyme protein reactivity.

beta-Hexosaminidase (Hex; EC 3.2.1.52) isoenzymes A and B were analyzed in sera from a control group of 22 apparently healthy subjects, 13 obligate carriers of Tay-Sachs disease (TSD), 10 obligate carriers of Sandhoff disease (SHD), and 4 affected TSD patients by enzyme immunoassay methods based on enzyme activity. No Hex A activity was detected in the sera of patients with TSD. The activities of Hex A in the obligate carriers of TSD and SHD tended to be lower (nonsignificantly) than in the control group. Hex B activities tended to be higher in TSD patients as well as in carriers of TSD, although the mean activities did not significantly differ from the corresponding mean for the control group. However, Hex B activities were decreased in the carriers of SHD in comparison with the other groups. Sera from 900 postmenopausal women, all of age 55 years, were also analyzed for Hex isoenzymes; the results indicated a carrier frequency of about 1 in 200 for both TSD and SHD. We also compared the enzyme immunoassay method based on enzyme activity with one based on the antigenic (enzyme protein) reactivity alone. Because both methods yielded similar information, we conclude that no significant amounts of inactive enzyme protein are present in the circulation.

Adolescent↗
PubMed1994

Final phase of enzyme reactions following a Michaelis-Menten mechanisms in which the free enzyme and/or the enzyme-substrate complex are unstable.

An important kinetic analysis of unstable enzyme systems was carried out by Duggleby (Duggleby, R.G. (1986) J. Theor. Biol. 123, 67-80). This author states that his results are of general validity in the sense that the instability rate constants may have any value. Later, Wang & Tsou (Wang and Tsou (1990) J. Theor. Biol. 142, 531-549) rediscovered Duggleby's results when they analyzed a scheme in which the inactivations were due to a non-complexing irreversible inhibitor, pointing out the need to assume an initial steady-state in the catalytic route of the reaction. In the present contribution we show that there are values of the instability rate constants for which the equations of Duggleby are not applicable. We propose, for these cases, an alternative equation, which relates the final substrate concentration with the initial ones of both the substrate and the enzyme. Based on this, an experimental design for the evaluation of kinetic parameters is suggested. The present work concerns enzyme reactions evolving according to a Michaelis-Menten mechanism, in which the free enzyme and/or the enzyme-substrate complex are unstable.

Algorithms↗
PubMed1981

The role of enzyme variants, polymorphisms and enzyme hybrids in enzyme deficiency conditions.

Based on the heterogeneity observed in two red cell enzymes, i.e. glucose 6-phosphate dehydrogenase (E.C. 1.1.1.49) and catalase (E.C.1.11.1.6), the role of different types of enzyme variants in enzyme deficiency conditions is discussed. For theoretical and practical reasons variants of unusually low specific activity and of low stability have to be distinguished. Whereas in the former type the activity level in blood more or less reflects the situation in other tissues, this is not the case for unstable mutants, e.g. the enzyme variant found in Swiss-type acatalasemia. In heterozygous carriers the situation can be complicated by the fact that variants of oligomer enzymes (e.g. catalase) are present as molecular hybrids exerting almost normal stability and activity.

Acatalasia↗
PubMed1977

Theoretical study of the effect of enzyme-enzyme interactions on steady-state enzyme kinetics.

Equilibrium statistical mechanics is much concerned with problems involving intermolecularinteractions, either in lattices or in pure fluids or solutions. The possibility of enzyme-enzyme interactions suggests that the same problems might be studied profitably at steady state as well as at equilibrium. In the systems we consider, each of the identical enzyme molecules of the system undergoes steady-state stochastic cycling among states i equal 1,....,n. But the molecules do not cycle independently. Two neghboring molecules, in states i and j, interact with a free energy wij (a function of the distance r in the solution case). The instantaneous transition probabilities between states for a given molecule will depend on the instantaneous interactions between the molecule in question and its neighbors. The primary question of interest is how the enzyme flux is influenced by the interactions. The general problem is outlined here and some simple special cases are treated. The discussion will be continued in a following paper [Hill, T. L. (1977) Proc. Natl. Acad. Sci. USA 74, in press]

Enzymes↗
PubMed1987

Ferredoxin-thioredoxin reductase, an iron-sulfur enzyme linking light to enzyme regulation in oxygenic photosynthesis: purification and properties of the enzyme from C3, C4, and cyanobacterial species.

Ferredoxin-thioredoxin reductase (FTR), an enzyme involved in the light regulation of chloroplast enzymes, was purified to homogeneity from leaves of spinach (a C3 plant) and corn (a C4 plant) and from cells of a cyanobacterium (Nostoc muscorum). The enzyme is a yellowish brown iron-sulfur protein, containing four nonheme iron and labile sulfide groups, that catalyzes the activation of NADP-malate dehydrogenase and fructose 1,6-bisphosphatase in the presence of ferredoxin and of thioredoxin m and f, respectively. FTR is synonymous with the protein earlier called ferralterin. FTR showed an Mr of about 30,000 (determined by sedimentation equilibrium ultracentrifugation, amino acid composition, gel filtration, and gradient gel electrophoresis) and was composed of two dissimilar subunits (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). One of the FTR subunits from each source was similar both in Mr (about 13,000) and immunological properties, while the other subunit (of variable molecular weight) was characteristic of a particular organism. The similar subunit contained a disulfide group that was rapidly reduced by a dithiol (dithiothreitol) but not by monothiols (2-mercaptoethanol or reduced glutathione). Homogeneous FTR formed a tight noncovalent complex with ferredoxin on affinity columns. The basis for the structural variation in the different FTR enzymes remains to be determined.

Chloroplast Thioredoxins↗
PubMed1987

A simple approach to detect active-site-directed enzyme-enzyme interactions. The aldolase/glycerol-phosphate-dehydrogenase enzyme system.

A novel approach has been elaborated to identify the mechanism of intermediate transfer in interacting enzyme systems. The aldolase/glycerol-3-phosphate-dehydrogenase enzyme system was investigated since complex formation between these two enzymes had been demonstrated. The kinetics of dihydroxyacetone phosphate conversion catalyzed by the dehydrogenase in the absence and presence of aldolase was analyzed. It was found that the second-order rate constant (kcat/Km) of the enzymatic reaction decreases due to the formation of a heterologous complex. The decrease could be attributed to an increase of the Km value since kcat did not change in the presence of aldolase. In contrast, an apparent increase in the second-order rate constant of dihydroxyacetone phosphate conversion by the dehydrogenase was observed if the triose phosphate was produced by aldolase from fructose 1,6-bisphosphate (consecutive reaction). Moreover, no effect of dihydroxyacetone phosphate on the dissociation constant of the heterologous enzyme complex could be detected by physico-chemical methods. The results suggest that the endogenous dihydroxyacetone phosphate produced by aldolase complexed with dehydrogenase is more accessible for the dehydrogenase than the exogenous one, the binding of which is impeded due to steric hindrance by bound aldolase.

Binding Sites↗
PubMed1996

Different exon-intron organizations of the genes for two astacin-like proteases, high choriolytic enzyme (choriolysin H) and low choriolytic enzyme (choriolysin L), the constituents of the fish hatching enzyme.

The hatching enzyme of the teleost, Oryzias latipes, is composed of two proteases, high choriolytic enzyme (choriolysin H, HCE) and low choriolytic enzyme (choriolysin L, LCE), which are similar in some enzymological characteristics and protein structure (55% identity in amino acid sequence) and belong to the astacin family. Two isoforms of HCE are detected. In the present study, the genes for HCE and LCE were isolated from the genomic library constructed from DNA of the inbred drR strain fish. In contrast to the close similarity of the enzymes, there was a marked difference in their gene organization. The LCE gene was a single copy gene and composed of eight exons interrupted by seven introns. The HCE genes were multicopy genes and lacked introns. In the haploid genome of the drR strain fish, there are eight HCE genes, seven of which were cloned. Each HCE gene was identified as that for either of the two isoforms of HCE. 5' flanking regions of the LCE gene and the HCE genes had consensus TATA box sequences, but not CAT box nor GC box sequences. The big difference in the exon-intron organization between the HCE genes and the LCE gene is discussed from an evolutionary viewpoint.

Amino Acid Sequence↗
PubMed2002

Biosynthesis in an on-line immobilized-enzyme reactor containing phenylethanolamine N-methyltransferase in single-enzyme and coupled-enzyme formats.

An immobilized-enzyme reactor (IMER) based upon phenylethanolamnie N-methyltransferase (PNMT) has been developed. The activity of the PNMT-IMER and its applicability for on-line N-methylation of normetanephrine was investigated. The reactor was connected through a switching valve to a cyano (CN) and ODS stationary phase connected in series. The substrate was injected onto the PNMT-IMER and the unreacted substrate and product were eluted and transported via a switching valve onto the analytical columns. The results from the PNMT-IMER/CN-ODS chromatographic system demonstrate that the enzyme retained its catalytic activity. Known substrates and inhibitors for PNMT were examined and the chromatographic system was utilized to carry out both quantitative and qualitative determinations. The PNMT-IMER/ CN-ODS system proves to be useful in basic biochemical studies, an ideal for the high throughput screening of substances for PNMT substrate-inhibitor properties. The PNMT-IMER was then coupled in series using switching-valve technology with a previously developed dopamine beta-hydroxylase immobilized-enzyme reactor and used to carry out the on-line two-step synthesis of epinephrine from dopamine.

Animals↗
PubMed2002

Enzyme activation in organic solvents: co-lyophilization of subtilisin Carlsberg with methyl-beta-cyclodextrin renders an enzyme catalyst more active than the cross-linked enzyme crystals.

In this study we explored the efficiency of the additive methyl-beta-cyclodextrin (M beta CD) to enhance the activity and enantioselectivity of the serine protease subtilisin Carlsberg in organic solvents. These two parameters, measured for different transesterification reactions and in several solvents, are compared with results obtained by using two additional preparations of the same enzyme: lyophilized powder and cross-linked enzyme crystals (CLEC). The results suggest that co-lyophilization of subtilisin with M beta CD preserves the enzyme's active site tertiary structure rendering a highly active and enantioselective catalyst.

Catalysis↗
PubMed1977

Further study of the effect of enzyme-enzyme interactions on steady-state enzyme kinetics.

This paper continues an earlier one [Hill, T.L. (1977) Proc. Natl . Acad. Sci. USA 74, 3632-3632] and presents further introductory examples. Most attention is devoted to a closed linear chain of two-state enzyme molecules with nearest-neighbor interactions. The one-dimensional Ising theory can be used here. The Bragg-Williams (mean field) approximation is introduced to deal with a one-, two-, or three-dimensional lattice of enzyme molecules, at steady state, with an arbitrary kinetic diagram. The behavior of the flux in a phase transition is noted. Finally, a treatment is given for the first effect (second "viral" coefficient) of interactions on the flux in a dilute solution of two-state enzyme molecules.

Enzymes↗
PubMed1978

Unsymmetrical and concerted examples of the effect of enzyme--enzyme interactions on steady-state enzyme kinetics.

In previous papers of this series, emphasis has been placed on the steady-state phase transition and critical properties of large lattices of interacting, symmetrical, and identical enzyme molecules. The present paper is concerned with a number of examples of enzyme--enzyme interactions that do not belong to the class of models of the earlier papers. These are more biochemically oriented and include heterologous dimers, a linear chain with unsymmetrical interactions, and concerted isologous dimers (half-the-sites reactivity).

Catalysis↗
PubMed1993

Control of glycolytic enzyme binding: effect of changing enzyme substrate concentrations on in vivo enzyme distributions.

The effect of changing concentrations of glycolytic intermediates on the binding of phosphofructokinase, aldolase and pyruvate kinase to cellular particulate matter was investigated. Concentrations of glycolytic intermediates were altered by adding 2 mM iodoacetic acid (IAA) to an incubation medium containing tissues isolated from the channelled whelk Busycon canaliculatum. Iodoacetic acid inhibited glyceraldehyde 3-phosphate dehydrogenase activity causing a 100-400 fold increase in the concentration of fructose 1,6-bisphosphate as well as 3-20 fold increases in glucose 6-phosphate, fructose 6-phosphate, and dihydroxyacetone phosphate levels depending on the experimental protocol. Cellular pH values were not statistically different in the presence of IAA. Measurement of enzyme binding to particulate matter showed that the binding of phosphofructokinase, aldolase and pyruvate kinase was unaffected by iodoacetic acid under any experimental condition. These results show that changes in the tissue concentrations of enzyme substrates and products do not regulate enzyme binding to particulate matter in the cell.

Animals↗