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A B Frey

Publications and source records attributed to A B Frey.

43 records · Page 3Linked to original sources
PubMed1986

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine photolabels a substrate-binding site of rat hepatic cytochrome P-450 form PB-4.

Hepatic microsomes isolated from untreated male rats or from rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) were labeled with the hydrophobic, photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID incorporation into 3-MC- and PB-induced liver microsomal protein was enhanced 5- and 8-fold, respectively, relative to the incorporation of [125I]TID into uninduced liver microsomes. The major hepatic microsomal cytochrome P-450 forms inducible by PB and 3-MC, respectively designated P-450s PB-4 and BNF-B, were shown to be the principal polypeptides labeled by [125I]TID in the correspondingly induced microsomes. Trypsin cleavage of [125I]TID-labeled microsomal P-450 PB-4 yielded several radiolabeled fragments, with a single labeled peptide of Mr approximately 4000 resistant to extensive proteolytic digestion. The following experiments suggested that TID binds to the substrate-binding site of P-450 PB-4. [125I]TID incorporation into microsomal P-450 PB-4 was inhibited in a dose-dependent manner by the P-450 PB-4 substrate benzphetamine. In the absence of photoactivation, TID inhibited competitively about 80% of the cytochrome P-450-dependent 7-ethoxycoumarin O-deethylation catalyzed by PB-induced microsomes with a Ki of 10 microM; TID was a markedly less effective inhibitor of the corresponding activity catalyzed by microsomes isolated from uninduced or beta-naphthoflavone-induced livers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

The structure of phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4. Production and characterization of site-specific antibodies.

Fifteen peptides corresponding in sequence to segments of the major phenobarbital-inducible forms of rat hepatic cytochrome P-450 (termed P-450 PB-4 and P-450 PB-5) were chemically synthesized, conjugated to carrier proteins, and used to prepare site-specific rabbit and/or mouse antipeptide antibodies. Four of the synthetic peptides were recognized by rabbit heterosera raised against purified P-450 PB-4. The titer of these heterosera measured against P-450 PB-4 was only partially reduced upon complete adsorption of antipeptide activity suggesting that these peptides represent minor antigenic determinants. Each of the antipeptide antibodies recognized purified P-450 PB-4 and the highly homologous P-450 PB-5 as demonstrated by a solid-phase enzyme-linked immunosorbent assay. Although each antipeptide immunoprecipitated both purified 125I-labeled P-450 PB-4 and also in vitro-synthesized apo-P-450 PB-4, the yields of immunoprecipitation were low relative to that obtained using anti-P-450 heterosera. Only one of the antipeptide antibodies gave a good signal in an immunoblot analysis of either microsomal or purified P-450s PB-4 and PB-5. Three antipeptide antibodies raised against hydrophilic segments located in the amino-terminal one-third of P-450 PB-4 markedly inhibited the P-450 PB-4-catalyzed O-deethylation of the model substrate 7-ethoxycoumarin. Four of the antipeptide antibodies were found to cross-react with P-450 beta NF-B, the major aromatic hydrocarbon-inducible rat hepatic P-450, suggesting that certain amino acid sequences or regions of secondary structure are conserved between the major phenobarbital-induced and polycyclic-induced rat liver P-450 isoenzymes. These studies demonstrate the utility of antipeptide antibodies for evaluation of antigenic sites exposed in native P-450 PB-4, for identification of specific amino acid sequences important for the interaction of P-450 PB-4 with its substrate and/or with cytochrome P-450 reductase in a reconstituted system and for elucidation of structural and immunochemical homologies between P-450 PB-4 and other P-450 isoenzymes present in rat liver endoplasmic reticulum.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1984

Induction of cytochrome P-450 isozymes in rat hepatoma-derived cell cultures.

We have investigated the responsiveness of established rat hepatocyte cell cultures to inducers of cytochrome P-450. One Reuber hepatoma-derived line (Fu5-C8), which under normal culture conditions produces no detectable cytochrome P-450(MC) or cytochrome P-450(PB)--the major cytochrome P-450 isozymes induced by 3-methylcholanthrene and phenobarbital, respectively--was tested for the ability to accumulate either cytochrome P-450 isozyme in response to treatment with various xenobiotics. By immune-precipitation from [35S]-methionine-labeled cell extracts, using monospecific anticytochrome P-450(MC) antibody or monoclonal anticytochrome P-450(PB) antibody, it was demonstrated that these cells possess the capability to synthesize cytochrome P-450(MC) in response to 3-methylcholanthrene treatment, while none of the drug treatments caused the synthesis of detectable quantities of cytochrome P-450(PB). RNA extracted from Fu5-C8 cells directed the in vitro synthesis of immune-precipitable cytochrome P-450(MC) only after treatment of the cells with 3-methylcholanthrene. Kinetic analysis of the response of these cells to 3-methylcholanthrene induction revealed detectable levels of immune-precipitable cytochrome P-450(MC) 2 h after drug treatment with maximal induction occurring between 12 and 16 h of exposure. Another cell line (HF 1.5), obtained originally by hybridization of Fao X H5 variants of a Reuber H35 hepatoma, produces cytochrome P-450(MC) and also cytochrome P-450(PB) constitutively, as determined by specific immune-precipitation from labeled cell extracts. Exposure of confluent monolayers to either phenobarbital or 3-methylcholanthrene resulted in an induction of cytochrome P-450(PB) or cytochrome P-450(MC), respectively. Double-labeling immunofluorescence studies indicate that all cells in the culture produce albumin and most of the cells produce cytochrome P-450(MC), but only a subset of cells synthesize cytochrome P-450(PB). Our results demonstrate that some continuously dividing hepatocyte cell cultures retain the capacity to respond to xenobiotics, including phenobarbital, a response which is typically exhibited by fully differentiated liver cells. Such established hepatocyte cell cultures should prove useful for investigating the mechanism of induction of cytochrome P-450(PB).

Animals↗
PubMed1983

Studies on the subunit composition of rat liver glutathione S-transferases.

Native glutathione S-transferases are composed of subunits with apparent molecular weights of 25,000, 23,500, or 22,000 which form either homo- or heterodimers. Glutathione S-transferases A, C, and X which contain two subunits with molecular weights of 23,500 yielded similar but nonidentical proteolytic fragmentation patterns. Fragments unique to the subunits of the homodimers A and X were present in decreased intensities in the patterns of form C. Two-dimensional electrophoresis under denaturing conditions showed single nonoverlapping spots for transferases A and X, while form C yielded two spots corresponding in position to those obtained from forms A and X. Renaturation of dissociated glutathione S-transferase C yielded enzymatically active transferases A, C, and X. These results indicate that form C is a heterodimer composed of one subunit from the homodimeric transferases A and X. This was substantiated by NH2-terminal sequence analysis showing extensive NH2-terminal homology amongst all three forms. However, in the positions where forms A and X yielded different residues, both amino acids were detected in the sequence of form C, indicating that the two subunits of Mr = 23,500 are the products of two different genes. NH2-terminal sequence analysis of the heterodimeric glutathione S-transferase B which is composed of subunits with molecular weights of 22,000 and 25,000 revealed a single unique sequence which bore no resemblance to the sequences of either forms A or X. Despite the identical NH2-terminal sequences, proteolytic fragmentation of the separated subunits showed markedly different fragmentation patterns. This indicates that two different mRNAs code for these two subunits.

Amino Acid Sequence↗
PubMed1982

Synthesis and incorporation of myelin polypeptides into CNS myelin.

The distribution of newly synthesized proteolipid protein (PLP, 23 kdaltons) and myelin basic proteins (MBPs, 14-21.5 kdaltons) was determined in microsomal and myelin fractions prepared from the brainstems o1 10-30 d-old rats sacrificed at different times after an intracranial injection of 35S-methionine. Labeled MBPs were found in the myelin fraction 2 min after the injection, whereas PLP appeared first in the rough microsomal fraction and only after a lag of 30 min in the myelin fraction. Cell-free translation experiments using purified mRNAs demonstrated that PLP and MBPs are synthesized in bound and free polysomes, respectively. A mechanism involving the cotranslational insertion into the ER membrane and subsequent passage of the polypeptides through the Golgi apparatus is consistent with the lag observed in the appearance of the in vivo-labeled PLP in the myelin membrane. Newly synthesized PLP and MBPs are not proteolytically processed, because the primary translation products synthesized in vitro had the same electrophoretic mobility and N-terminal amino acid sequence as the mature PLP and MBP polypeptides. It was found that crude myelin fractions are highly enriched in mRNAs coding for the MBPs but not in mRNA coding for PLP. This suggests that whereas the bound polysomes synthesizing PLP are largely confined to the cell body, free polysomes synthesizing MBPs are concentrated in oligodendrocyte processes involved in myelination, which explains the immediate incorporation of MBPs into the developing myelin sheath.

Animals↗