Studies on the maintenance of cytochromes P-450 and b5, monooxygenases and cytochrome reductases in primary cultures of rat hepatocytes

The cytochrome P-450 content of rat hepatocytes declined rapidly over 72 h in culture, due primarily to denaturation to cytochrome P-420. Six different media were investigated for their ability to conserve cytochrome P-450 during culture, and the most successful was a modified Earle's medium. After 72 h culture in this medium, cytochromes P-450 and b5, NADH-cytochrome b5- and NADPH-cytochrome c-reductases were maintained at 40, 100, 35 and 52% of fresh cell values, respectively. Cytochrome P-450 showed differential functional stability during culture with ethoxyresorufin O-deethylation being more stable than either pentoxyphenoxazone O-depentylation or biphenyl 4-hydroxylation. Monooxygenase than did cytochrome P-450 content. This discrepancy was not explained by loss of flavin nucleotides, FMN or FAD.     

Nucleotide sequence analysis of a variant human T-cell leukemia virus (HTLV-Ib) provirus with a deletion in pX-I.

A variant of human T-cell leukemia virus subgroup I (HTLV-I), designated HTLV-Ib, has been isolated from a transformed T-lymphocytic cell line established from a Zairian patient with adult T-cell lymphoma. A recombinant phage clone of the variant provirus, denoted lambda MC-1, hybridizes under high stringency to HTLV-I DNA probes, but 17 of 43 restriction enzyme sites differ from those of HTLV-I, 10 of them clustering within 1.5 kilobases in the env-pX region. Since this variant virus retains its capacity to transform T-cells in vitro, and since a pX product is suspected to be important in transformation, we have determined the nucleotide sequence of the entire pX region of this virus for comparison to the prototype HTLV-I. In addition, the region between the gag and pol genes, parts of the pol and env genes, and a portion of the U3 region of the long terminal repeat sequence were also analyzed. We noted 141 single-base-pair changes among 3,897 base pairs, which were relatively well distributed over those portions of the provirus that were examined. In addition, an 11-base-pair deletion was found which included the potential initiator ATG codon of the first open reading frame of pX (pX-I). The next potential initiator codon predicted by the sequence is followed by 10 codons and then a termination codon. An identical deletion was also demonstrated in the only provirus present in another cell line established from the same patient on a different occasion after transformation in vitro of normal human umbilical cord blood cells. These results indicate that pX-I is not required for transformation.     

The amino acid sequence of equine milk lysozyme

The amino acid sequence of equine milk lysozyme has been elucidated. The study involves the determination of the sequence of the N-terminal region of the whole protein, cyanogen bromide fragments, tryptic and chymotryptic peptides and fragments produced by chemical cleavage after tryptophan residues. The protein consists of a single chain of 129 amino acid residues and has a Mr of 14647. While equine milk lysozyme has the essential features of a c(chick)-type lysozyme, there is only 51% sequence homology with human milk lysozyme and 50% with domestic hen egg white lysozyme. Some of the implications of the large number of differences are discussed.     

HTLV-III env gene products synthesized in E. coli are recognized by antibodies present in the sera of AIDS patients

The envelope gene of HTLV-III, the retrovirus directly linked to AIDS, encodes a protein of 856 amino acids. Our sequence analysis of the cloned HTLV-III (HXB-3) env gene and its comparison with other isolates reveal significant divergence, especially in the external portion of this protein. A large segment of the env gene (1800 bp) was inserted into the expression vector pEV-vrf3, and a corresponding 68 kd protein, which encompasses both the extracellular and the membrane-associated regions of the native protein, was produced in E. coli. Several smaller polypeptides, which appear to be internal initiation products, were also produced. All 50 AIDS patient sera obtained from different locations in the United States specifically recognized the bacterially synthesized envelope proteins, as judged by Western blots. This suggests that these proteins will be useful for the diagnosis of HTLV-III infection and possibly as a vaccine against AIDS.     

Metabolism and gastric acid secretion. Substrate-dependency of aminopyrine accumulation in isolated rat parietal cells.

The substrate-dependency of gastric acid secretion was investigated in isolated rat parietal cells by using the accumulation of the weak base aminopyrine as an index of acid secretion. Exogenous substrates enhanced accumulation of aminopyrine in rat parietal cells stimulated by secretagogues, and this effect was probably directly related to the provision of energy for acid secretion. At physiological concentrations, certain of the substrates (glucose, oleate, lactate, D-3-hydroxybutyrate, L-isoleucine, L-valine and acetoacetate) could support acid secretion, with glucose being the most effective. L-Leucine and acetate were only effective stimulators of parietal-cell aminopyrine accumulation at high concentrations (5mM). L-Glutamine was unable to stimulate aminopyrine accumulation even at high concentrations, and glutaminase activity in parietal cells was estimated to be low by comparison with small-intestinal epithelial cells. Variation in the concentrations of D-3-hydroxybutyrate and L-isoleucine, but not of glucose, within the physiological range affected their ability to support aminopyrine accumulation. The presence of 5 mM-L-isoleucine, 5 mM-lactate and combinations of certain substrates at physiological concentrations produced aminopyrine accumulation in stimulated parietal cells that was greater than that obtained in cells incubated with 5 mM-glucose alone. In conclusion, fulfillment of the metabolic requirements of the acid-secreting parietal cell under physiological circumstances requires a combination of substrates, and integration of the results with previous data [Anderson & Hanson (1983) Biochem. J. 210, 451-455; 212, 875-879] suggests that after overnight starvation in vivo metabolism of glucose, D-3-hydroxybutyrate and L-isoleucine may be of particular importance.     

The suicide inactivation of ox liver short-chain acyl-CoA dehydrogenase by propionyl-CoA. Formation of an FAD adduct.

R-Phycoerythrin contains two covalently bound bilin prosthetic groups, phycoerythrobilin and phycourobilin. The two chromophore types were separated as their peptide-bound derivatives by subjecting tryptic digests of R-phycoerythrin to adsorption chromatography on Sephadex G-25. The structure and apoprotein linkages of the bound phycoerythrobilin were found to be identical with those previously reported for this phycobilin [Killilea, O'Carra & Murphy (1980) Biochem. J. 187, 311-320]. Phycourobilin is a tetrapyrrole, containing no oxo bridges and has the same order of side chains as IX alpha bilins. The chromophore is linked to the peptide through two and possibly three of its pyrrole rings. One linkage possibly consists of an ester bond between the hydroxy group of a serine residue and the propionic acid side chain of one of the inner rings. The second linkage is a labile thioether bond between a cysteine residue and the C2 side chain of pyrrole ring A. The third linkage is a stable thioether bond between a cysteine residue and the alpha-carbon atom of the C2 side chain of pyrrole ring D. Ring D is unsaturated and is attached to ring C through a saturated carbon bridge. Rings B and C have a conjugated system of five bonds, as found in other urobilinoid pigments. Ring A is attached to ring B via a saturated carbon bridge. Both of the alpha-positions of ring A are in the reduced state, but the ring does contain an unsaturated centre (probably a double bond between the beta-carbon and the ring nitrogen atom). The presence of this double bond and its isomerization into the bridge position between rings A and B would explain the extension of the conjugated system of phycourobilin to that of a phycoerythrobilinoid/rhodenoid pigment in acid or alkali.     

Evaluation of inhibitor constants and alkylation rates for a series of thrombin affinity labels.

The kinetics for the inactivation of thrombin (EC 3.4.21.5) by a series of peptides containing C-terminal arginyl chloromethane in the presence of substrate were determined. The inhibitor effectiveness was analysed so as to allow for both the evaluation of the affinity with which the enzyme binds the inhibitor before irreversible modification and also the rate of covalent-bond formation between enzyme and inhibitor. The results obtained show that the observed large range in inhibitor effectiveness can be accounted for almost entirely by marked differences in affinity, with only small variations in rates of formation of covalent complex.