4-Dimethylaminoazobenzenes: carcinogenicities and reductive cleavage by microsomal azo reductase

Twenty-four 4-dimethylaminoazobenzenes (DABs) in which systematic structural modifications have been made in the prime ring have been studied for substrate specificity for microsomal azo reductase. The DABs were also evaluated for carcinogenicity and it was found that there was no correlation between carcinogenicity and extent of azo bond cleavage by azo reductase. While any substituent in the prime ring reduces the rate of cleavage of the azo bond relative to the unsubstituted dye, there is a correlation between substituent size and susceptibility to the enzyme. Substituent size was also found to be a significant factor in the induction of hepatomas by the dyes. Preliminary studies have shown that there appears to be a positive correlation between microsomal riboflavin content and the activity of the azo reductase.     

Partial purification and characterization of a bacterial enzyme catalyzing reductive cleavage of anthracycline glycosides.

A bacterial enzyme catalyzing the NADH-dependent reductive cleavage of certain anthracycline glycosides has been partially purified. The enzyme is acidic, stable in solution and has an estimated molecular weight of 35,000. The enzyme activity is strongly inhibited by molecular oxygen but not by cyanide or EDTA. No evidence has been found for an enzyme system or associated elements of electron transport.     

Oxidative and reductive cleavage of folates--a critical appraisal.

A critical analysis has been made of the oxidative and reductive techniques employedfor cleavage of the C⁹-N¹⁰ bond of folic acid and its derivaatives. The assumption has previously been made that these cleavage reactions reduce folates to a common family of p-aminobenzoylglutamate derivatives varying only in the lengths of γ-polyglutamyl peptide side chains which are readily subjected to quantitative and qualitative analysis. This assumption is incorrect. Oxidation by potassium permanganate effectively cleaved folic acid, dihydrofolic acid, tetrahydrofolic acid, and 5-formyltetrahydrofolic acid to yield p-aminobenzoylglutamate. 5-Methyltetrahydrofolic acid was merely oxidized to 5-methyldihydrofolic acid while 5,10-methenyltetrahydrofolic acid and 10-formyltetrahydrofolic acid were oxidized to 10-formylfolate which was stable to further attack. Of all the folate derivatives tested only folic acid and dihydrofolic acid were cleaved to p-aminobenzoylglutamate by the zinc-hydrochloric acid reduction method. Both tetrahydrofolic acid and 5-methyltetrahydrofolic acid were stable under fully reducing conditions. 5,10-Methenyl-,10-formyl-, and 5-formyltetrahydrofolic acid yielded N-methyl-p-aminobenzoylglutamate. It is evident, therefore, that not only is the dominant mammalian tissue folate derivative, 5-methyltetrahydrofolate, resistant to cleavage by either method, but that a common family of p-aminobenzoylglutamate derivatives is not the end product of those folate compounds that are susceptible. While this may not invalidate the reports of the relative polyglutamate chain lengths of tissue folates such data should be regarded with some caution.     

The role of glandular kallikrein in the formation of a salivary proline-rich protein A by cleavage of a single bond in salivary protein C.

An enzyme was purified from human parotid saliva that can cleave a single arginine-glycine peptide bond between residues 106 and 107 in human salivary proline-rich protein C, hereby giving rise to another proline-rich protein A, which is also found in saliva. The enzyme was purified 2400-fold. It cleaved salivary protein C at the rate of 59 micrograms of protein/h per microgram of enzyme and had amino acid composition, molecular weight and inhibition characteristics similar to those reported for human salivary kallikrein. Confirmation that the enzyme was kallikrein was demonstrated by its kinin-generating ability. Histochemical evidence indicates that a post-synthetic cleavage of protein C by kallikrein would have to take place during passage of saliva through the secretory ducts. In secreted saliva, cleavage of salivary protein C can only be observed after 72 h incubation. In addition, there is no effect of salivary flow rate on the relative amounts of proteins A and C in saliva. On the basis of the experimental observations, it is proposed that in vivo it is unlikely that kallikrein secreted from ductal cells plays a significant role in converting protein C into protein A.     

Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly

The hepatitis C virus (HCV) is a flavivirus replicating in the cytoplasm of infected cells. The HCV genome is a single-stranded RNA encoding a polyprotein that is cleaved by cellular and viral proteases into 10 different products. While the structural proteins core protein, envelope protein 1 (E1) and E2 build up the virus particle, most nonstructural (NS) proteins are required for RNA replication. One of the least studied proteins is NS2, which is composed of a C-terminal cytosolic protease domain and a highly hydrophobic N-terminal domain. It is assumed that the latter is composed of three trans-membrane segments (TMS) that tightly attach NS2 to intracellular membranes. Taking advantage of a system to study HCV assembly in a hepatoma cell line, in this study we performed a detailed characterization of NS2 with respect to its role for virus particle assembly. In agreement with an earlier report ( Jones, C. T., Murray, C. L., Eastman, D. K., Tassello, J., and Rice, C. M. (2007) J. Virol. 81, 8374-8383 ), we demonstrate that the protease domain, but not its enzymatic activity, is required for infectious virus production. We also show that serine residue 168 in NS2, implicated in the phosphorylation and stability of this protein, is dispensable for virion formation. In addition, we determined the NMR structure of the first TMS of NS2 and show that the N-terminal segment (amino acids 3-11) forms a putative flexible helical element connected to a stable alpha-helix (amino acids 12-21) that includes an absolutely conserved helix side in genotype 1b. By using this structure as well as the amino acid conservation as a guide for a functional study, we determined the contribution of individual amino acid residues in TMS1 for HCV assembly. We identified several residues that are critical for virion formation, most notably a central glycine residue at position 10 of TMS1. Finally, we demonstrate that mutations in NS2 blocking HCV assembly can be rescued by trans-complementation.     

A new catalyst for reductive cleavage of methylated glycans

Several per-O-methylated D-glucans and D-fructans were used as models in an attempt to identify new catalysts for carrying out reductive cleavage. Included in these model studies were several D-glucans that contained 4-linked D-glucopyranosyl residues as well as one having a 4-linked D-glucitol residue, as both types of residue had previously been found to give rise to substantial proportions of artifactual products. These studies led to the development of a new catalyst for carrying out reductive cleavage, namely, a mixture of 5 equivalents of trimethylsilyl methanesulfonate (Me3SiOSO2Me) and 1 equivalent of boron trifluoride etherate (BF3 . Et2O) per equivalent of acetal. This new catalyst was found to accomplish the reductive cleavage of per-O-methylated, 4-linked D-glucopyranosyl residues and 4-linked D-glucitol residues, to give only the expected derivatives of 1,5-anhydro-D-glucitol and D-glucitol, respectively. The mixture of Me3SiOSO2Me and BF3 . Et2O also catalyzed reductive cleavage of the D-fructofuranosyl residues of per-O-methylated sucrose and inulin, to give only the expected derivatives of 2,5-anhydro-D-mannitol and 2,5-anhydro-D-glucitol. Indeed, when used alone, Me3SiOSO2Me also rapidly catalyzed the reductive cleavage of D-fructofuranosyl residues, but, under the same conditions, D-glucopyranosyl residues were unaffected. The results of these and other model studies demonstrated that catalysis of reductive cleavage by the mixture of Me3SiOSO2Me and BF3 . Et2O occurs in a synergistic manner. Examination of the mixture of Me3SiOSO2Me and BF3 . Et2O by 1H-n.m.r. spectroscopy demonstrated that a reaction occurs to generate trimethylsily fluoride and species of the type F2BOSO2Me, FB(OSO2Me)2, or B(OSO2Me)3 via ligand exchange.     

Hydrogen carrier protein from chicken liver: purification, characterization, and role of its prosthetic group, lipolic acid, in the glycine cleavage reaction.

Hydrogen carrier protein (H-protein), a component of the glycine cleavage system, has been purified to homogeneity from chicken liver mitochondria. The molecular weight and the partial specific volume determined by two different methods were 14,500 and 0.724 ml/g, respectively. The protein has an isoelectric point of 4.0. Amino acid analysis revealed 131 residues, about one-third of which are acidic residues. Evidence is presented indicating that the protein contains one lipoic acid moiety per molecule. In the decarboxylation of glycine the disulfide of the lipoyl moiety is cleaved and one of the resultant sulf-hydryl groups receives an intermediate derived from glycine.     

Escherichia coli nitrate reductase subunit A: its role as the catalytic site and evidence for its modification.

Subunits A and B were isolated from purified nitrate reductase by preparative electrophoresis in low levels of sodium dodecyl sulfate. Nonheme iron and low levels of molybdenum were associated with isolated subunit A but not with isolated subunit B. After dialysis against a source of molybdenum cofactor, subunit A regained tightly bound molybdenum and concomitantly regained enzyme activity and reactivity with anti-nitrate reductase antiserum. Subunit B neither bound cofactor nor regained activity or reactivity with antiserum. These data indicate that subunit A contains the active site of the enzyme. Subunit A was also found to be modified posttranslationally in a similar fashion as is subunit B. This was determined by comparison of partial proteolytic digests and amino acid analyses of A subunits from precursor and membrane-bound forms of nitrate reductase.     

Kinetic analysis of the individual reductive steps catalyzed by beta-hydroxy-beta-methylglutaryl-coenzyme A reductase obtained from yeast.

The mechanism of action of yeast beta-hydroxy-beta-methylglutaryl-coenzyme A reductase has been investigated through kinetic studies on the oxidation of mevaldate by nicotinamide adeninine dinucleotide phosphate (NADP) in the presence of coenzyme A (CoA) and on the reduction of mevaldate by reduced NADP (NADPH) in the absence of presence of CoA or acetyl-CoA. NADP and mevalonate were also used as product inhibitors of the reduction of mevaldate. In the reduction of mevaldate to mevalonate, coenzyme A and acetyl-CoA decreased the Km for mevaldate 30- and 3-fold, respectively. Both compounds increased the Vmax 1.5-fold. These results suggest that CoA is an allosteric activator for the second reductive step and that it acts by enhancing the binding of mevaldate. The intersecting patterns obtained from initial velocities and the patterns produced by product inhibitions suggest the following features of the mechanism. The binding of substrates and release of products proceeds sequentially in both reductive steps, and is ordered throughout or random with respect to the binding of the beta-hydroxy-beta-methylglutaryl-coenzymeA and the first NADPH. The binding of NADPH enhances the binding of the beta-hydroxy-beta-methylglutaryl portion of the CoA ester and the binding of free mevaldate, whereas the binding of NADP leads to an increased affinity of the enzyme for the hemithioacetal (of mevaldate and CoA) and for mevalonate. Thus, the replacement of NADP by NADPH after the first reductive step promotes the conversion of the hemithioacetal to the free carbonyl form, which is then rapidly reduced. The products, CoA and mevalonic acid, of the second reductive step leave the enzyme before the release of the second NADP. This release of the last product is probably the rate-limiting step for the overall process.     

Purification of protein components of the clostridial glycine reductase system and characterization of protein A as a selenoprotein

A procedure for the isolation in nearly homogeneous form of protein A, a low molecular-weight, acidic, protein component of clostridial glycine reductase, is described. The yield of protein A is high only in early log phase cells of Clostridium sticklandii grown under standard laboratory conditions in a rich tryptone-yeast extract-distilled water medium but, when selenite (1 μm) is added, the levels of protein A remain high throughout the entire log phase of growth. Addition of ⁷⁵Se-labeled selenite to the culture medium results in the highly selective incorporation of radioactive selenium into protein A. The procedure for isolation of protein A results in about a 700-fold enrichment when extracts prepared from cells that actively catalyze glycine reduction are used. However, the catalytic activity of the purified protein varies considerably from preparation to preparation. The molecular weight of protein A, estimated by sucrose density-gradient centrifugation, is approximately 12,000.The other higher molecular-weight components of glycine reductase are associated with the membrane fraction of the cell and are released as soluble proteins by sonic disruption of the membrane. After purification by ion-exchange and molecular sieve chromatography, these components are separated by DEAE-cellulose chromatography into two protein fractions both necessary for glycine reductase activity in protein A-supplemented assays. One of these fractions consists of a major protein component, protein B, also nearly homogeneous as determined by polyacrylamide gel electrophoresis. The other protein fraction still is heterogeneous.     

Purification and characterization of an enzymically active cleavage product of pig kidney aldehyde reductase

During the purification of pig kidney aldehyde reductase by an established procedure [Flynn, Cromlish & Davidson (1982) Methods Enzymol. 89, 501-506] a second enzyme with aldehyde reductase activity may be purified. When the procedure was performed in the presence of 5 mM-EDTA, only traces of the second reductase, pig kidney aldehyde reductase (minor form), were present. By the criterion of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, pig kidney aldehyde reductase (minor form) had Mr 35 000, in comparison with Mr 40 200 found for pig kidney aldehyde reductase. Amino acid analysis of both enzymes and tryptic-peptide-map comparisons indicated differences in primary structure. The N-terminus of pig kidney aldehyde reductase (minor form) had the sequence Lys-Val-Leu, in contrast with the blocked (acetylated) N-terminus of pig kidney aldehyde reductase. The C-terminal sequence of both enzymes was the same. Both reductases were immunologically identical by double immunodiffusion and rocket immunoelectrophoresis. Pig kidney aldehyde reductase (minor form) had 50% of the specific activity of pig kidney aldehyde reductase when tested with a variety of aldehyde substrates. Michaelis constants of both enzymes for these substrates and for NADPH were similar, but values for kcat. and kcat./Km indicated that catalytically pig kidney aldehyde reductase was the more efficient enzyme. Typical aldehyde reductase inhibitors, such as phenobarbital and sodium valproate, had the same effect on both enzymes. It was concluded that pig kidney aldehyde reductase (minor form) is an enzymically active cleavage product of pig kidney aldehyde reductase which is formed when the latter is purified in the absence of the metalloproteinase inhibitor EDTA.     

Covalent molecular imprinting of bisphenol A using its diesters followed by the reductive cleavage with LiAlH(4)

Bisphenol A (BPA) recognition materials were synthesized by a covalent imprinting technique using BPA-dimethacrylate or BPA-diacrylate as the template monomer. Binding sites in the polymers consisted of two hydroxyl groups that are generated by reducing the ester bonds of the template monomer with lithium aluminum hydride. The polymers strongly adsorbed BPA and structurally related compounds, however, other endocrine disruptors were hardly adsorbed. The BPA-dimethacrylate-based polymer interacted with the samples more strongly than the BPA-diacrylate-based polymer.     

Regulation of cleavage by protein kinase C in Chaetopterus

We report that protein kinase C (PKC) plays a regulatory role in early cleavage in Chaetopterus eggs. Using Western blotting, we assayed the expression patterns of conventional PKCs (cPKC), novel PKCs (nPKC), and atypical PKCs (aPKC). During early development after fertilization, PKC protein levels varied independently by isoform. PKC protein expression during differentiation, without cleavage and after parthenogenetic activation, was very similar to that during normal development indicating that PKC gene expression does not require cellularization. Since PKC has been shown to regulate meiosis in this organism, we also assayed the membrane association of these isoforms as an indicator of their activation during meiosis and early cleavage. PKC-gamma transiently associated with membranes and therefore became activated before meiotic division and cleavage, whereas PKC-alpha and -beta transiently dissociated from membranes and therefore became inactivated at these times. Inhibition of these PKC isoforms by bisindolylmaleimide I had no effect on cleavage or early development to the trochophore larva, indicating that PKC-gamma activation is not essential for cleavage or early development. However, their persistent activation by thymeleatoxin blocked cleavage. The results indicate that the dissociation of PKC-alpha and/or -beta from the membrane fraction, and therefore their inactivation, is essential for normal cleavage. Elevated PKC activity is essential for nuclear envelope breakdown and spindle formation at meiosis I. By contrast, down-regulation of this activity is essential for cleavage after fertilization.     

Glycine metabolism by rat liver mitochondria. Reconstruction of the reversible glycine cleavage system with partially purified protein components

An enzyme system which catalyzes the degradation of glycine to one carbon unit, ammonia, and carbon dioxide and the synthesis of glycine from these three substances has been isolated from rat liver mitochondria. The reversible glycine cleavage system is composed of four protein components named as P-, H-, L-, and T-protein, respectively. A procedure is described for the purification of P-protein which catalyzes the decarboxylation of glycine or its reverse reaction in the presence of H-protein, and for T-protein which participates in the formation of one carbon unit and ammonia or the reverse reaction. The procedure described leads to the isolation of a nearly homogeneous form of T-protein but P-protein still is heterogeneous. The molecular weight of T-protein, estimated by molecular sieve chromatography, is 33,000. Properties of the synthesis and cleavage reactions and the exchange of carboxyl group of glycine with bicarbonate are also presented.