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J M Kittler

Publications and source records attributed to J M Kittler.

9 recordsLinked to original sources
PubMed1993

Interaction of non-myristoylated NADH-cytochrome b5 reductase with cytochrome b5-dimyristoylphosphatidylcholine vesicles.

An expression vector for NADH-cytochrome b5 reductase containing a thrombin cleavage site directly before the N-terminal glycine residue of the flavoprotein was used to isolate the non-myristoylated enzyme by thrombin cleavage of the initial fusion protein of a short segment of the multiple cloning site of the plasmid vector and the reductase. This flavoprotein preparation, containing only the 28-residue N-terminal peptide segment of the membrane-binding domain of the mammalian enzyme, binds to phospholipid vesicles and interacts with membrane-bound cytochrome b5. The effect of N-myristoylation of the enzyme therefore appears to be limited to facilitating and stabilizing interactions with phospholipid vesicles. However, the relatively short intervening peptide sequence that separates the crucial peptide membrane-binding domain from lysine 41, which has been implicated in the active-site interaction with cytochrome b5 (Strittmatter, P., Kittler, J. M., Coghill, J. E., and Ozols, J. (1992) J. Biol. Chem. 267, 2519-2523), provides some limitation of the distance from the membrane surface for the interactions required for rapid electron transfer from the flavin of the reductase to the heme of cytochrome b5.

Base Sequence↗
PubMed1992

Characterization of the role of lysine 110 of NADH-cytochrome b5 reductase in the binding and oxidation of NADH by site-directed mutagenesis.

An expression vector for bovine NADH-cytochrome b5 reductase was used for site-directed mutagenesis of lysine 110, the residue previously implicated in NADH interactions with this flavoprotein. Replacement of this basic residue with an uncharged glutamine resulted in an increase of 3 orders of magnitude in the Km for NADH and a decrease in kcat of an order of magnitude, strongly implicating lysine 110 in both binding of NADH to the reductase and the orientation of the reduced nicotinamide group for rapid hydride ion transfer to the flavin. Substitution of lysine 110 by histidine, to provide a pH-sensitive positive charge at this position in the neutral pH range, exhibited only a moderate 25-fold increase in Km and a normal kcat at pH 6.0, whereas at pH 8.5 the Km for NADH rose to 238 microM with a decrease of 45% over unmodified enzyme in the kcat. A similar pH sensitivity in the inhibition constant for adenosine diphosphate ribose, lacking only the nicotinamide moiety of NADH, emphasizes the crucial role of the positive charge at this locus and is consistent with charge-pairing of lysine 110 with the pyrophosphate group of NADH or adenosine diphosphate ribose.

Adenosine Diphosphate Ribose↗
PubMed1992

Characterization of lysyl residues of NADH-cytochrome b5 reductase implicated in charge-pairing with active-site carboxyl residues of cytochrome b5 by site-directed mutagenesis of an expression vector for the flavoprotein.

An expression vector for bovine NADH-cytochrome b5 reductase was constructed from two DNA fragments that were derived from beef liver poly(A+) RNA using the polymerase chain reaction. Site-directed mutagenesis of the 3 lysine residues of the reductase, previously implicated in the formation of active-site charge pairs with carboxylate residues of cytochrome b5, was then used to obtain the purified catalytic domains of flavoproteins modified at each of these sites. The observed marked decreases in catalytic efficiencies of substitutions of a negative charge at the normally positively charged residues with the catalytic domain of cytochrome b5 are consistent with their participation in the formation of charge pairs with carboxylate groups of the hemeprotein to optimize rapid electron transfer from the reductase flavin to the heme of the cytochrome.

Amino Acid Sequence↗
PubMed1986

Reactivity of a functional carbonyl moiety in bovine aortic lysyl oxidase. Evidence against pyridoxal 5'-phosphate.

Previous studies have pointed towards a cofactor role for pyridoxal 5'-phosphate (PLP) in lysyl oxidase, the enzyme that generates the peptidyl aldehyde precursor to the lysine-derived cross-linkages in elastin and collagen. The nature of a carbonyl moiety in purified bovine aortic lysyl oxidase was explored in the present study. A PLP dinitrophenylhydrazone could not be isolated from lysyl oxidase, although corresponding preparations of aspartate aminotransferase, a PLP-dependent enzyme, yielded this derivative, as revealed by h.p.l.c. Analysis of lysyl oxidase for PLP after reduction of the enzyme by NaBH4, a procedure that converts PLP-protein aldimines into stable 5'-phosphopyridoxyl functions, also proved negative in tests using monoclonal antibody specific for this epitope. Lysyl oxidase was competitively inhibited by phenylhydrazine, and inhibition became irreversible with time at 37 degrees C, displaying a first-order inactivation rate constant of 0.4 min-1 and KI of 1 microM. [14C]Phenylhydrazine was covalently incorporated into the enzyme in a manner that was prevented by prior modification of the enzyme with beta-aminopropionitrile, a specific active-site inhibitor, and which correlated with functional active-site content. The chemical stability of the enzyme-bound phenylhydrazine exceeded that expected of linkages between PLP and proteins. The absorption spectrum of the phenylhydrazine derivative of lysyl oxidase was clearly distinct from that of the phenylhydrazone of PLP. It is concluded that lysyl oxidase contains a carbonyl cofactor that is not identical with PLP and that is bound to the enzyme by a stable chemical bond.

Amino Acid Oxidoreductases↗
PubMed1986

Azo dye-induced alterations in vitamin B-6 metabolism and in pyridoxal 5'-phosphate-binding proteins in rat liver.

The effects of the hepatocarcinogen, 3'-methyl-4-dimethylamino-azobenzene, on vitamin B-6 metabolism in rat liver were studied. The following parameters were measured: pyridoxal 5'-phosphate (PLP) concentrations in plasma, brain, liver and azo dye-induced hepatomas, as well as the activities of pyridoxine (PN) kinase, pyridoxine 5'-phosphate (PNP) oxidase, PNP phosphatase and PLP-dependent ornithine decarboxylase. Hepatomas more closely resembled fetal than normal adult rat liver with respect to their ability to convert vitamer forms such as PN to coenzymatically active PLP. Microtiter plate enzyme-linked immunosorbent analyses revealed that the absence of PNP oxidase activity in a dissectable hepatoma was attributable to the absence of enzyme protein. In addition, monoclonal antibodies to vitamin B-6 were used in a Western immunoblot technique to examine the effects of azo dye ingestion on the pattern of PLP-binding proteins in cytosolic extracts of liver and hepatomas. Nitrocellulose blots of electrophoretically resolved cytosolic extracts probed for PLP-binding proteins showed increasing complexity with development; hepatomas bore a striking resemblance to fetal liver. The data indicate that hepatomas lose the properties of terminally differentiated hepatic tissue and take on the properties of fetal hepatic tissue characterized by lower concentrations of PLP, selective use of the coenzyme, and a lowered-to-absent capability to convert precursor vitamer forms to PLP. Therefore, with respect to vitamin B-6 metabolism and use, it appears likely that azo dye-induced hepatocarcinogenesis involves proliferation of a stem cell type(s) having the phenotypic characteristics of fetal hepatic tissue.

Animals↗
PubMed1984

A general immunochemical method for detecting proteins on blots.

Following horizontal electroelution, or blotting, of proteins from polyacrylamide gels to immobilizing matrices, such as nitrocellulose or Zeta-bind paper, the transferred proteins can be derivatized in situ with pyridoxal 5'-phosphate and sodium borohydride. After a quenching step to eliminate nonspecific binding of antibody to the protein-binding matrix, the blot is incubated with a solution containing a mouse monoclonal antibody specific for the 5'-phosphopyridoxyl group. The transferred proteins can then be located on the blot with second antibody staining procedures employing either a peroxidase-linked goat anti-mouse F(ab')2 antibody or a peroxidase-linked avidin/biotin system. The solid-phase enzyme-linked immunosorbent assay method described in this report is a mild, general, and sensitive immunochemical method for the detection of proteins on protein-binding matrices.

Animals↗
PubMed1983

Immunoblot detection of pyridoxal phosphate binding proteins in liver and hepatoma cytosolic extracts.

A monoclonal antibody, highly selective for the 5'-phosphopyridoxyl group, can be used to detect cytosolic pyridoxal-5'-phosphate binding proteins by an immunoblot procedure. This technique, when applied to sodium borohydride-treated cytosolic extracts obtained from normal rat liver at various stages of development as well as several liver-derived Morris hepatomas, reveals patterns of pyridoxal-5'-phosphate binding proteins that are characteristic of the various sources of cytosol. These findings suggest that there are developmental and tumor-specific requirements for pyridoxal-5'-phosphate, the coenzymatically active form of vitamin B-6.

Aging↗
PubMed1983

Preparation, characterization, and use of monoclonal antibodies to vitamin B6.

Monoclonal antibodies exhibiting various specificities for B6 vitamer forms have been prepared. The antigen preparation employed was a partially purified mixture of human placental proteins that had been derivatized by reaction with pyridoxal 5'-phosphate and sodium borohydride. Spleen cells obtained from mice immunized with the phosphopyridoxyl protein preparation were fused with the mouse myeloma cell line designated X63-Ag8.653. The resulting hybridomas were screened for production of antibodies to the haptenic phosphopyridoxyl group using an enzyme-linked immunosorbent assay. Clones producing such antibodies were isolated by limiting dilution methods. The monoclonal antibodies obtained in this fashion have been characterized with respect to their ability to interact with various forms of vitamin B6. In addition, these antibodies have been shown to be useful in the detection of cellular pyridoxal phosphate binding components using immunoblot techniques. Monoclonal antibodies to vitamin B6 derivatives are potentially powerful tools in the assessment of vitamin B6 nutritional status and in the study of the roles of pyridoxal phosphate binding components in relation to growth, differentiation, carcinogenesis, and steroid hormone action.

Animals↗