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PubMed1985

Reduced 6,6,8-trimethylpterins. Preparation, properties and enzymic reactivities with dihydropteridine reductase, phenylalanine hydroxylase and tyrosine hydroxylase.

The substrates of dihydropteridine reductase (EC 1.6.99.7), quinonoid 7,8-dihydro(6 H)pterins, are unstable and decompose in various ways. In attempting to prepare a more stable substrate, 6,6,8-trimethyl-5,6,7,8-tetrahydro(3 H)pterin was synthesised and the quinonoid 6,6,8-trimethyl-7,8-dihydro(6 H)pterin derived from it is extremely stable with a half-life in 0.1 M Tris/HCl (pH 7.6, 25 degrees C) of 33 h. Quinonoid 6,6,8-trimethyl-7,8-dihydro(6 H)pterin is not a substrate for dihydropteridine reductase but it is reduced non-enzymically by NADH at a significant rate and it is a weak inhibitor of the enzyme: I50 200 microM, pH 7.6, 25 degrees C when using quinonoid 6-methyl-7,8-dihydro(6 H)pterin as substrate. 6,6,8-Trimethyl-5,6,7,8-tetrahydropterin is a cofactor for phenylalanine hydroxylase (EC 1.14.16.1) with an apparent Km of 0.33 mM, but no cofactor activity could be detected with tyrosine hydroxylase (EC 1.14.16.2). Its phenylalanine hydroxylase activity, together with the enhanced stability of quinonoid 6,6,8-trimethyl-7,8-dihydro(6 H)pterin, suggest that it may have potential for the treatment of variant forms of phenylketonuria.

Animals↗
PubMed1979

Subunits of RNA polymerase in function and structure. 8. Catalytic properties of self-reactivated core enzyme.

As an attempt to identify the maturation pathway of Escherichia coli RNA polymerase, the catalytic properties of core enzyme reactivated in the absence of maturation-promoting factors (sigma subunit or DNA) (that is, of self-reactivated core enzyme) were compared with those of native core enzyme. Differences have been found in the intrinsic activities such as in the template specificity, Km value of DNA template for the polymerase, activation energy for RNA synthesis, and increment of enzyme activity by sigma subunit. These observations imply that the transcription initiation by self-reactivated core enzyme is inaccurate and, therefore, more strict conditions including the presence of maturation-promoting factors are required for premature core be activated to the genuine function with the transcription specificity of native core enzyme.

DNA-Directed RNA Polymerases↗
PubMed1999

Hydrogen peroxide production from reactive liposomes encapsulating enzymes.

Reactive cationic and anionic liposomes have been prepared from mixtures of dimyristoylphosphatidylcholine (DMPC) and cholesterol incorporating dimethyldioctadecylammonium bromide and DMPC incorporating phosphatidylinositol, respectively. The liposomes were prepared by the vesicle extrusion technique and had the enzymes glucose oxidase (GO) encapsulated in combination with horseradish peroxidase (HRP) or lactoperoxidase (LPO). The generation of hydrogen peroxide from the liposomes in response to externally added D-glucose substrate was monitored using a Rank electrode system polarised to +650 mV, relative to a standard silver-silver chloride electrode. The effects of encapsulated enzyme concentration, enzyme combinations (GO+HRP, GO+LPO), substrate concentration, electron donor and temperature on the production of hydrogen peroxide have been investigated. The electrode signal (peroxide production) was found to increase linearly with GO incorporation, was reduced on addition of HRP and an electron donor (o-dianisidine) and showed a maximum at the lipid chain-melting temperature from the anionic liposomes containing no cholesterol. To aid interpretation of the results, the permeability of the non-reactive substrate (methyl glucoside) across the bilayer membranes was measured. It was found that the encapsulation of the enzymes effected the permeability coefficients of methyl glucoside, increasing them in the case of anionic liposomes and decreasing them in the case of cationic liposomes. These observations are discussed in terms of enzyme bilayer interactions.

Cholesterol↗
PubMed2005

Dioxygen activation by copper, heme and non-heme iron enzymes: comparison of electronic structures and reactivities.

Enzymes containing heme, non-heme iron and copper active sites play important roles in the activation of dioxygen for substrate oxidation. One key reaction step is CH bond cleavage through H-atom abstraction. On the basis of the ligand environment and the redox properties of the metal, these enzymes employ different methods of dioxygen activation. Heme enzymes are able to stabilize the very reactive iron(IV)-oxo porphyrin-radical intermediate. This is generally not accessible for non-heme iron systems, which can instead use low-spin ferric-hydroperoxo and iron(IV)-oxo species as reactive oxidants. Copper enzymes employ still a different strategy and achieve H-atom abstraction potentially through a superoxo intermediate. This review compares and contrasts the electronic structures and reactivities of these various oxygen intermediates.

Binding Sites↗
PubMed1997

Denaturation of uridine phosphorylase from Escherichia coli K-12 with guanidine hydrochloride: kinetics of inactivation, dissociation, and reactivation of the enzyme.

Denaturation of uridine phosphorylase from Escherichia coli K-12 by guanidine hydrochloride is accompanied by the displacement of the maximum in the protein fluorescence spectrum (lambda max) from 331 to 348 nm. The half-maximal change in the lambda max position is observed at 1.18 M guanidine hydrochloride. For this concentration of denaturant, the sedimentation pattern consists of two boundaries, one of which corresponds to the motion of the hexameric enzyme form (s20,w = 8.2 S) and other represents a monomer (s20,w = 2.6 S). In the presence of 2 M guanidine hydrochloride the enzyme moves as a monomer. The kinetics of inactivation of uridine phosphorylase by guanidine hydrochloride are complex (minima and maxima are observed on the kinetic curves). The initial rate of the enzyme reactivation after dilution of the enzyme preincubated with guanidine hydrochloride is second order with respect to protein. It is assumed that the rate of the reactivation process is limited by the reassociation of low-activity monomers into dimers followed by a rapid hexamer formation. The second-order rate constant for the reassociation of the enzyme is 3.0.10(4) M-1.sec-1 (50 mM borate buffer, pH 7.7, containing 100 mM inorganic phosphate; 20 degrees C). Thiol groups become accessible to titration by 5,5'-dithiobis-(2-nitrobenzoic acid) after treatment of uridine phosphorylase with guanidine hydrochloride. Uridine and uracil inhibit the unfolding of the protein globule by guanidine hydrochloride.

Enzyme Reactivators↗
PubMed1974

Chick-erythrocyte nucleus reactivation in heterokaryons: suppression by inhibitors of proteolytic enzymes.

Reactivation of chick-erythrocyte nuclei in heterokaryons (obtained by Sendai virus-induced fusion of chick erythrocytes with HeLa cells) is suppressed by specific inhibitors of trypsin and trypsin-like enzymes. N-alpha-tosyl-L-lysyl-chloromethane and N-alpha-tosyl-L-arginine methylester inhibit erythrocyte nuclear enlargement and suppress RNA and DNA synthesis in nuclei of erythrocytes and HeLa cells in heterokaryons at concentrations that only minimally influence individual HeLa cells or HeLa homokaryons. Although other unknown mechanisms of action cannot be formally excluded, the data are interpreted as fitting best with an intracellular site of action of the protease inhibitors studied, and as suggesting a role for cellular proteases in reactivation of chick-erythrocyte nuclei in heterokaryons.

Animals↗
PubMed1986

Comparisons of antibody reactivity and enzyme sensitivity between small proteoglycans from bovine tendon, bone, and cartilage.

Preparations of small proteoglycans from bovine tendon, bone, and cartilage have been compared for sensitivity to various enzymes and reactivity with different polyclonal antibodies. Chondroitinase ABC digestion of all proteoglycans generated a core protein preparation that migrated similarly in sodium dodecyl sulfate-polyacrylamide electrophoresis as a doublet band with Mr approximately equal to 45,000. The small proteoglycans of cartilage were divided into two populations based upon electrophoretic migration of the intact molecules (Rosenberg, L. C., Choi, H. U., Tank, L-H., Johnson, T. L., Pal, S., Webber, C., Reiner, A., and Poole, A. R. (1985) J. Biol. Chem. 260, 6304-6313). The core preparations of tendon, bone, and the faster-migrating (PG II) proteoglycans of cartilage all interacted in Western blot/enzyme-linked immunosorbent assay analysis with polyclonal antibody raised against either the tendon or bone proteoglycans. The slower-migrating (PG I) proteoglycans of cartilage did not react with these antibodies. Digestion of the tendon small proteoglycan with Staphylococcus aureus V8 protease released glycosaminoglycan chains from the molecule and generated a 40-kDa protein fragment that was resistant to further rapid degradation by the enzyme. This large digestion fragment was also prominent following V8 protease digestion of the faster-migrating (PG II) population of small cartilage proteoglycans, but not the small proteoglycan of bone. The N-terminal amino acid sequence of the tendon (PG II) proteoglycan was determined. These observations provide additional evidence for heterogeneity among the chemically similar small proteoglycans from different tissues.

Amino Acid Sequence↗
PubMed1982

Ultrastructural study of enzymes in reactive astrocytes: clarification of astrocytic activity.

Enzymes in reactive of the cerebral cortex were examined at the ultrastructural level in an attempt to resolve some conflicting aspects of astrocytic activity. Correlations between morphological and enzyme changes after injury established that the apparent increase in oxidative enzyme activity was exclusively mitochondrial and not an artefactual reaction product resulting from anoxic cellular damage. Pronounced glucose-6-phosphatase activity within cisternae of an increased amount of the granular endoplasmic reticulum was related to increased glycogen. Further evidence from acid phosphatase activity indicated that astrocytes played a minimal role in phagocytosis.

Acid Phosphatase↗
PubMed1994

Donor follow-up of influenza vaccine-related multiple viral enzyme immunoassay reactivity.

False-positive enzyme immunoassay (EIA) tests in blood donors receiving influenza vaccine were first reported in 1991. We conducted follow-up testing for 6 months of those donors with multiply reactive, but unconfirmed EIA (at least 2 positives in anti-HCV-1.0, anti-HIV-1, and anti-HTLV-I assays) with a history of recent flu vaccine to determine the duration of false positivity. Of 133,000 donors tested, 16 met study criteria; all 16 were reactive for anti-HCV, 10 were reactive for anti-HIV-1, and 12 were reactive for anti-HTLV-I. Fifteen donors were available for follow-up testing (using the original screening and supplemental tests): 10 (67%) reverted to negative for the 3 tests and 5 remained false positive for various markers at last sampling (3-6 months after vaccination). The mean duration of false positivity for those reverting to negative EIA test status, was 4.2 months (range 2-7 months) indicating a transient phenomenon and supporting studies which suggest a role for IgM in the mechanism.

Adolescent↗
PubMed1987

Species differences in enzymes controlling reactive epoxides.

Activities of enzymes involved in the metabolic formation and catabolism of epoxides were determined in liver subcellular preparations from 11 mammalian species and various strains of mice. The most conspicuous finding was that the activities of the microsomal epoxide hydrolase were clearly lower in the mouse than in the other species. This invited the working hypothesis that epoxides may be involved in mouse liver carcinogenesis. The carcinogens may be metabolised themselves to reactive epoxides or they may modify the metabolism of epoxides formed from endogenous or other foreign compounds. To examine the former point, phenobarbital, DDT (1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane), lindane and benzo(a)pyrene were investigated for mutagenicity in Salmonella typhimurium using as the carcinogen-metabolising system subcellular liver preparations from animals in which these compounds efficiently induce liver tumours and from resistant animals. Phenobarbital, DDT and lindane were not mutagenic under any conditions, including those where microsomal epoxide hydrolase was also inhibited. However, a DDT metabolite, 1,1-bis(p-chlorophenyl)-2,2-dichloroethane was mutagenic in strain TA98, when norharman was added to the metabolising system, rat liver postmitochondrial fraction. Benzo(a)pyrene, which efficiently induces liver tumours in male but not in female newborn C3HeB/FeJ X A/J mice, was similarly activated by liver preparations from male and female animals. This was true with and without pretreatment of the mice with an inducer of cytochrome P-448. Also, activities and inducibilities of monooxygenase, epoxide hydrolase and glutathione transferase (toward benzo(a)pyrene and benzo(a)pyrene 4,5-oxide, respectively) were indistinguishable between males and females. Therefore, differences in the metabolism of benzo(a)pyrene do not appear to be the reason for the sex difference in tumour susceptibility. Likewise, mouse strains with high and low frequencies of spontaneous and chemically-induced liver tumours did not appreciably differ in their hepatic microsomal epoxide hydrolase activities. The low level of this activity therefore cannot constitute the critical factor for the high tumour susceptibility of certain strains of mice. However the statement does not preclude potentiation of the susceptibility toward particular carcinogens owing to this metabolic trait of the mouse.

Animals↗
PubMed1978

An immunological determination of specific activities of enzymes: its use in quantification of cross reactivity between enzymes of different origins.

An immunological method is presented which enables the determination of the specific activity of a pure enzyme without its extensive purification. The method consists essentially in the specific fixation of immunologically related enzymes to an immunoadsorbent containing specific antibodies raised against the wild-type form of the enzyme. We applied this method to determine the specific activity of plasmid-coded beta-galactosidases and to quantify the extent of cross-reaction between these enzymes and Escherichia coli beta-galactosidase.

Animals↗