Biomedical subjects
Publications and source records attributed to J Wolff.
At least in boys hypogenitalism is a constant feature of the Prader-Labhart-Willi Syndrome. Our patient, a 16 10/12 years old girl had the characteristic symptoms of the syndrome and precocious puberty. Menarche had occurred at the age of 8 6/12 years. Precocious puberty does not exclude the diagnosis of a Prader-Labhart-Willi syndrome.
A variety of proteins and tissue preparations (rabbit erythrocyte lysate, catalase, peroxidase, creatine phosphokinase, and lima bean trypsin inhibitor) contain protein activator(s) of the extracellular adenylate cyclase of intact Bordetella pertussis organisms. Stimulation of adenylate cyclase activity of up to 1000-fold over basal activity can be obtained. Activation of the adenylate cyclase is due to the presence of calmodulin in these protein preparations. The criteria to establish this were: Ca2+ dependence of the activation, inhibition by trifluoperazine, heat stability of the activator, chromatographic behavior like authentic calmodulin, and stimulation of cyclic nucleotide phosphodiesterase by the activators. The great sensitivity of the B.pertussis adenylate cyclase assay makes this and ideal system for the detection of trace amounts of calmodulin, in the presence of large amounts of other proteins.
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Brain tubulin binds to lactoperoxidase coupled to Affigel 10 through a 10 A succinylated aminoalkyl spacer and can be eluted by an ionic strength gradient. The tubulin can be obtained about 90% electrophoretically pure in 2 to 3 h without glycerol or GTP. It retains its ability to bind colchicine. Compared to tubulin purified by the assembly-disassembly procedure, affinity-purified tubulin has a higher critical concentration for polymerization and the purified protein appears to be free of high molecular weight microtubule-associated proteins. Tubulin binding to the lactoperoxidase affinity column protects the colchicine binding site against decay at 4 degrees C, whereas the interaction of tubulin with soluble lactoperoxidase does not.
Antibodies to colchicine have been prepared by injecting rabbits with an antigen prepared by coupling deacetylcolchicine to bovine serum albumin with a water-soluble carbodiimide. Sera of high titer were obtained with apparent dissociation constants for colchicine ranging from 5 to 17 nM. Binding was rapid and occurred at low temperatures. The chemical specificity of the binding site(s) resembled that in tubulin for the A and B rings of colchicine. Unlike tubulin, the antibody binding site tolerated numerous changes in the tropolone moiety (C ring). In contrast to tubulin, binding of colchicine to the antibody site did not promote fluorescence. The colchicine binding capacity of the antibody was high enough to prevent colchicine fluorescence in the presence of tubulin and to restore polymerization in colchicine-inhibited tubulin preparations.
The molecular weight of NaF-activated, Triton N-101-solubilized adenylate cyclase from bovine thyroid membranes has been measured by a combination of sucrose density centrifugation and gel exclusion chromatography. The physical parameters are: sedimentation coefficient, 6.6 S; Stokes radium 41 A; partial specific volume, 0.75 ml/g; molecular weight, 119,000. This is in contrast to the molecular weight (159,000) of the enzyme from the same source activated with guanosine 5'-(beta, gamma-imido)triphosphate. Both soluble adenylate cyclase enzymes are subject to cholera toxin-mediated ADP-ribosylation. This implies that the diminished molecular weight of the NaF-activated solubilized adenylate cyclase is a consequence of one of the following: loss of a GTP-binding protein and labeling of some other protein in the cyclase complex; loss of another protein not subject to cholera toxin labeling; or that two GTP-binding proteins normally reside within the catalytic complex and upon NaF activation one is lost.
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Self-iodinated lactoperoxidase co-polymerizes with brain microtubules to constant specific activity and a stoichiometry of 0.2 to 0.3 lactoperoxidase molecule/tubulin heterodimer polymerized in the presence of 4 M glycerol. By contrast, iodinated tubulin loses its competence to polymerize. The lactoperoxidase-microtubule association is salt- and temperature-sensitive, shows considerable specificity, is saturable, and is reversible. Lactoperoxidase does not displace the microtubule-associated proteins from microtubules, does not promote polymerization, and binds to preformed microtubules. Self-iodinated lactoperoxidase also binds to tubulin oligomers at 0 degrees C and in the presence of CaCl2. The stoichiometry for this interaction is 0.6 to 0.8 molecules of enzyme/dimer. Lactoperoxidase forms a complex with soluble brain tubulin prepared by two cycles of polymerization and depolymerization or by phosphocellulose chromatography. The interaction was studied by sucrose gradient analysis, gel filtration, and spectral analysis based on the finding that tubulin binding to lactoperoxidase leads to a red shift in the Soret spectrum, yielding a difference spectrum with a minimum of 410 nm and maximum at 430 nm. This interaction involves one or more sulfhydryl groups of tubulin. Complex formation is relatively slow, is retarded by 0.6 M NaCl, and is accelerated by diiodotyrosine. By all three methods of analysis, the stoichiometry approaches a value of 2 lactoperoxidase molecules/tubulin dimer. There is a single class of binding sites in pig, beef, or rat tubulin with an apparent overall affinity constant of approximately 2.0 x 10(6) M-1. The molecular weight of the complex by sucrose gradient or gel filtration is approximately 140,000 i.e. half of the expected value for a 2:1 adduct. Since both alpha and beta subunits are present in the complex, we propose that the complex consists of a mixture of equal parts of presumably native alpha-tubulin-lactoperoxidase and beta-tubulin-lactoperoxidase.
Studies conducted with virus-infected monolayer cell cultures have demonstrated the feasibility of producing several tumor-associated viruses in microcarrier (mc) cultures (Sephadex G50 beads treated with DEAE-chloride). The efficiency of cell adherence to mc varied with the cell type, the pH of the growth medium, and the stirring force applied to keep the mc in suspension. Most cells attached firmly to the mc and could not be removed easily with routine trypsinization procedures. Techniques using Enzar-T and Pronase were effective in detaching cells from mc in 10 to 15 min while retaining 95% cell viability. After detachment, Ficoll gradients were used for rapid and complete separation of viable cell suspensions from the mc. Retrovirus production in large volumes of mc cultures was investigated with periodic harvesting of growth fluids. Physical, biochemical, and biological properties of the Mason-Pfizer monkey virus and the RD114 virus recovered from the mc cultures were identical to those produced in conventional cultures. The utilization of mc has several applications in research and short-term cultures, but the as-yet-unsolved technical problems met were found to be serious limitations when attempting mass cell culturing on a long-term basis.
Cell surface adenosine receptors mediate either stimulation or inhibition of adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], and the receptors that mediate these different responses can be discriminated with selected adenosine analogs. 5'-N-Ethylcarboxamide-adenosine is a more potent agonist at stimulatory receptors (Ra) than is N6-phenylisopropyladenosine, whereas the reverse potency order is seen with inhibitory receptors (Ri). The potency of adenosine is intermediate between the potencies of these two analogs. The relative potencies of adenosine receptor agonists are maintained in physiological responses in intact cells, such as steroidogenesis and inhibition of lipolysis. As with adrenergic receptors, subclasses of adenosine receptors differ functionally and pharmacologically.
The adenylate cyclase of Bordetella pertussis is stimulated 100- to 1000-fold in a dose-dependent manner by calf brain calmodulin. The system has the following properties. (i) The activation is prevented by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and restored by Ca2+. (ii) Oxidation of the methionine residues of calmodulin abolishes the ability to activate the cyclase. (iii) Trifluoperazine inhibits calmodulin-activated cyclase. (iv) A troponin C preparation stimulates the B. pertussis cyclase with < 0.01 the potency of calmodulin. Although calmodulin has not been demonstrated in prokaryotes, this is an example of a (eukaryotic) calmodulin effect in a prokaryote.
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The activity of Bordetella pertussis extracytoplasmic adenylate cyclase is 100-fold higher in organisms grown on blood agar than in those grown in synthetic medium. This increase in activity is due to in vivo activation of the enzyme by a factor present in erythrocytes. Activation also occurs in killed or disrupted organisms. The activator can be separated from heme proteins and has been purified approximately 100-fold from erythrocytes, yielding material of approximately 105,000 daltons. It is sensitive to trypsin and alpha-chymotrypsin and exhibits considerable heat stability. Activation of cyclase in intact B. pertussis organisms exhibits a lag of 3 to 4 min and is not reversed by washing. Response to the activator decreases with increasing purification of the adenylate cyclase and is absent in the pure enzyme. The activation does not appear to be proteolytic and does not appear to change access to the substrate, ATP. The activator has no effect on a number of eukaryotic cyclases. We conclude that this is a new type of activation and that the activator differs from all those previously described.
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Familial hyperproinsulinemia is an autosomal dominant defect that is associated with strikingly elevated levels of serum proinsulin-like material. Our studies show that trypsin converts familial hyperproinsulinemia proinsulin to insulin more slowly than it converts a 131I-labeled porcine proinsulin marker. Molar yields of insulin indicated that the material may be an intermediate proinsulin. Studies with two human C-peptide antisera that differ in their relative immunoreactivity with human C-peptide and proinsulin showed that the two antisera reacted equally with familial hyperproinsulinemia proinsulin, suggesting that it is a partially cleaved proinsulin intermediate. Sulfitolysis of highly purified material to break the inter- and intra-chain disulfide bridges and subsequent adsorption on a specific B-chain antibody covalently bound to Sepharose beads showed that the C-peptide was still connected to the B-chain. These data indicate that familial hyperproinsulinemia proinsulin is normally cleaved at the C-peptide-A-chain linkage site. A structural abnormality appears to underlie familial hyperproinsulinemia proinsulin, which impairs its cleavage at the B-chain-C-peptide linkage site.