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Biomedical subjects

J Wolff

Publications and source records attributed to J Wolff.

At least 253 records · Page 14Linked to original sources
PubMed1976

Extracytoplasmic adenylate cyclase of Bordetella pertussis.

Soluble adenylate cyclase [EC 4.6.1.1] accumulates in the culture medium of exponentially growing Bordetella pertussis (300-900 pmol of cAMP formed/min per ml of 24 hr culture supernatant). In addition, there is an extracytoplasmic adenylate cyclase which enables the intact organisms to form [32P] cAMP (adenosine 3':5'-cyclic monophosphate) from exogenous [alpha-32P] ATP (200-1200 nmol of cAMP formed/min per g wet weight of cells) and which comprises 20-45% of the total adenylate cyclase activity. In contrast, only 1.7 and 2.4% of the total cell malate dehydrogenase [EC 1.1.1.37] and alkaline phosphatase [EC 3.1.3.1], respectively, are detectable in the intact cell. Trypsin treatment of intact organisms destroys 96% of the extracytoplasmic adenylate cyclase, but does not reduce the total cell malate dehydrogenase or a small pool of intracellular adenylate cyclase. Four compartments of adenylate cyclase in B. pertussis are proposed; (A) soluble enzyme in the culture supernatant (up to 20% of the total activity); (B) enzyme associated with intact cells and measurable without cell disruption (20-45%); (C) extracytoplasmic enzyme sensitive to trypsin, but not measurable in intact cells at standard substrate concentrations (40-60%); and (D) intracellular enzyme (7-9%). In comparison with previously studied bacterial adenylate cyclases, the extracytoplasmic location appears to be unique to the B. pertussis enzyme.

Adenylyl Cyclases↗
PubMed1976

Tubulin aggregation and disaggregation: mediation by two distinct vinblastine-binding sites.

Rat brain tubulin possesses two distinct binding sites for vinblastine per molecule: a high-affinity site with an affinity constant of 6.2 x 10(6) M-1 and a low-affinity site with an affinity constant of 8 x 10(4) M-1. The high-affinity site is labile, with a t1/237 degrees of 3.5 hr, is protected by colchicine, and is unaffected by salt, whereas the low-affinity site is stable but is inhibited by salt. Binding to both sites is rapid. The high-affinity binding constant of vinblastine to tubulin (6.2 x 10(6) M-1) corresponds to the half-maximal concentration of vinblastine needed to prevent polymerization of tubulin in vitro, whereas the low-affinity binding constant (8 x 10(4) M-1) corresponds to the half-maximal concentration of vinblastine required to aggregate tubulin. We conclude that vinblastine binding to the high- and low-affinity sites, respectively, accounts for the depolymerization and aggregation behavior of tubulin.

Animals↗
PubMed1976

Soluble adenylate cyclase from the culture medium of Bordetella pertussis: purification and characterization.

Culture medium of exponentially growing Bordetella pertussis (strain 114) contains significant quantities of soluble (100,000 X g for 1 h) adenylate cyclase. The enzyme was purified by chromatography on diethylaminoethyl-cellulose and Sephadex G-200. The purest material yielded a single band on sodium dodecyl sulfate-disc gel electrophoresis. It is heat labile, has a temperature optimum of 30 degrees C, a pH optimum of pH 7 to 8, and a Km for adenosine 5'-triphosphate of 0.4 mM, and requires Mg2+ for maximum activity. The molecular weight, by sodium dodecyl sulfate-disc gel electrophoresis and sucrose density gradient, is approximately 70,000. The enzyme is markedly inhibited by fluoride and weakly inhibited by monovalent salts, but its activity is not altered by alpha-keto acids of nonsubstrate nucleoside triphosphates. Thus, but its presence in the culture supernatant, its smaller molecular weight, and its insensitivity to alpha-keto acids and nucleotides, this enzyme differs from the bacterial adenylate cyclases previously described.

Adenosine Triphosphate↗
PubMed1976

Regulation of adenylate cyclase in two rat liver cell lines, 3C4 and 62.

Adenylate cyclase (EC 4.6.1.1) activities were examined in membrane preparations from two rat liver cell lines (62 and 3C4) which were grown in monolayer cultures. The cells were epithelial-like in growth character. Adenylate cyclase from the line 62 was stimulated by epinephrine, Gpp(NH)p, and prostaglandins A1,A2,E1,E2, and F2alpha, but not by glucagon. Arrhenius plots of adenylate cylase activity from line 62 gave straight lines, except when epinephrine was present in the assay; epinephrine-stimulated activity gave a distinct break at 20 degrees C. Adenylate cyclase activity in line 3C4 was stimulated by glucagon ten times greater than by epinephrine. It was responsive to Gpp(NH)p and all the prostaglandins. Arrhenius plots of adenylate cyclase activity of line 3C4 always gave straight line curves. Prostaglandins flattened the straight line curves (allowed temperature independence) of adenylate cyclase activity in membranes from both cell lines.

Adenylyl Cyclases↗
PubMed1976

Thyroid-pituitary feedback during iodine repletion.

The changes in serum triiodothyronine (T3), thyroxine (T4), and thyrotropin (TSH) were measured during iodine repletion in a woman who was severely iodine-deficient because of a congenital iodide-trapping defect. Serum T3 became detectable 12 h after iodine was begun and reached 68 ng/dl, a level within the normal range, by 36 h. It rose progressively during the first 9 days reaching a supra-normal level (200 ng/dl) and then fell slowly to 130 ng/dl. Serum T4 was not detected (less than 1 mug/dl) until the 9th day and then rose to 6.8 mug/dl. Serum TSH fell rapidly during the first 9 days (disappearance rate was 0.17/day); there was a significant negative correlation with serum T3 (P less than 0.01). From the 10th through the 32nd day serum TSH fell more slowly (disappearance rate was 0.05/day) and correlation was with serum T4 (P less than 0.001), not T3. It appears that either T3 or T4 may regulate TSH secretion by direct effects on the thyrotroph, although their intracellular mode of action has not been defined.

Adolescent↗
PubMed1975

Choleragen stimulates steroidogenesis and adenylate cyclase in cells lacking functional hormone receptors.

Choleragen stimulates steroid secretion and adenylate cyclase in three cell lines, adrenal tumor line (Y-1), a corticotropin-resistant mutant derived from Y-1 called OS-3, and a receptor-deficient Leydig tumor line (I-10). Sensitivity for half-maximal stimulation varies from 3 to 36 pM choleragen, the I-10 line being the most sensitive. Latency before the onset of steroidogenesis is longer in OS-3 and I-10 cells than in the Y-1 line. In both OS-3 and I-10 cells choleragen stimulates adenylate cyclase whether ITP or 5'-guanylylimidodiphosphate is the regulatory cofactor used. In addition to the responses of the receptor-deficient lines, choleragen does not, during its latency, block the response to corticotropin in Y-1 cells; corticotropin does not block binding of 125I-labeled choleragen to Y-1 cells; gangliosides do not interfere with the corticotropin-induced stimulation of Y-1 cells. We conclude that the corticotropin and choleragen receptors are different.

Adenylyl Cyclases↗
PubMed1975

Endotoxic lipopolysaccharides stimulate steroidogenesis and adenylate cyclase in adrenal tumor cells.

Lipopolysaccharides (endotoxins) from Escherichia coli, Serratia marcesens and Salmonella typhosa stimulated steroid production in Y-1 adrenal tumor cells in culture with a latent period of 3-4 h. Lipid A, derived from Escherichia coli lipopolysaccharide, also stimulated steroidogenesis. Lipopolysaccharides and lipid A also stimulate adenylate cyclase activity and cause rounding of the cells. In contrast, lipopolysaccharides do not stimulate steroidogenesis in receptor-deficient adrenal tumor cells (OS-3) or Leydig tumor cells (I-10). This tends to rule out contamination by enterotoxin to which these lines respond. Although both hormone and lipopolysaccharide responses are lost in these lines, there was no interaction between these sites as judged by the failure of lipopolysaccharides to block, during their latency, the response to corticotropin in Y-1 cells. The possibility that the lipopolysaccharide effect is one on membrane conformation is discussed.

Adenylyl Cyclases↗
PubMed1975

Charge effects in the activation of adenylate cyclase.

Polycations, including ribonuclease A, ribonuclease S protein and peptide, spermine, spermidine, and polylysines, enhance unstimulated and stimulated adenylate cyclase activity of beef thyroid membranes at low concentrations and inhibit these activities at high concentrations. Peak polylysine stimulation occurs with degrees of polymerization of 6 to 14, and for large polymers a potency limit for this maximum is reached at 4 X 10(-5) M expressed as lysine residues. Both enhancement and inhibition appear to be due to charge-charge interactions and are abolished by KC1. Polyanions are inhibitory only. The biphasic effect of polycations is seen on basal cyclase activity, occurs with prostaglandin E1- and 5'-guanylyl-imidodiphosphate-stimulated cyclase, but is most striking with thyrotropin. There is little enhancement of F--activated cyclase. The enhancement is not sensitive to changes in pH, Mg2+, or regenerating system and does not correlate with the stability constants between polycations and ATP. We suggest that the polycation effect is a general, electrostatic effect on membrane conformation and is not restricted to a particular receptor domain.

Adenylyl Cyclases↗
PubMed1975

Interactions of bovine neurophysins with neurohypophyseal hormones. On the role of tyrosine-49.

Reaction of tetranitromethane with the lone tyrosine residue of bovine neurophysin I and II, tyrosine-49, gave nitro derivatives of these proteins which were obtained in a highly purified form by preparative electrophoresis. Equilibrium dialysis experiments indicated clearly that oxytocin binding remained essentially unaffected by the chemical modification of tyrosine-49. However, in the case of (8-lysine)vasopressin, the nitrated protein was found to bind only 1 hormone molecule in contrast to the 2 vasopressin molecules bound by the native protein. Ultraviolet absorption difference spectroscopy measurements between 250 nm and 300 nm indicated that upon binding of (2-phenylalanine, 8-lysine)vasopressin, tyrosine-49 of native neurophysin undergoes a change of microenvironment from less to more polar surroundings. Studies of the nitrotyrosyl-49 chromophore of neurophysin by ab sorption spectroscopy in the absence and presence of oxytocin or (8-lysine)vasopressin confirmed this finding. Since dimethylsulfoxide solvent perturbation studies suggested that in the Cys(Me)-Phe-Ile-NH2-neurophysin I complex, tyrosine-49 is more exposed to solvent than in neurophysin I alone, it is concluded that this residue is unmasked by conformational changes upon complex formation.

Amino Acids↗
PubMed1975

New method for the removal of extraneous proteins from purified oncornaviruses.

Conventional methods (i.e. gradient centrifugation) for the purification of oncornaviruses are usually not effective in complete removal of nonviral proteins. Such contaminants often prove to be a nuisance in subsequent immunological or biochemical studies. Hyperimmune sera prepared from these viruses must be absorbed to assure specificity; cell-derived proteins can be shown to interfere with studies of virus structural proteins, nucleic acids, or viral enzymes. Herein is described a method for removal of most of these contaminants. Viruses are diluted in a high concentration of NaCl to achieve a final concentration of 15%, incubated for 30 min, sedimented, and resuspended in buffer. This procedure results in reductions of up to 48% of the protein without affecting particle count. Immunological, biochemical, and biological properties are not adversely affected. Of the proteins removed, fetal calf serum components and a ribonuclease (presumably cell-derived) were identified. This technique differs significantly from other high-salt methods in that the virus is not precipitated from suspension. It is believed that absorbed proteins are desorbed and left in solution (or suspension) as the virus is sedimented by centrifugation.

Cell Line↗