Isolation of 3 beta,20 alpha-hydroxysteroid oxidoreductase from sheep fetal blood

3 beta,20 alpha-Hydroxysteroid oxidoreductase has been isolated from ovine fetal blood by a 2,370-fold purification scheme of ammonium sulfate fractionation, calcium phosphate gel adsorption, affinity chromatography, and fast performance liquid chromatography. A new high performance liquid chromatography-based assay for measuring 20 alpha-reductase activity is described. The enzyme is a monomer with a molecular weight of 35,000 and uses NADPH as a cofactor for reductase activity. It reduces progesterone to 4-pregnen-20 alpha-ol-3-one or 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol with kinetic characteristics of Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1 or Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1, respectively. 5 alpha-Dihydrotestosterone competitively inhibits 20 alpha-reductase activity with a Ki value of 102 microM.     

Effects of electrical stimulation upon post-hatching development of fibre types in normally innervated fast and slow latissimus dorsii muscles of the chicken

In chicken, the main characteristic properties of muscle fibre types in slow anterior (ALD) and fast posterior (PLD) latissimus dorsii are acquired during post-hatching development. At day 4 it becomes possible to distinguish between alpha' and beta' fibre types in ALD muscle. At the same time, mATPase staining and NADH-TR activity permit recognition of alpha w and alpha R fibres within PLD muscle. During further development, muscle fibre typology progressively changes towards the adult slow and fast type. Chronic stimulation at a slow rhythm (5 Hz) of PLD prevents the change in relative proportions of alpha R and alpha W fibres within the muscle that occurs in normal post-hatching development and increases the number of beta R fibres. Moreover, oxidative activity is increased in all muscle fibre types following stimulation. In ALD muscle, chronic stimulation at a fast rhythm (40 Hz) results in a decrease in oxidative activity and inhibits the differentiation of alpha' and beta' muscle fibre types. This study demonstrates that in young chicken, the pattern of activity influences the differenciation of fibre types in slow and fast muscles.     

The reaction of cytochromes c and c2 with the Rhodospirillum rubrum reaction center involves the heme crevice domain

In order to define the interaction domain on Rhodospirillum rubrum cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged carboxydinitrophenyl lysines. The reaction mixture was separated into six different fractions by ion exchange chromatography on carboxymethylcellulose and sulfopropyl-Sepharose. Peptide mapping studies indicated that fraction A consisted of a mixture of singly labeled derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2. Fractions C1, C2, C3, and C4 were found to be mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The photooxidation of the carboxydinitrophenyl-cytochrome c2 derivatives by reaction centers purified from R. rubrum was measured following excitation with a laser pulse. The second-order rate constant of fraction A modified at backside lysines was found to be 2.3 X 10(7) M-1 s-1, nearly the same as that of native cytochrome c2, 2.6 X 10(7) M-1 s-1. However, the rate constants of fractions C1-C4 were found to be 6 to 12-fold smaller than that of native cytochrome c2. These results indicate that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 27, 72, 79, or 87 surrounding the heme crevice was found to significantly lower the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.     

Production and characterization of human renin antibodies with region-oriented synthetic peptides

Polyclonal and monoclonal antibodies were raised against pure human renin, but nothing was known about the regions against which they were directed. Using a three-dimensional model of mouse submandibular renin, we selected seven peptide sequences as belonging to potential epitopes. The main criteria for their choice were the location of the peptide sequences near the catalytic region and on the surface of the renin molecule and their hydrophilicity. After transposition of the regions to the 340-amino acid sequence of human renin, the seven peptides (corresponding to amino acids 50-60, 63-71, 81-90, 118-126, 162-169, 247-255, and 287-295) were synthesized, coupled to bovine serum albumin, and injected into rabbits. Five of these peptides elicited antibodies, and 50-68% binding of the corresponding iodinated peptide was obtained with a 1:25 dilution of antiserum. The antisera titers ranged from 1:5,000 to 1:100,000 when tested by enzyme-linked immunosorbent assay. The same antisera bound 15-65% of labeled pure human renin at a final dilution of 1:2.5, the highest percentage being obtained with peptide 81-90 antiserum. At a 1:5 dilution, the five antisera inhibited renin activity by 23-68% in human plasma with a high renin activity (40 ng of angiotensin I/h/ml). At a final dilution of 1:50, peptide 81-90 antiserum was still capable of producing 25% inhibition. Purified IgG (0.6 mg) from this antiserum inhibited pure human renin activity by up to about 40%, as measured by its reaction with pure synthetic human tetradecapeptide substrate. Antigenic peptides that mimic a part of the human renin sequence, especially peptide 81-90 representing the "flap" covering the cleft between the two renin lobes, constitute promising tools for the development of a synthetic antirenin vaccine.     

Elevation of glutathione in phenylalanine mustard-resistant murine L1210 leukemia cells

Murine L1210 leukemia cells resistant to the antineoplastic agent L-phenylalanine mustard have a 1.5-2.0-fold elevation in their cellular GSH and GSSG content as compared to drug-sensitive cells. Cellular uptake of L-[U-14C]cystine and its incorporation into GSH of the resistant tumor are correspondingly elevated. Synthesis of gamma-glutamylcysteine, GSH, and GSSG is elevated 1.5-2.0-fold in cell-free preparations of the resistant tumor. This increased synthesis of GSH is attributed to increased cellular content (1.6-fold) of gamma-glutamylcysteine synthetase. GSH synthetase activity is equivalent in both drug-sensitive and -resistant cells. Investigation into the hydrolysis of selected peptides by cell-free preparations of both sensitive and resistant tumors suggest that aminopeptidase M participates in the formation of L-cysteine from L-Cys-Gly. This is supported by the observation that these preparations readily degrade L-Leu-p-nitroanilide and L-Ala-L-Ala-L-Ala, known substrates for aminopeptidase M, but not dipeptidase. The failure of the tumors to degrade Gly-D-Ala, a dipeptidase substrate, and the marked inhibition of L-Ala-Gly, L-Cys-Gly, and L-Ala-L-Ala-L-Ala hydrolysis by Bestatin further support a role for aminopeptidase M in the generation of L-cysteine from L-Cys-Gly. These results suggest that the drug-resistant tumor cell has developed an efficient mechanism for maintenance of elevated GSH which involves both gamma-glutamyl transpeptidase-initiated catabolism of GSH to cysteine and its reutilization by gamma-glutamylcysteine synthetase.     

Isolation and reconstitution of the dicyclohexylcarbodiimide-sensitive proton pore of the clathrin-coated vesicle proton translocating complex

The clathrin-coated vesicle proton translocating complex is composed of a maximum of eight polypeptides. The function of the components of this system have not been defined. Proton pumping catalyzed by the reconstituted, 200-fold purified proton translocating complex of clathrin-coated vesicles is inhibited 50% at a dicyclohexylcarbodiimide (DCCD)/protein ratio of 0.66 mumol of DCCD/mg of protein. At an identical DCCD/protein ratio, the 17-kDa component of the proton pump is labeled by [14C]DCCD. Through toluene extraction, the 17-kDa subunit has been isolated from the holoenzyme. The 17-kDa polypeptide diminished proteoliposome acidification when coreconstituted with either bacteriorhodopsin or the intact clathrin-coated vesicle proton translocating ATPase. In both instances, treatment of the 17-kDa polypeptide with DCCD restored proteoliposome acidification. Moreover, the proton-conducting activity of the 17-kDa polypeptide is abolished by trypsin digestion. These results demonstrate that the 17-kDa polypeptide present in the isolated proton ATPase of clathrin-coated vesicles is a subunit which functions as a transmembranous proton pore.     

Properties of bovine heart mitochondrial cytochrome b560

A large-scale preparation of the two-subunit protein complex (QPs) that converts succinate dehydrogenase into succinate-ubiquinone reductase from cytochrome b-c1 particles is achieved by a procedure involving Triton X-100 solubilization and calcium phosphate column chromatography at different pH values. The isolated two-subunit QPs contains 25 nmol of cytochrome b560/mg of protein and is able to reconstitute with soluble succinate dehydrogenase to form a TTFA-sensitive succinate-ubiquinone reductase. The maximum reconstitutive activity is 100 mumol of succinate oxidized per min per mg of QPs protein at 23 degrees C. Although cytochrome b560 in isolated QPs is not succinate reducible and its dithionite reduced form is reactive to carbon monoxide, cytochrome b560 is shown to be physically associated with succinate dehydrogenase by the following observations. The dithionite reduced form of cytochrome b560 in isolated QPs has a symmetrical alpha-absorption peak, which upon reconstitution with succinate dehydrogenase becomes slightly broadened and shows a shoulder at around 553 nm, identical to that of cytochrome b560 in succinate-ubiquinone reductase. Upon addition of succinate dehydrogenase to QPs, about 50% of the reduced form of cytochrome b560 in the QPs becomes insensitive to carbon monoxide treatment. The redox potential of cytochrome b560 in QPs is -144 mV which is higher than that of cytochrome b560 in succinate-ubiquinone reductase (-185 mV). Upon addition of succinate dehydrogenase, the redox potential of about 46% of the cytochrome b560 in QPs preparation becomes identical to that of cytochrome b560 in succinate-ubiquinone reductase. Cytochrome b560 in the QPs preparation shows two epr signals, g = 3.07 and g = 2.92, whereas cytochrome b560 in succinate-ubiquinone reductase exhibits only one epr signal at g = 3.46. When QPs is reconstituted with succinate dehydrogenase to form succinate-ubiquinone reductase, the g = 3.46 epr signal reappears at the expense of the g = 3.07 signal. Based on epr measurement at liquid helium temperature, about 18% of the total cytochrome b in the isolated active succinate-cytochrome c reductase is cytochrome b560, indicating that cytochrome b560 is indeed a unique cytochrome b and not a denatured product of cytochrome b562 or b565.     

A soluble interleukin 2 receptor produced by a normal alloreactive human T cell clone binds interleukin 2 with low affinity

Several alloreactive human T cell clones derived from a rejected kidney graft were found to produce in their culture supernatants soluble interleukin 2 receptors (IL-2R) upon specific antigenic challenge (irradiated B cell line from the graft's donor). Among them, the 2B11, a high producer clone, was used to purify a soluble IL-2R preparation which was analyzed, in comparison with the high and low affinity cell-surface IL-2R expressed by 2B11 cells, for its parameters of interaction with a set of anti-IL-2R monoclonal antibodies (mAb) and IL-2. This soluble receptor purified by affinity chromatography (anti-IL-2R mAb column) and sodium dodecyl sulfate gel electrophoresis is composed of a single chain of 35,000 to 45,000 Da. Immunoradiometric assays (IRMA) at equilibrium were set up, using pairs of mAb directed against two separate epitopes on the Tac antigen of the human IL-2R, to measure the respective dissociation constant of these mAb for the soluble IL-2R. They were found to be identical to those found on the cell-surface IL-2R. A 1:1 stoichiometry between the two epitopes were found both on the membrane and soluble species. Competition experiments between membrane and soluble IL-2R for binding the mAb allowed the quantitative analysis of the concentration of soluble IL-2R without the need of amino acid analysis on purified material and set up a quantitative IRMA for the human soluble IL-2R (detection limit 5 pM). The affinity of the soluble IL-2R for IL-2 was determined by various techniques including an IRMA using an anti-IL-2R mAb and radiolabeled IL-2. The results obtained led us to conclude that the soluble IL-2R binds IL-2 with a dissociation constant (KD = 30 nM) identical to that found for the binding of IL-2 to low affinity cell-surface IL-2R (Tac antigen). Whereas 2.5% of cell-surface IL-2R expressed 2 days after antigenic stimulation of 2B11 cells were of high affinity for IL-2 (KD = 25 pM), no (less than 0.07%) high affinity binding sites could be detected on the purified soluble IL-2R. This soluble IL-2R therefore likely corresponds to a truncated, extracellular part of the membrane Tac antigen. The amounts of soluble Tac antigen produced by the 2B11 alloreactive human T cell clone did not exceed 1 nM and, as expected from the binding studies, did not affect IL-2-induced T cell proliferation. The physiologic and pathologic implications of our results are discussed.     

Myasthenogenicity of human acetylcholine receptor synthetic alpha-subunit peptide 125-147 does not require intramolecular disulfide cyclization

This study reports the synthesis of a disulfide-looped peptide corresponding to residues 125-147 (Cys 128-Cys 142) of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle, H alpha 125-147 (Lys-Ser-Tyr-Cys-Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Glu-Gln- Asn-Cys-Ser-Nle-Lys Leu-Gly), and a nondisulfide-looped analogue, H alpha 125-147(S) (Lys-Ser-Tyr-Ser-Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Glu- Gln-Asn-Cys-Ser-Nle-Lys-Leu-Gly), in which the amino acid Cys 128 was replaced with serine. Both peptides induced antigen-specific helper T cell responses, as evidenced in vitro by lymph node cell proliferation and in vivo by production of anti-AChR antibodies. Rats immunized with 100 micrograms of either synthetic peptide, without conjugation to a carrier, produced anti-peptide antibodies which bound to native AChR in immunoprecipitation assays and induced modulation of membrane-bound AChR from cultured human myotubes. Both peptides also induced electrophysiologic and biochemical signs of experimental autoimmune myasthenia gravis. Thus, region 125-147 of the AChR alpha-subunit is at least partly exposed extracellularly in human muscle and contains one or more autoantigenic sites capable of stimulating T cells and B cells. Disulfide-linkage between residues Cys 128 and Cys 142 is not essential for myasthenogenicity.     

Diastereomers of thymidine 3'-O-(methanephosphonothioate): synthesis, absolute configuration and reaction with 3'-methoxyacetylthymidine under conditions of triester approach to oligonucleotide synthesis

Diastereomers of the title compound were obtained and absolute configuration was assigned by means of stereochemical correlation. Their reaction with 3'-O-methoxyacetylthymidine in the presence of triisopropylbenzenesulphonyl (4-nitro) triazole is neither chemo- nor stereo-selective and leads to diastereomeric pairs of dithymidyl (3',5')methanephosphonate and -methanephosphonothioate. Obtained results are discussed in terms of mechanism of activation of phosphodiesters under conditions known as "phosphotriester approach to oligonucleotide synthesis".