Frederick G. Walz

Liver endoplasmic reticulum polypeptides resolved by two-dimensional gel electrophoresis

Abstract Several modifications of the two-dimensional electrophoretic technique of P. H. OFarrell (i.e., first-dimensional isoelectric focusing followed by second-dimensional sodium dodecyl sulfate electrophoresis: 1975, J. Biol. Chem. 250 , 4007) were required for maximum resolution of rat liver smooth microsomal polypeptides without significant artifact. All substantial changes were in isoelectric focusing which gave ∼55 bands with 130 μg of microsomal protein; subsequent electrophoresis in the second dimension yielded ∼230 polypeptide spots. Mild deoxycholate treatment to lyse microsomes suggested that ∼170 spots represent integral membrane proteins. Comparisons of two-dimensional polypeptide patterns for liver microsomes from phenobarbital-induced, 3-methylcholanthrene-induced, and noninduced rats revealed differences mainly in putative cytochrome P -450 polypeptides.

Base-group specificity at the primary recognition site of ribonuclease T1 for minimal RNA substrates☆

Abstract Kinetic studies on the RNase T 1 -catalyzed transesterification of 12 dinucleoside monophosphates, N p 1 N 2 (N 1 = A, C, and U; N 2 = A, C, G, and U) at pH 5, 25 °C, and 0.2 m ionic strength, revealed that the catalytic efficiency ( k cat K m ) for GpN substrates (H. L. Osterman, and F. G. Walz, Jr., 1978, Biochemistry , 17 , 4142) was ~10 6 -fold greater than corresponding ApNs and at least 10 8 -fold greater than corresponding CpNs and UpNs. The catalytic activity with ApN substrates survives phenol extraction which indicates (along with other criteria) that it is intrinsic to RNase T 1 and is not due to trace contamination by other nucleases. Circumstantial evidence is presented which suggests that homologous GpN and ApN substrates bind productively at different sites on the enzyme. The results of steady-state kinetic studies of RNase T 1 with IpNs (N = C and U) were compared with those for GpNs and indicated that the primary effect of the guanine 2-NH 2 group is to enhance substrate binding at the primary recognition site by ~2.6 kcal/mol. Values of ( k cat K m ) showed the order NpC > NpU (N = A, G, and I) which evidences the existence of a subsite for the leaving nucleoside group that prefers cytidine: interactions at this subsite are reflected in k cat rather than K m .

Liver microsomal polypeptides from Fischer-344 rats affected by age, sex, and xenobiotic induction

Abstract In order to investigate age and sex as determinants of hepatic cytochromes P-450, the polypeptide compositions of liver smooth microsomes from Fischer-344 rats were examined using two-dimensional gel electrophoresis ( G. P. Vlasuk and F. G. Walz, Jr. (1980) Anal. Biochem. 105, 112). The effects of phenobarbital and 3-methylcholanthrene treatments were investigated using sexually immature (1 month), young adult (3 months), middle aged (12 months), and senescent (26 months) animals of both sexes. The appearance of five major microsomal polypeptides characterized sexual maturation in males. The only qualitative difference in the patterns of xenobiotic-induced polypeptides were found for young adult and middle-aged males where cytochrome P-450a ( D. Ryan, P. E. Thomas, D. Korzeniowski, and W. Levin (1979) J. Biol. Chem. 254, 1365) was not induced by phenobarbital. A number of major microsomal polypeptides which might represent unidentified forms of cytochrome P-450 in untreated males and females were markedly decreased in a specific manner as a result of phenobarbital and/or 3-methylcholanthrene treatments. Microsomes from females of all ages tested and immature males were essentially indistinguishable on the basis of their total cytochrome P-450 contents and polypeptide patterns. Untreated senescent males were characterized by a reversion of their microsomal polypeptide patterns and total cytochrome P-450 contents to those for females and sexually immature males. In addition, phenobarbital-induced levels of total cytochrome P-450 for senescent males were the lowest observed for all of the groups tested even though their pattern of induced polypeptides was qualitatively the same as that for females.

Comparisons of highly purified hepatic microsomal cytochromes P-450 from holtzman and long-evans rats

The present study describes the purification and characterization of strain variant forms of a major phenobarbital-inducible microsomal hemoprotein, cytochrome P-450b, from Holtzman and Long-Evans rats. The strain variant hemoproteins cannot be resolved by sodium dodecyl sulfate gel electrophoresis, but can be partially separated in two-dimensional isoelectric focusing SDS gels. If, however, sodium tetradecyl sulfate is incorporated into the one-dimensional gel system, separation of the cytochromes P-450b is achieved. Minor structural differences are detected in the peptides of the cytochromes P-450b following limited proteolysis by Staphylococcus aureus V8 protease, cleavage by cyanogen bromide, or reverse-phase high-pressure liquid chromatography of tryptic peptides. The strain variant cytochromes P-450b are immunochemically and spectrally indistinguishable. The optical spectra of the ferric and ferrous hemoproteins are identical, as are the CO- and ethylisocyanide-reduced difference spectra. Ferrous cytochromes P-450b from both rat strains effectively bind metyrapone with equivalent affinities. In addition, the cytochromes P-450b do not differ in their catalytic activities toward benzphetamine, hexobarbital, benzo [a]pyrene, zoxazolamine, 7-ethoxycoumarin, estradiol-17 beta and testosterone. Cytochrome P-450c, the predominant isozyme inducible in rat liver by 3-methylcholanthrene, was purified from Holtzman and Long-Evans rats. Cytochromes P-450c from both rat strains are indistinguishable based on electrophoretic, immunological, spectral and catalytic properties. Minor structural differences in the cytochromes P-450c were revealed in the reverse-phase high-pressure liquid chromatographic profiles of the tryptic peptides of these hemoproteins, but not in the peptides generated by limited proteolysis or cleavage with cyanogen bromide.

Proalbumin is bound to the membrane of rat liver smooth microsomes.

Summary Rat serum albumin and its immediate biosynthetic precursor, proalbumin, were resolved from rat liver smooth microsomes using two dimensional gel electrophoresis. These polypeptides were identified by considering their charge properties (in first dimensional isoelectric focusing), apparent molecular weights (in second dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis), and in situ radio-iodinated peptide fingerprints in comparisons with pure albumin. Tryptic digestion or alkaline-high salt treatment of microsomes did not affect their content of either polypeptide, while 0.05% deoxycholate completely removed proalbumin and decreased albumin. Lactoperoxidase catalyzed radio-iodination of intact microsomes resulted in significant labelling of proalbumin but virtually no labelling of albumin. These results suggest that a short, internal polypeptide segment of proalbumin is exposed on the outer microsomal surface, whereas the bulk of the protein chain is entrapped by the membrane. It is concluded that some, if not all, of proalbumin in liver smooth endoplasmic reticulum is associated with the membrane which may be important for its subsequent processing.

Microheterogeneity of a male-specific rat hepatic cytochrome P-450: existence of three allozymic forms.

Preparations of hepatic cytochrome P-450 h [D. E. Ryan, et al. (1984) J. Biol. Chem. 259, 1239] and cytochrome P-450 2c [D. J. Waxman (1984) J. Biol. Chem. 259, 15481] from outbred Sprague-Dawley rats were analyzed using two-dimensional electrophoresis and in situ peptide mapping. Both preparations consisted of the same isozyme which was previously characterized as a developmentally regulated, male-specific cytochrome P-450 active in the 16 alpha-hydroxylation of steroids. Each preparation evidenced microheterogeneity which was shown, in part, to result from the existence of two genetically determined variant forms of cytochrome P-450 h/2c. Analyses of hepatic microsomes from several inbred strains of rat revealed that each was characterized by a single variant form of this isozyme, with some strains expressing a variant that was not present in Sprague-Dawley rats. Genetic crosses indicated that these electrophoretic variants represent allozymic forms of cytochrome P-450 h/2c which are codominantly expressed at a single autosomal locus. Additional microheterogeneity of each allozymic form of cytochrome P-450 h/2c was shown to result from a specific in vitro modification that may involve limited proteolysis near its C terminus by a microsome-bound protease.

A regulatory polymorphism for rat hepatic cytochrome P-450g

Rat hepatic cytochrome P-450g is a male-specific hemoprotein found at significant levels only in adult animals. In the present study, two-dimensional gel electrophoretic and immunochemical methods were used to study a polymorphism of this isozyme and its ontogenetic regulation. Inbred ACI/Hsd and WF/Hsd rats were found to express high and low levels of cytochrome P-450g, respectively. F1 hybrids of these strains showed additive inheritance for this trait and the responsible gene was found to be autosomal. Cytochrome P-450g and another male-specific form of the enzyme, cytochrome P-450h, were characterized by a similar time-course for their ontogenetic expressions. However, unlike cytochrome P-450g, the level of cytochrome P-450h was indistinguishable in hepatic microsomes from mature ACI/Hsd and WF/Hsd rats. Considering these results, we tentatively conclude that the gene regulating the level of cytochrome P-450g is Cis-acting.

Cytochrome P-450 polypeptides in pulmonary microsomes from rats

Pulmonary microsomal polypeptides from different strains of rats were resolved using two-dimensional electrophoresis and were further characterized by in situ peptide mapping. Triton X-114 detergent separation was used to enrich cytochromes P-450 (P-450) and other integral membrane proteins from pulmonary microsomes, and these were directly compared with corresponding polypeptides from hepatic microsomes. The results demonstrated that P-450b and epoxide hydrolase were present in the lungs of male and female rats and that their expression in this tissue was independent of phenobarbital treatment. P-450e, which is co-induced with P-450b in the liver, was not detected in pulmonary microsomes under any condition. Four other pulmonary microsomal polypeptides were characterized and preliminary evidence suggested that they represent unique isozymic forms of P-450 with three of them being related to P-450b.

Exocyclic-keto reductase activities for progesterone and S-warfarin in hepatic microsomes from adult male rats.

Hepatic microsomes from adult male rats representing six inbred strains catalyzed quantitatively significant, NADPH dependent reductions of progesterone to the 20 beta (20R) alcohol and S-warfarin to its 11S-OH product. Microsomes from mature females and immature rats of both sexes were essentially devoid of these activities. Two strains of rat evidenced about 21% of these activities compared with the other strains and both activities were 25-81% repressed by treatment of rats with phenobarbital (PB). An excellent linear correlation was demonstrated for the two activities considering sex, age, NADPH much greater than NADH preference, PB-repression and strain differences. However, detergent latency (71%) and resistance to trypsinolysis were only observed for the keto-reductase activity with S-warfarin. Microsomes also catalyzed the reduction of progesterone to its 20 alpha-OH derivative but this activity preferred NADH greater than NADPH, was induced 2.7-fold by PB and was essentially independent of age, sex and animal strain. Furthermore, unlike the 20 beta-OH activity, this reduction was resistant to proteolytic inactivation.

Adult-male-specific S-warfarin (11S-OH) and progesterone (20β-OH) keto-reductases in rat hepatic microsomes are not identical

Adult-male-specific reductase activities in rat hepatic microsomes use NADPH to reduce S-warfarin and progesterone to their 11S-OH and 20 beta-OH products, respectively (Apanovitch et al. (1992) Biochem. Biophys. Res. Commun. 184, 338-346). When microsomes were treated with increasing concentrations of detergent, S-warfarin (11S-OH) reductase (SW(11S)R) activity was subject to monophasic activation by Triton X-100, monophasic inhibition by sodium cholate, and, activation followed by inhibition with either CHAPS or dodecyl-beta-D-maltoside. A non-dialyzable, heat-sensitive factor in rat and rabbit sera activates microsomal SW(11S)R activity six- to eight-fold. Similar detergent inhibitions but no detergent or serum activations were observed for progesterone (20 beta-OH) reductase (P(20 beta)R) activity. A significant amount of SW(11S)R activity was lost during purification regardless of whether the detergent used for solubilization was activating or inhibiting. Octyl-Sepharose, hydroxyapatite, DEAE-cellulose and carboxymethyl matrices were used to partially purify SW(11S)R. P(20 beta)R activity co-purified with SW(11S)R and the most purified fraction contained two major and several minor polypeptides. Partially purified SW(11S)R is activated by detergents, serum, and salt. These and previous results indicate that SW(11S)R and P(20 beta)R are not identical even though they are both adult male-specific, integral membrane proteins apparently having their active sites exposed on the cytoplasmic surface of the endoplasmic reticulum.