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Biomedical subjects

F G Walz

Publications and source records attributed to F G Walz.

At least 37 records · Page 2Linked to original sources
PubMed1982

Polypeptide patterns of hepatic microsomes from Long-Evans rats treated with different xenobiotics.

Two-dimensional gel electrophoresis was used to analyze hepatic microsomal polypeptides after treatment of immature, male Long-Evans rats with 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile, isosafrole, SKF-525A, Aroclor-1254, gamma-chlordane, or trans-stilbene oxide. Epoxide hydrolase and cytochromes P-450a, P-450bLE, P-450c, P-450d, and P-450e were all identified as resolved polypeptides in these electrophoretograms. Idiosyncratic polypeptide patterns characterized the microsomal preparations following treatment of rats with each inducing agent. Immunochemically identical cytochromes P-450bLE and P-450e were always present at the same relative levels even though their total amount varied 3-fold after induction by isosafrole, SKF-525A, Aroclor-1254, gamma-chlordane, and trans-stilbene oxide. Cytochromes P-450c and P-450d were coinduced by 3-methylcholanthrene, isosafrole, and Aroclor-1254, but their relative amounts varied. Pregnenolone-16 alpha-carbonitrile treatment resulted in an increase of a single major microsomal polypeptide which was also induced by phenobarbital, isosafrole, SKF-525A, Aroclor-1254, and trans-stilbene oxide. Only one polypeptide was identified as epoxide hydrolase in all of the microsomes analyzed. The results suggest that the levels of cytochromes P-450bLE and P-450e may be subject to coordinate control, whereas the other cytochromes P-450 are independently regulated.

Animals↗
PubMed1982

Multiple, immunoidentical forms of phenobarbital-induced rat liver cytochromes P-450 are encoded by different mRNAs.

It was shown previously that four immunochemically identical forms of phenobarbital-induced hepatic cytochrome P-450 exist in unique combinations which characterize different strains and colonies of rats (Vlasuk, G. P., Ghrayeb, J., Ryan, D. E., Reik, L., Thomas, P. E., Levin, W., and Walz, F. G., Jr. (1982) Biochemistry 21, 789-798). One colony of Long-Evans rats exhibited only cytochromes P-450bLE and P-450e among these immunorelated enzymes; whereas, one colony of Holtzman rats was characterized by cytochromes P-450bH and P-450e. After phenobarbital treatment, hepatic poly(A)+-mRNA was isolated from these groups of rats and translated in vitro. The 35S-labeled products were immunoisolated with antibody to cytochrome P-450bLE and analyzed by two-dimensional gel electrophoresis. The results indicated that the products synthesized in vitro correspond exactly to the two particular forms of the enzyme that characterize liver microsomes from each of these groups of rats. It is concluded that different structural genes encode these immunorelated forms of cytochrome P-450 and that significant post-translational processing of their polypeptide products does not occur in vivo.

Animals↗
PubMed1982

Multiplicity strain differences, and topology of phenobarbital-induced cytochromes P-450 in rat liver microsomes.

The multiplicity of phenobarbital-induced cytochromes P-450 in live microsomes from male rats was investigated by using two-dimensional gel electrophoresis, peptide fingerprinting, and immunoaffinity chromatography. Two colonies each of Holtzman and Long-Evans rats were studied. Four molecular forms of phenobarbital-induced cytochromes P-450 were distinguished as polypeptides (designated PB3, variant PB3, PB4, and PB5) which showed apparent immunochemical identity and greater than or equal to 95% fingerprint homology. Two of these polypeptides corresponded to cytochrome P-450b [Ryan, D., Thomas, P. E., Korzeniowski, D., & Levin, W. (1979) J. Biol. Chem. 254, 1365-1374] and cytochrome P-450e [Ryan, D., & Levin, W. (1981) Fed. Proc., Fed. Am. Soc. Exp. Biol. 40, 1640] which had been purified from Long-Evans rats (variant PB3 and PB5, respectively). Each rat colony was characterized by unique combinations of two or three of these immunochemically related forms of cytochromes P-450. Cytochrome P-450e was present in rats from all four colonies, but cytochrome P-450b was only found in Long-Evans rats. Polypeptide PB3 was only found in the two colonies of Holtzman rats, whereas polypeptide PB4 was present in one colony each of Holtzman and Long-Evans rats. In addition to these forms of cytochrome P-450, rats from each colony also evidenced three other major phenobarbital-induced polypeptides which gave unique fingerprints, and one of these was identified as representing epoxide hydrolase. Proteolytic digestion studies of intact microsomes demonstrated that the four immunochemically identical forms of cytochrome P-450 were partially exposed on the outer (cytoplasmic) surface of microsomes. However, polypeptide PB3 was characterized by the greatest rate of proteolytic degradation. These results clearly demonstrate that phenobarbital-induced cytochromes P-450 include microheterogeneous proteins which show remarkable variations related to rat strains and/or colony.

Animals↗
PubMed1977

Spectrophotometric titration of a single carboxyl group at the active site of ribonuclease T1.

Low-pH-induced difference spectra for ribonuclease T1, which were determined using a reference solution at pH 6, consisted of a shorter wavelength component from 270 to 285 nm that relfected an ionization having a pKa of 3.54 and a longer wavelength component above 285 nm that reflected an ionization having a pKa of 4.29. The temperature dependence of the pKa value for data at 300 nm is consistent with its representing the dissociation of a carboxyl group. In addition, the pKa determined at this wavelength significantly decreased at lower ionic strength. Similar experiments which were conducted using catalytically inactive gamma-carboxymethyl-Glu-58-ribonuclease T1 gave difference spectra having only the shorter wavelength component and were characterized by a single pKa of 3.53. It is concluded that the longer wavelength component of the difference spectra is due to the ionization of Glu-58. The pKa determined for this residue in the present study agrees with one found previously from kinetic studies which supports a role for Glu-58 in catalysis. Furthermore, the results suggest a model for the interaction of Glu-58 with histidine and tryptophan residues at the active site.

Binding Sites↗
PubMed1976

Ribose recognition by ribonuclease T1: difference spectral binding studies with guanosine and deoxyguanosine.

The binding of ribonuclease T1 with guanosine (Guo) and deoxyguanosine (dGuo) was studied in experiments employing ultraviolet difference spectroscopy in the pH range 3-9 at 0.2 M ionic strength and 25 degrees C. Similar experiments were also conducted with psi-carboxymethyl-glutamate-58 ribonuclease T1 at pH 5.0. At most pH values the characteristic difference spectrum and association constant were obtained. The binding constant for dGuo was approximately 550 M-1 and did not significantly vary in the pH range 3.5-9.0. The binding constant for Guo increased from pH 3.5 to 5.0, was constant between pH 5.0 and 7.0 (approximately 3200 M-1), and decreased at higher pH values. The binding of Guo and dGuo with ribonuclease T1 could also be distinguished in terms of the wavelength for maximal difference absorbance, lambdamax, between pH 5.0 and 7.0. At higher and lower pH values, lambdamax for Guo approached that found fr dGuo. On the other hand, the value of the binding constant (approximately6500 M-1) and the nature of the difference spectra for Guo and dGuo binding with lambdamax-carboxymethyl-glutamate-58-ribonuclease T1 at pH 5.0 were identical. These results suggest that the discrete interaction of the Guo 2'-hydroxyl group with ribonuclease T1 involves the lambda-carboxylate of glutamate-58 and an imidazolium group at the active site.

Binding Sites↗
PubMed1976

Subsite interactions of ribonuclease T1: binding studies of dimeric substrate analogues.

Ultraviolet difference spectral binding studies of ribonuclease T1 with pGp, ApG, CpG, UpG, DGpdA, dGpdC, dGpdG, dGpdT, dTpdG, pdApdG, pdTpdG, pdGpdA, pdGpdG, pdGpdT, c(pdGpdA), and c(pdGpdG) were conducted at pH 5.0, 0.2 M ionic strength and 25 degrees C. Under these conditions, the characteristic difference spectrum and association constant for (1:1) ribonuclease T1 binding were determined for each ligand. The binding of guanosine and deoxyguanosine containing ligands could be distinguished by the shapes of their difference spectra. The results indicated that the guanine moiety of each ligand was bound at the enzyme's primary recognition site. Evidence of a specific enzyme subsite for binding the adenine moiety of ApG and pdApdG is presented. The proposal of a specific enzyme subsite for binding the 5'-phosphate group of a complexed guanosine moiety (Sawada, F., Samejima, T., and Saneyoshi, M. (1973), Biochim. Biophys. Acta 299, 596) is not supported in the present work. Preliminary evidence for the existence of two additional enzyme subsites and the effect of oligomer conformation on enzyme binding are also discussed.

Kinetics↗
PubMed1975

The interaction of ribonuclease T 1 with DNA.

The interaction of RNase T1 with calf thymus DNA was studied using uv difference spectroscopy and the effect of the enzyme on DNA melting. There was no indication of RNase T1 binding with native DNA. A prominent difference spectrum for RNase T1 binding with denatured DNA (d-DNA) was observed at pH 5, 25 degrees and low ionic strength (mu = .01 M) which was depressed at higher ionic strength and pH. The normalized difference spectrum at mu = .01 M, pH 5 and 25 degrees can be interpreted as indicating an interaction of an exposed guanine residue directly with the enzyme and a coupling of this process with the "melting" of short folded segments of d-DNA. The apparent association constant calculated per M guanine residues was 2.4 X 10-4 M-1 under these conditions. The results are discussed in reference to comparable studies on the interaction of RNase T1 with RNA and small guanine ligands.

Animals↗