Reconstitution of the 9 S estrogen receptor with heat shock protein 90

As a first step in the investigation of the reconstitution of steroid hormone receptor systems, we studied the reconstitution of 9 S estrogen receptor (ER) from purified vero ER, which is the estradiol binding subunit, and heat shock protein 90 (hsp 90). By using a phosphate buffer containing molybdate, thiocyanate, dimethylformamide, glycerol, etc., vero ER could be converted to 9 S ER with hsp 90, but not with the control protein, ovalbumin. Inactivation of ER during the reconstitution was suppressed partially by hsp 90, but not by ovalbumin. Like native 8 S ER, the reconstituted ER was sedimented at about 8.9 S and 4.6 S on glycerol gradient centrifugation in low and high salt buffers, respectively.     

Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae

A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is responsive to DNA alkylation. Reaction rates were stimulated by 7,8-benzoflavone and inhibited by rabbit anti-P-450 IIIA (anti-P-450NF), troleandomycin, gestodene, and cimetidine. Evidence was obtained that rates of reduction of ferric P-450 IIIA4 in yeast microsomes and the reconstituted systems are slow and at least partially responsible for the lower rates of catalysis seen in these systems (relative to liver microsomes). The results of these studies with a defined protein clearly demonstrate the ability of P-450 IIIA4 to catalyze regio- and stereoselective oxidations with a diverse group of substrates, and this enzyme appears to be one of the most versatile catalysts in the P-450 family.     

Conversion of dNTP to dNMP dependent on DNA synthesis in isolated Yoshida sarcoma nuclei

Nuclei isolated from Yoshida sarcoma cells had activity for conversion of dGTP to dGMP dependent on DNA synthesis. The ratio of nucleotide generation/generation + incorporation was 0.4 ± 0.1, indicating that approx. 40% of the incorporated dGMP was excised. Two lines of evidence indicated the dependence of this activity on DNA synthesis. (1) The activity was observed only in the presence of ATP, which is essential for nuclear DNA synthesis. (2) Inhibitors of DNA synthesis, such as N-ethylmaleimide, aphidicolin, spermine and KCl, also inhibited ATP- or DNA synthesis-dependent dGMP generation. Although nuclei contain nucleoside triphosphatase (N-nucleotidase), this enzyme was not involved appreciably in DNA synthesis-dependent dGMP generation. The reason for this was explained by the following findings. (a) Inhibitors did not decrease dGMP production in the complete absence of DNA synthesis. (b) Inhibitors did not inactivate N-nucleotidase to the same degree as they inhibited DNA synthesis-dependent dGMP generation. (c) Addition of ATP reduced dGTP hydrolysis catalyzed by N-nucleotidase. (d) GDP had no appreciable effect on DNA synthesis-dependent dGMP generation, but had a diluting effect on dGMP production catalyzed by N-nucleotidase. These results show that the pathway of dGMP generation in isolated nuclei was switched on addition of ATP from a N-nucleotidase-catalyzed one to a DNA polymerase-exonuclease-catalyzed one.     

ADP-ribosylation of nuclear protein A24

Nuclear protein A24, which is composed of histone H2A and ubiquitin, a nonhistone protein, joined by an isopeptide linkage [Goldknopf and Busch (1977) $\text{Proc}$. $\text{Natl}$. $\text{Acad}$. $\text{Sci}$. $\text{USA}$$\text{74}$, 864–868], is found to be ADP-ribosylated in isolated rat liver nuclei.     

Heterogeneity of mouse lymphocytes in interferon production upon influenza virus challenge in culture

Heterogeneity of mouse lymphocytes with respect to interferon production upon influenza virus challenge in culture was studied. Spleen cells produced much more interferon than thymocytes or mesenteric lymph node cells. Spleen cells mainly responsible for interferon production belonged to the hydrocortisone-sensitive population. When spleen lymphocytes were separated into seven fractions by centrifugation on a serum albumin density gradient, they were found to differ greatly in interferon producing capacity; a small fraction of intermediate density, representing a few percent or less of the total lymphocytes, produced markedly high levels of interferon.     

Interaction of the low molecular weight form of elongation factor 1 with guanine nucleotides and aminoacyl-tRNA.

A low molecular weight form of the eukaryotic polypeptide chain elongation factor 1 (EF-1α) has been extensively purified from pig liver to give an apparently homogeneous preparation, which seemed to be analogous to the bacterial elongation factor, EF-Tu (Iwasaki, K., Nagata, S., Mizumoto, K., and Kaziro, Y. (1974) J. Biol. Chem. 249, 5008). Thus, the interaction of the purified EF-1α with guanine nucleotides as well as aminoacyl-tRNA has been investigated and the following results have been obtained. (1) EF-1α when kept in the absence of glycerol lost its activity to promote the binding of aminoacylt-RNA to ribosomes though it retained the ability to bind guanine nucleotides. However, the former activity could be stabilized by the addition of 25% ($\text{v}\text{v}$) glycerol to the solution. (2) EF-1α formed a binary complex with guanine nucleotides such as GTP, GDP, 5′-guanylyl methylenediphosphonate or 5′-guanylyl imidodiphosphate. The molar ratio of EF-1α to GTP or GDP in the binary complex was shown to be 1. (3) The presence of a ternary complex containing EF-1α, GTP and aminoacyl-tRNA was demonstrated by several methods, i.e., (i) an increased heat stability of EF-1α in the presence of GTP and Phe-tRNA, (ii) a decrease in the amount of the EF-1α·GTP complex in the presence of aminoacyl-tRNA, (iii) a protection of the ester linkage of Phe-tRNA from hydrolysis at alkaline pH by the presence of both EF-1α and GTP, and (iv) the isolation of the complex by gel filtration.     

Transplacental transmission of BK virus in human.

The prevalance and distribution of BK virus antibody in women during pregnancy and the occurrence of transplacental transmission of BK virus was determined by measurement of IgM antibody in the serum. Sera were collected from 63 nonpregnant women, 71 women who had experienced spontaneous abortion, 80 in the first trimester of pregnancy and the same 80 at delivery. Umbilical cord blood was also taken at delivery. Hemagglutination inhibition (HI) tests for BK virus used the micromethod of Gardner. Results indicate that a significant level of HI antibody was present in 70% of sera from all 4 experimental groups. This showed that BK virus infection was not limited to cases of spontaneous abortion. Of the 80 pregnant mothers, 6 showed a 4-fold or greater HI antibody seroconversion to BK virus after delivery. Of these 6 seroconversion patients, sensitive antibody was detected in 3 umbilical cords. Umbilical cords of those without seroconversion had no sensitive antibody. As evidenced by 2-NE-sensitive antibody, BK virus infections were also recognized in 6 of 71 women who aborted, 4 of 80 in the first trimester of pregnancy and 2 collected after delivery. The 2-ME-sensitive antibody was not found in any of 63 samples from nonpregnant women. Data indicate that 2-ME-sensitive antibody was present only in sera of women during pregnancy and after abortion. It may be possible that BK virus persists in a latent form in many healthy women and becomes activated during pregnancy.     

Amino acid sequence of an active fragment of potato proteinase inhibitor IIa.

The complete amino acid sequence of an active fragment of potato proteinase inhibitor IIa has been established by the Edman degradation procedure and the carboxypeptidase technique. Sequence analyses were carried out on the reduced and carboxymethylated active fragment and its tryptic peptides. To aid in the alignment of some tryptic peptides, the partial sequences of two fragments obtained by selective tryptic cleavage of the reactive site peptide bond of inhibitor IIa at acidic pH, with subsequent reduction and carboxymethylation, were also analyzed. The active fragment consisted of 45 amino acid residues including 6 half-cystine residues. Degradation of the intact active fragment by subtilisin [EC 3.4.21.14.] at pH 6.5. yielded 3 cystine-containing peptides. Sequence analyses of these peptides revealed that the 3 disulfide linkages were located between Cys(10) and Cys(24), Cys(14) and Cys(35), and Cys(20) and Cys(43). The reactive site peptide bond of inhibitor IIa, a Lys-Ser bond, was located between positions 32 and 33 of the active fragment. The overall sequence of the active fragment was quite different from those of potato chymotrypsin inhibitor I (subunit A) and potato carboxypeptidase inhibitor.