PubMed Health⌕ Search

Biomedical subjects

J W Thanassi

Publications and source records attributed to J W Thanassi.

At least 19 recordsLinked to original sources
PubMed1998

Absence of pyridoxine-5'-phosphate oxidase (PNPO) activity in neoplastic cells: isolation, characterization, and expression of PNPO cDNA.

Major differences in the metabolism of vitamin B6 in various cancers compared to their normal cellular counterparts have been documented. In particular, pyridoxine- 5'-phosphate oxidase (PNPO), the rate-limiting enzyme in pyridoxal 5'-phosphate (PLP) biosynthesis, is absent in liver and neurally-derived tumors. We show that the expression of PNPO is developmentally regulated not only in liver but also in brain. Specifically, PNPO activity in fetal brain tissue is 7.5-fold lower than that found in adult brain tissue. Furthermore, the isolation and characterization of a PNPO cDNA are described. The isolated cDNA was verified to be the authentic PNPO cDNA on the basis of two criteria. First, the translated product from the PNPO cDNA is immunologically reactive to a polyclonal PNPO antibody. Second, PNPO negative hepatoma cell lines stably transfected with the PNPO cDNA express enzymatically active PNPO protein. The availability of these biological reagents will not only facilitate in depth investigations of the reasons for the absence of PNPO in liver and brain malignancies but also aid in an understanding of the biochemical regulation of B6 metabolism in development.

Amino Acid Sequence↗
PubMed1990

Pyridoxine-derived B6 vitamers and pyridoxal 5'-phosphate-binding proteins in cytosolic and nuclear fractions of HTC cells.

The nuclear fraction of rat hepatoma-derived HTC cells contained approximately 8% of the total cellular pyridoxal 5'-phosphate. HTC cells were able to metabolize [3H]pyridoxine to coenzymatically active pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate. As HTC cells did not have any demonstrable pyridoxine-5'-phosphate oxidase activity, the conversion of pyridoxine to pyridoxal 5'-phosphate must have taken place by a nonconventional route. The ratio of pyridoxal 5'-phosphate to pyridoxamine 5'-phosphate in the nonnuclear fraction of HTC cells was approximately 1:1, whereas in the nuclear fraction it was approximately 17:1, indicating that there was selective acquisition of pyridoxal 5'-phosphate by the nucleus. With the aid of a monoclonal antibody specific for the 5'-phosphopyridoxyl group, it was shown that there was one major pyridoxal 5'-phosphate-binding protein in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-resolved nucleoplasmic extract of HTC cells. This finding was confirmed by radioautography of an SDS-PAGE-resolved nucleoplasmic extract obtained from cells grown in a medium containing [3H]pyridoxine. Isoelectric focusing followed by SDS-PAGE also indicated the presence of one major pyridoxal 5'-phosphate-binding protein in the nucleoplasmic extract of HTC cells having a relatively high isoelectric point (approximately 7). Data were obtained indicating that the protein might exist in a higher molecular weight form, probably a dimer. Currently, these findings constitute virtually all of the available information on vitamin B6 and the cell nucleus.

Animals↗
PubMed1988

Vitamin B6 metabolism in McA-RH7777 cells.

The metabolism of vitamin B6 in McA-RH7777 cells has been characterized with respect to pyridoxal 5'-phosphate (PLP) levels, and the activities of pyridoxine (PN) kinase (EC 2.1.7.35) and pyridoxine 5'-phosphate (PNP) oxidase (EC 1.4.3.5). PLP levels (12.4 +/- 4.4 ng/mg protein) were at the lower end of the range found for Morris hepatomas, carcinogen-induced rat hepatomas, and liver from rats fed a PN-deficient diet. PN kinase activity was about one-third of that found in normal rat liver. PNP oxidase appeared to be absent in high-speed supernatants of homogenates prepared from McA-RH7777 cells. The absence of PNP oxidase was supported by enzymatic and immunological data. These findings resemble those found previously for Morris hepatoma 7777. In contrast to rat liver, such preparations caused little or no release of volatile counts upon incubation with either [3H-C4']PN or [3H-C4']PNP. High-speed supernatants of homogenates prepared from both McA-RH7777 cells and Morris hepatoma 7777 were very much less capable than similar preparations from rat liver in converting [G-3H]PN to PLP and pyridoxamine 5'-phosphate. Despite the apparent absence of PNP oxidase, intact confluent or log-phase McA-RH7777 cells were capable of converting [G-3H]PN to PLP and pyridoxamine 5'-phosphate. These findings are discussed in terms of tumor nutrition and vitamin B6 metabolism in a rat hepatoma cell line.

Animals↗
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

Absence of pyridoxine- (pyridoxamine-) 5'-phosphate oxidase in Morris hepatoma 7777.

Morris hepatoma 7777 previously has been shown to have no detectable pyridoxine- (pyridoxamine-) 5'-phosphate oxidase activity [Thanassi, J. W., Nutter, L. M., Meisler, N. T., Commers, P., & Chiu, J.-F. (1981) J. Biol. Chem. 256, 3370-3375]. In order to determine if this enzyme was missing in the hepatoma, we purified rat liver oxidase and raised antibodies to it in rabbits. Final purification of rat liver oxidase for use as an antigen was accomplished by affinity chromatography and gel electrophoresis. The rat liver enzyme is similar to rabbit liver oxidase [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436-3442] having two noncovalently linked subunits with molecular weights in the range of 25 000-28 000. Evidence indicating that inactive enzyme was simultaneously purified with native enzyme was obtained. The IgG fraction was purified from the serum of a rabbit that had been immunized with rat liver oxidase. This was used in the development of ELISA and immunoblot analyses for the presence of antigenically active pyridoxine- (pyridoxamine-) 5'-phosphate oxidase in cytosolic preparations from normal rat liver and Morris hepatoma 7777. The results indicated that there was no immunologically detectable oxidase protein in the tumor. An alternate pathway of pyridoxal 5'-phosphate synthesis, involving oxidation of pyridoxine to pyridoxal followed by phosphorylation, was ruled out. The implications of these findings with respect to acquisition of nutrients by tumors are discussed.

Animals↗
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↗
PubMed1982

Vitamin B-6 metabolism and its relation to ornithine decarboxylase activity in regenerating rat liver.

The metabolism of vitamin B-6 regenerating rat liver and liver from sham-operated control animals fed either a pyridoxine-sufficient or pyridoxine-deficient was investigated. The pyridoxal phosphate levels in plasma, regenerating liver and control liver were determined as were the activities of three enzymes involved in the metabolism of the vitamin, namely, pyridoxine kinase, pyridoxine phosphate oxidase, and pyridoxine phosphate phosphatase. In addition, total and holo-ornithine decarboxylase activities in the livers were measured. The results indicate that the metabolism of vitamin B-6 regenerating rat liver is different from that observed in Morris hepatomas (Thanassi et al. (1981) J. Biol. Chem. 256, 3370-3375). Vitamin B-6 metabolism in Morris hepatomas is concluded to be characteristic of the tumors rather than a property common to rapidly proliferating hepatic tissue. Regenerating liver ornithine decarboxylase holoenzyme activity in pyridoxine deprived rats was maintained at the same level as that in regenerating liver of pyridoxine-sufficient animals. The mechanism behind this maintenance of holo-enzyme activity appears to involve a pronounced increase in the amount of apoornithine decarboxylase. The time-dependent peak of ornithine decarboxylase activity following partial hepatectomy was shifted from four hours to twelve hours by vitamin B-6 deficiency.

Animals↗
PubMed1982

Vitamin B6 metabolism in liver and liver-derived tumors.

Vitamin B6 metabolism has been investigated in several highly and well-differentiated Morris hepatomas. Comparisons have been made with two poorly differentiated Morris hepatomas, with host livers obtained from tumor-bearing animals, and with fetal, neonatal, and adult rat liver. The pyridoxal phosphate content and the activities of pyridoxine kinase and pyridoxine phosphate oxidase of all Morris hepatomas examined were significantly less than those in adult host or control livers and generally fell in the range determined for fetal and neonatal liver. A similar pattern was not evident for the activity of pyridoxine phosphate phosphatase. Relative to control and host livers, the activity in hepatomas of the pyridoxal phosphate (PLP)-dependent enzyme, ornithine decarboxylase, was generally elevated. Dexamethasone, at a dose which caused an elevation in the activity of PLP-dependent tumor tyrosine aminotransferase, had no effect on PLP metabolism. The data indicate that tumor progression in the Morris hepatoma spectrum in relation to vitamin b6 metabolism falls into an onco-developmental pattern characterized by a diminished amount of tissue PLP and a diminished capability to metabolize precursor vitamer forms to PLP.

Animals↗
PubMed1981

Hormonal modulation of alpha-fetoprotein gene expression in newborn rat livers.

Suppression of serum alpha-fetoprotein (AFP) levels in glucocorticoid treated newborn rats was investigated. Daily intraperitoneal injection of 2 micrograms/g body weight of dexamethasone into newborn rats greatly reduced the concentration of AFP in the serum and liver cytosol. In contrast, this treatment stimulated liver ornithine decarboxylase activity. The reduction in AFP levels is not due to a change of distribution of AFP molecular variants, inhibition of secretion of synthesized AFP by the liver or disruption of liver polysomes. Glucocorticoids decrease the AFP levels in hormone-treated rats by supressing the synthesis of AFP. The size of AFP polysomes isolated from the livers of dexamethasone-treated rats were as large as those from normal rats. However, the amount of AFP-producing polysomes in hormone-treated rat liver is only 14% of the controls. By hybridization assays, it was found that dexamethasone treated livers contained decreased amounts of AFP mRNA sequences in liver cytoplasmic and nuclear RNAs. The decreased amounts of AFP mRNA sequences in hormone-treated liver are caused by both a decrease in the rate of AFP mRNA transcription and in AFP mRNA stability.

Animals↗
PubMed1981

Vitamin B6 metabolism in Morris hepatomas.

The enzymes involved in the metabolism of vitamin B6 were measured in Morris hepatomas and livers of female Buffalo rats fed pyridoxine-sufficient and deficient diets. Pyridoxal phosphate levels in plasmas hepatomas, and livers were also determined. Nontumor-bearing animals were maintained as controls. Regardless of the B6 nutritional status, the concentration of pyridoxal phosphate was lower in the hepatomas than in the livers of the host animals. The apoenzyme levels of ornithine decarboxylase, a pyridoxal phosphate-dependent enzyme, were higher in the hepatomas from animals fed the B6-deficient diet. Liver pyridoxine kinase activity was higher in B6-sufficient animals. In contrast, tumor pyridoxine kinase activity was influenced by B6 intake and was significantly lower than that in host liver. Liver pyridoxine phosphate oxidase activity was not significantly affected by B6 intake or by the presence of tumor. In contrast, hepatomas had little or no pyridoxine phosphate oxidase activity. Pyridoxine phosphate phosphatase activity was elevated in tumors relative to livers. These data indicate that the metabolism of vitamin B6 is markedly different in the hepatomas than in host or control livers and suggest that the tumor is apparently incapable of the complete synthesis of co-enzymatically active pyridoxal phosphate from inactive precursor forms such as pyridoxine.

Animals↗