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At least 19 recordsLinked to original sources
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed1977

"Viral" expansion of enzyme flux and use of quasi-chemical approximation for two-state enzymes with enzyme-enzyme interactions.

Two examples of enzyme systems with interactions, at steady state, are treated here. In both cases, the enzyme cycle has two states and quasi-equilibrium in spatial distributions obtains at steady state (because f alpha + f beta = 1). The first example is a dilute solution of enzyme molecules in a solvent. The flux (turnover) per molecule is expanded in powers of the enzyme concentration (a "viral" expansion). Aggregation of the enzyme molecules in solution is considered as a special case. In the second example, we treat an arbitrary lattice of enzyme molecules, with nearest-neighbor interactions, using the well-known quasi-chemical approximation. The flux per molecule is obtained. Critical behavior and hysteresis are illustrated.

Enzymes↗
PubMed1979

Characterization of "thyroliberin-deamidating enzyme" as a post-proline-cleaving enzyme. Partial purification and enzyme-chemical analysis of the enzyme from anterior pituitary tissue.

An enzyme which catalyzes the deamidation of thyroliberin (TRF; less than Glu-His-Pro-NH2) has been purified 110-fold from extracts of bovine anterior pituitary by ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and gel filtration. This enzyme of 76,000 molecular weight (as estimated by gel filtration) exhibits maximal activity at neutral pH (optimum pH 7.4 to 7.6) in buffers of high ionic strength supplemented with thiol-protecting agents. As indicated by the strong inhibition of the enzymatic activity by N-ethylmaleimide and Hg2+, as well as by the extreme sensitivity toward diisopropyl fluorophosphate, -SH, and -OH residues apparently represent essential functional groups of the enzyme. The stereospecific deamidation of TRF (Km = 4.1 . 10(-4) M) is inhibited competitively by TRF analogues which contain proline or by the proline containing biologically active peptides luliberin (LH-RF), oxytocin, vasopressin, angiotensin II, and Substance P. TRF analogues without proline or peptide amides without proline are ineffective. This enzyme cleaves the appropriate Pro-X bonds in luliberin, angiotensin II, pyroGlu-His-Pro-Gly-NH2, and the collagenase substrate Z-Gly-Pro-Leu-Gly-Pro. Thus, it may be characterized as a post-proline-cleaving enzyme.

Amidohydrolases↗
PubMed1994

The modulation of enzyme reaction rates within multi-enzyme complexes. 2. Information transfer within a chloroplast multi-enzyme complex containing ribulose bisphosphate carboxylase-oxygenase.

Octameric ribulose bisphosphate carboxylase-oxygenase binds in an independent manner its substrate (ribulose bisphosphate) and a substrate analog (6-phosphogluconate). The eight active sites of the free enzyme are thus independent. The kinetic behaviour of the active site becomes different if ribulose bisphosphate carboxylase-oxygenase is inserted in the five-enzyme complex previously isolated from chloroplasts. Ribulose bisphosphate carboxylase-oxygenase then becomes more active than the corresponding free enzyme form. By comparing the behaviour of the same enzyme in the free state and in the associated state it then becomes possible to study how the thermodynamics of protein-protein interactions alters the kinetic behaviour of ribulose bisphosphate carboxylase-oxygenase. This alteration may be expressed in terms of stabilization-destabilization energies exerted upon the various intermediate states of the enzyme reaction, within the multi-protein complex. Heterologous interactions within this complex exert a constant stabilization energy on the enzyme ground states along the reaction co-ordinate of -1.68 kJ/mol and a constant stabilization energy of -3.79 kJ/mol on the enzyme transition states. These stabilization energies express how information propagates within the multi-enzyme complex as to increase the apparent affinity of the substrate for the active sites of ribulose bisphosphate carboxylase-oxygenase, as well as to increase the catalytic rate constant. The binding of the substrate analog 6-phosphogluconate to free ribulose bisphosphate carboxylase-oxygenase is non-co-operative. It becomes positively co-operative if this enzyme is inserted in the multi-protein complex. Under these conditions, only one type of enzyme-inhibitor complex is detected experimentally. Here again heterologous interactions stabilize this enzyme-inhibitor complex relative to that expected if ribulose bisphosphate carboxylase oxygenase is free. The extent of stabilization is -1.03 kJ/mol. Neither free nor associated ribulose bisphosphate carboxylase-oxygenase display any co-operativity relative to substrate binding. However, in the presence of the substrate analog 6-phosphogluconate, this enzyme displays positive co-operativity relative to substrate, although not if it is naked. These results can be explained theoretically and show that the maximum value of the Hill coefficient is < or = 2. As 6-phosphogluconate and other substrate analogs are present in chloroplasts under normal conditions, this co-operativity might be of functional importance in vivo.

Binding Sites↗
PubMed1978

An alternative hypothesis of cellular transport of lysosomal enzymes in fibroblasts. Effect of inhibitors of lysosomal enzyme endocytosis on intra- and extra-cellular lysosomal enzyme activities.

Recapture of lysosomal enzymes secreted by fibroblasts was inhibited by growing the cells in the presence of either free or immobilized antibodies against lysosomal enzymes or in the presence of phosphorylated carbohydrates known to interact with the cell-surface receptors for lysosomal enzymes. The following results were obtained. 1. Conditions that prevent recapture of released lysosomal enzymes increase the rate of extracellular accumulation of these enzymes up to twice that of controls. 2. Growing cells for 12 days in the presence of 0.5mm-mannose 6-phosphate, which decreases beta-N-acetylglucosaminidase endocytosis to less than 10% of that of controls, has no effect on the intracellular activity of this and four other lysosomal enzymes. 3. Growing cells for 4 days in the presence of 50mm-mannose 6-phosphate, which is a 1000-fold higher concentration than that required for 50% inhibition of lysosomal enzyme endocytosis, leads to a 4-fold increase in extracellular beta-N-acetylglucosaminidase accumulation and a decrease in intracellular enzyme. These results give evidence that, in fibroblasts, transfer of lysosomal enzymes into lysosomes does not require secretion before a receptor-mediated recapture [Hickman & Neufeld (1972) Biochem. Biophys. Res. Commun.49, 992-999]. We propose that (a) lysosomal enzymes are present in a receptor-bound form in those vesicles that fuse with the cell membrane, (b) the major part of the lysosomal enzyme cycles via the cell surface in a receptor-bound form and (c) only a minor part of the lysosomal enzyme is released into the extracellular space during its life cycle.

Acetylglucosaminidase↗
PubMed1994

The modulation of enzyme reaction rates within multi-enzyme complexes. 1. Statistical thermodynamics of information transfer through multi-enzyme complexes.

There is now experimental evidence that association of different enzymes as a multi-enzyme complex may result in an alteration of the catalytic properties of the enzymes present in this complex. This effect is not related to the channelling of reaction intermediates between different active sites. It appears as a consequence of an information transfer that occurs within the multi-enzyme complex. A theory, based on statistical thermodynamics, has been developed which provides an understanding, on a physical basis, for how isologous as well as heterologous interactions between identical, or different, enzymes of the complex may modulate the catalytic properties of an oligomeric enzyme of that complex. The theory predicts three possible types of effects: an alteration, through heterologous interactions, of an already existing co-operativity of the oligomeric enzyme within the complex; a co-operativity, generated by heterologous interactions in the complex that could not occur if the oligomeric enzyme were isolated from the rest of the complex; a Michaelis-Menten character of the oligomeric enzyme within the complex, but with altered values of Vm and Km relative to what would have been observed with the naked enzyme. All these effects appear as a consequence of a transfer of information between different enzymes of the same multi-protein complex. The following paper in this journal shows how one can demonstrate and characterize experimentally these effects in a multi-enzyme complex containing ribulose bisphosphate carboxylase-oxygenase.

Catalysis↗
PubMed1993

Kinetic behaviour of a repressible acid phosphatase from the yeast Yarrowia lipolytica: a comparative study between the solubilized enzyme, the enzyme bound to cell-wall fragments and the enzyme bound to intact cells.

(1) The substrate specificities and types of inhibitors of a repressible acid phosphatase from the yeast Yarrowia lipolytica as solubilized enzyme, enzyme bound to cell-wall fragments and enzyme bound to the intact cell were found to be essentially the same. (2) A similar pattern for the activation of the enzymatic activity by ionic strength was found for solubilized enzyme, the enzyme in cell-wall fragments and the enzyme in intact cells. (3) v[S] studies with all three locations of the enzyme revealed non-linear Eadie-Hofstee plots with concave-up curves of the negative cooperativity type; these were correctly fitted with a rate equation of 2:2 degree polynomial quotient. In all cases, the same behaviour was obtained and no new kinetic properties were observed when the enzyme was bound to the cell-wall matrix with respect to the solubilized enzyme. (4) Inhibition by phosphate was characterized for the three locations of the enzyme by v[I] and v[S] studies. The same pattern of partial inhibition and non-Michaelian inhibition of 'non-competitive' nature was observed for all three forms. (5) The above results are interpreted in terms of the hypothesis that the cell wall of Y. lipolytica has a slight negative charge but behaves as a permeable matrix that does not lead to novel characteristics regarding the catalytic and regulatory properties shown by the enzyme molecule in free solution.

Acid Phosphatase↗
PubMed1991

Enzymic synthesis of 1-O-(indol-3-ylacetyl)-beta-D-glucose. Purification of the enzyme from Zea mays, and preparation of antibodies to the enzyme.

The enzyme indol-3-ylacetylglucose synthase (UDP-glucose:indol-3-ylacetate beta-D-glucosyltransferase) catalyses the reaction: [formula: see text] This is the first step in the series of reactions leading to the indol-3-ylacetic acid conjugates found in maize. Previous attempts to purify this enzyme from the liquid endosperm of kernels of Zea mays (sweet corn) were not entirely successful owing to the lability of partially purified preparations during column chromatography. Thus this enzyme has not previously been purified to homogeneity. During the present study it was found that retention of enzyme activity required the combined presence of glycerol and dithiothreitol. Adding these requirements permitted purification of the enzyme to homogeneity with retention of catalytic activity. These purified preparations were used for preparation of rabbit polyclonal antibodies to the enzyme. Antibodies to the Zea mays endosperm enzyme cross-react with the enzyme from Zea mays vegetative tissues and with an enzyme from the liquid endosperm of oak acorns (Quercus sp). In this paper we report a simplified purification procedure adaptable to the preparation of milligram amounts of the enzyme.

Antibodies↗
PubMed2002

A new conceptual framework for enzyme catalysis. Hydrogen tunnelling coupled to enzyme dynamics in flavoprotein and quinoprotein enzymes.

Recent years have witnessed high levels of activity in identifying enzyme systems that catalyse H-transfer by quantum tunneling. Rather than being restricted to a small number of specific enzymes as perceived initially, it has now become an accepted mechanism for H-transfer in a growing number of enzymes. Furthermore, H-tunneling is driven by the thermally induced dynamics of the enzyme. In some of those enzymes that break stable C-H bonds the reaction proceeds purely by quantum tunneling, without the need to partially ascend the barrier. Enzymes studied that fall into this category include the flavoprotein and quinoprotein amine dehydrogenases, which have proved to be excellent model systems. These enzymes have enabled us to study the relationship between barrier shape and reaction kinetics. This has involved studies with "slow" and "fast" substrates and enzymes impaired by mutagenesis. A number of key questions now remain, including the nature of the coupling between protein dynamics and quantum tunneling. The wide-ranging implications of quantum tunneling introduce a paradigm shift in the conceptual framework for enzyme catalysis, inhibition and design.

Catalysis↗
PubMed2002

Possible roles of angiotensin II-forming enzymes, angiotensin converting enzyme and chymase-like enzyme, in the human aneurysmal aorta.

Aortic aneurysm is a chronic degenerative condition associated with atherosclerosis. Recent studies have revealed that angiotensin (Ang) II plays important roles in atherosclerosis. In this study, to investigate the relationship between aortic aneurysm and Ang II, we measured the activities of the angiotensin (Ang) II-forming enzymes, angiotensin converting enzyme (ACE) and chymase-like enzyme, in human aneurysmal and control aortae. Aneurysmal aortic specimens were obtained from 16 aneurysm patients and control aortic specimens were obtained from 16 patients who underwent coronary artery bypass surgery (8 patients in each group were administered ACE inhibitors). The ACE and chymase-like enzyme activities were determined using extracts from vascular tissues. Both the ACE and chymase-like enzyme activities in the aneurysmal aortae were significantly higher than those in the control aortae (p < 0.01). In the patients treated with ACE inhibitors, the ACE activity in the aneurysmal aortae tended to be low, but the chymase-like enzyme activity tended to be high. In the aneurysmal aortae, the chymase-like enzyme activity in the adventitia was significantly higher than that in the intimal or medial layers (p < 0.01), while differences in ACE activity were not observed. Our results suggest that increases in local Ang II formation induced by chymase-like enzymes may play important roles in the pathogenesis of aneurysmal formation.

Aged↗
PubMed1982

[Adsorption of cellulolytic enzymes on cellulose and the kinetics of the adsorbed enzymes. Two modes for interaction of the enzymes with the insoluble substrate].

The affinity of cellulolytic enzymes of various origin for microcrystalline cellulose (MCC) in a column type reactor has been studied. It was shown that there exist two types of the enzymes differing in their ability to bind to cellulose and to degrade MCC. One group of cellulases is characterized by weak affinity for MCC and by a rather low degree of conversion of this substrate into soluble products, but shows a burst of soluble products during the initial period of the hydrolysis. On the contrary, the other group of the enzymes irreversibly binds to cellulose and shows a noticeable ability to solubilize MCC. The results obtained suggest that the weak binding reflects a productive enzyme--cellulose interaction by the enzyme active center, like its interaction with soluble polymeric substrates. The tight binding, on the other hand, is less productive and reflects the interaction of peripheral parts of the enzymes with the cellulose surface. The hydrolytic action of the tightly adsorbed cellulases proceeds on the substrate surface consecutively, without leaving the insoluble substrate between the catalytic acts and the enzymes might steadily penetrate into the cellulose matrix. This in turn might induce mechanical fragmentation of the substrate. The rate of action of adsorbed cellulases can be limited by their diffusion along the surface or into the cellulose matrix. A decrease of the ionic strength weakens the cellulase affinity for cellulose, which in its turn leads to an increase in the initial burst of the soluble products of cellulose hydrolysis and a respective decrease of efficiency of cellulose conversion into glucose. It is concluded that the principal factor which determines the ability of cellulases to degrade crystalline cellulose is the affinity (i. e. degree of adsorption) of the enzymes for the insoluble substrate.

Cellulase↗
PubMed1992

The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--I. Purification and properties of a nitroreductase enzyme from Escherichia coli--a potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT).

A nitroreductase enzyme has been isolated from Escherichia coli B. This enzyme is an FMN-containing flavoprotein with a molecular mass of 24 kDa and requires either NADH or NADPH as a cofactor. Partial protein sequence analysis showed extensive homology with the "classical nitroreductase" of Salmonella typhimurium and a nitroreductase induced in Enterobacter cloacae. In common with the Salmonella enzyme, the E. coli B enzyme is capable of reducing nitrofurazone. The E. coli nitroreductase is also capable of reducing the anti-tumour agent CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], a property shared with the mammalian enzyme DT diaphorase [NAD(P)H dehydrogenase (quinone)] as isolated from Walker cells. The reduction of CB1954 by the E. coli enzyme results in the generation of cytotoxic species. Both enzymes also share the properties of being able to reduce quinones and are both inhibited by dicoumarol. The nitroreductase is a more active enzyme against CB1954 (kcat = 360 min-1) than Walker DT diaphorase (kcat = 4 min-1) and also has a lower Km for NADH (6 vs 75 microM).

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of enzymes hydrolyzing chymotrypsin synthetic substrate (Enzyme I) and trypsin synthetic substrate (Enzyme II) from the envelope of Capnocytophaga gingivalis.

Enzymes hydrolyzing chymotrypsin synthetic substrate and trypsin synthetic substrate, referred to as Enzyme I and Enzyme II, respectively, were found in the envelope fraction of Capnocytophaga gingivalis (ATCC 33624). Detergent extraction of both enzymes were purified by gel filtration, ion exchange chromatography, and affinity chromatography. The Enzyme I was a serine-containing metallo enzyme with a molecular mass of 77 kDa. The molecular mass of the Enzyme II was 83 kDa, and it was inhibited by tosyl-L-lysine chloromethyl ketone and leupeptin, and thus may be related to trypsin.

Amino Acid Sequence↗
PubMed1984

A cloned cDNA for duck malic enzyme detects abnormally large malic enzyme mRNAs in a strain of mice (Mod-1n) that does not express malic enzyme protein.

Sensitive immunochemical assays were used to measure the mass and rate of synthesis of malic enzyme protein in wild-type and Mod-1n mutant mice fed a high carbohydrate/low fat diet supplemented with thyroid hormone. Malic enzyme activity in the fed, wild-type mice was 100-fold higher than in starved, wild-type mice. Neither activity, mass, nor synthesis of malic enzyme could be detected in fed, mutant mice. However, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase responded to these dietary manipulations with normal or supranormal increases in activities, respectively, in mutant mice. A cDNA clone containing an almost complete copy of the mRNA for malic enzyme from duck liver was used to analyze poly(A+) RNA from C57BL/6J-DBA/2J hybrid mice that had been fasted and refed a high carbohydrate/low fat diet supplemented with thyroid hormone. The 32P-cDNA probe hybridized to two RNAs of 2250 and 2950 nucleotides. The same two RNAs were detected in RNA from starved mice except at much lower concentrations. A similar analysis of RNA from Mod-1n mice fed the high carbohydrate-thyroid diet also revealed two hybridizing RNAs but each was 700-800 nucleotides longer than its counterpart in wild-type mice. The abundance of malic enzyme mRNA in the fed, mutant mice was about the same as that in fed, wild-type mice. The mutant malic enzyme mRNAs also were present in RNA from starved mice but at much lower concentrations. These results suggest that the mutation responsible for the Mod-1n phenotype is in the structural gene for malic enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
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↗