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Biomedical subjects

J Wolff

Publications and source records attributed to J Wolff.

At least 235 records · Page 13Linked to original sources
PubMed1978

Soluble adenylate cyclase from thyroid membranes.

Adenylate cyclase from purified beef thyroid membranes has been solubilized by the use of Triton N-101 after preactivation with guanosine 5'-(beta, gamma-imido)-triphosphate. The soluble activity passed a 0.22- micron filter, was not sedimented at 100,000 X g for 2 h, and behaved like aldolase in sucrose density gradients and on Sepharose 6B. From comparison of the sedimentation in D2O and H2O the partial specific volume was found to be like that of globular proteins (0.75 +/- 0.006), hence little detergent appeared to be bound to the enzyme. The sedimentation coefficient was 7.4 +/- 0.15, the Stokes radius 45 A, and the molecular weight 159,000. Prestimulation by thyrotropin did not survive solubilization. The stimulation produced by guanosine 5'-(beta, gamma-imido)triphosphate persisted as did the more active state resulting from pretreatment with both this nucleotide plus thyrotropin. Thyrotropin did not stimulate the solubilized enzyme. The Km for ATP, thermal stability, and inhibition by Ca2+ were identical for the membrane-bound and soluble enzyme, while the pH optimum was increased 0.5 unit in the latter. Polyanions and phenothiazines inhibited both preparations equally, whereas only membranes responded to stimulation by polylysine and ribonuclease.

Adenylyl Cyclases↗
PubMed1978

Synthesis and characterization of N-(2,4-diphosphobenzyl)-1-amino-5-naphthalenesulfonic acid, a new fluorescent analogue of diphosphoglyceric acid.

The synthesis of N-(2,4-diphosphobenzyl)-1-amino-5-naphthalenesulfonic acid (DIPANS) is described. It entails the synthesis of 2,4-diphosphobenzaldehyde from the action of POCl3 on 2,4-dihydroxybenzaldehyde. This is followed by coupling of the 2,4-diphosphobenzaldehyde to 1-amino-5-naphthalenesulfonic acid. Subsequent reduction with NaBH4 yields the desired product. The DIPANS exhibits an excitation maximum at 337 nm and a fluorescence emission maximum at 504 nm. This dye is quantitatively displaced by inositol hexaphosphate and is an effective analogus of diphosphoglyceric acid (DPG), possessing a KD at pH 7.0 in 0.05 M [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (bis-Tris) plus 0.1 M chloride of 6.88 microgram, with 1.0 molecule bound/hemoglobin tetramer. Like DPG its binding to deoxyhemoglobin decreases with increasing pH; in the presence of 0.1 M chloride it binds 0.031 times as tightly to CO hemoglobin and it yields a value for free energy coupling of 2.0 kcal/mol. The presence of 1 mM DIPANS decreases the affinity of hemoglobin for oxygen in the absence of salt from p1/2 of 0.8 mm Hg to 12.4 mm Hg. Using DPG as a competitor of DIPANS binding, a dissociation constant of 11.4 micrometer was calculated for DPG binding to deoxy-Hb at pH 7.0 in the presence of 0.05 M bis-Tris and 0.1 M chloride.

Binding, Competitive↗
PubMed1978

Bordetella pertussis does not induce beta-adrenergic blockade.

Bordetella pertussis organisms induce histamine sensitivity and diminish the normal hyperglycemic response to epinephrine in experimental animals. These effects have been attributed to beta-adrenergic blockade. However, under conditions in which the decrease in epinephrine-induced hyperglycemia after B. pertussis administration was demonstrable, there was no change in rat reticulocyte beta-adrenergic receptor number or affinity measured by iodohydroxybenzylpindolol binding or in isoproterenol-stimulated adenylate cyclase activity. Therefore, there was no generalized beta-adrenergic blockade induced by B. pertussis. The observed effects can be explained by the hypersecretion of insulin resulting from B. pertussis administration.

Adenylyl Cyclases↗
PubMed1977

Melittin interactions with adenylate cyclase.

Melittin, a basic polypeptide from bee venom, inhibits basal and thyrotropin-stimulated adenylate cyclase of beef thyroid membranes with a Ki approximately 10 micron. Although this property resides in the basic C-terminal and not the N-terminal portion of the molecule, inhibition is due primarily to its detergent-like nature rather than charge effects. There is also a small enhancing effect of both basal and thyrotropin-stimulated adenylate cyclase of 0.3-3 micron melittin.

Adenylyl Cyclases↗
PubMed1977

Podophyllotoxin as a probe for the colchicine binding site of tubulin.

The binding of [3H]podophyllotoxin to tubulin, measured by a DEAE-cellulose filter paper method, occurs with an affinity constant of 1.8 X 10(6) M-1 (37 degrees at pH 6.7). Like colchicine, approximately 0.8 mol of podophyllotixin are bound per mol of tubulin dimer, and the reaction is entropy-driven (43 cal deg-1 mol-1). At 37 degrees the association rate constant for podophyllotoxin binding is 3.8 X 10(6) M-1 h-1, approximtaely 10 times higher than for colchicine; this is reflected in the activation energies for binding which are 14.7 kcal/mol for podophyllotoxin and 20.3 kcal/mol for colchicine. The dissociation rate constant for the tubulin-podophyllotoxin complex is 1.9 h-1, and the affinity constant calculated from the ratio of the rates is close to that obtained by equilibrium measurements. Podophyllotxin and colchicine are mutually competitive inhibitors. This can be ascribed to the fact that both compounds have a trimethoxyphenyl ring and analogues of either compound with bulky substituents in their trimethoxyphenyl moiety are unable to inhibit the the binding of either of the two ligands. Tropolone, which inhibits colchicine binding competitively, has no effect on the podophyllotoxin/tubulin reaction. Conversely, podophyllotoxin does not influence tropolone binding. Moreover, the tropolone binding site of tubulin does not show the temperature and pH lability of the colchicine and podophyllotoxin domains, hence this lability can be ascribed to the trimethoxyphenyl binding region of tubulin. Since podophyllotoxin analogues with a modified B ring do not bind, it is concluded that both podophyllotoxin and colchicine each have at least two points of attachment to tubulin and that they share one of them, the binding region of the trimethoxyphenyl moiety.

Animals↗
PubMed1977

Activation of steroidogenesis and adenylate cyclase by adenosine in adrenal and Leydig tumor cells.

Steroidogenesis by Y-1 adrenal tumor cells in culture is stimulated by ATP, adenyl-5'-yl imidodiphosphate (App(NH)), adenosine 5'(beta, alpha-methylene)triphosphate (App(CH2)p), ADP, AMP, NAD, FAD, and adenosine but not by adenine or other nucleoside triphosphates. ATP, App(NH)p, App(CH2)p, and adenosine are active in the micromolar range. Like adrenocorticotropic hormone (ACTH), the onset of stimulation is immediate and occurs to the same extent. Also active are 2'- and 5'-deoxyadenosine and 2-chloroadenosine whereas adenine xyloside, L-riboside, or arabinoside have very low activity. Stimulation is accompanied by rounding of the cells. Dipyridamole, an inhibitor of adenosine transport, increased the response to low concentrations of adenosine, suggesting that adenosine acts externally. Stimulation of steroidogenesis by adenosine or phosphorylated adenosine compounds fails to occur in the presence of crystalline adenosine deaminase, and the effect of the enzyme on adenosine, ATP, or NAD stimulation is reversed by the competitive inhibitor erythro-9-[3-(nonane-2-ol)]adenine. This suggests that the enzyme acts specifically on adenosine and a requirement for the conversion of the above compounds to adenosine seems probable. The inhibition of cAMP effects by adenosine deaminase suggests that some of its effects are also mediated by conversion to adenosine. Similar stimulation is seen in I-10 Leydig tumor cells, but an ACTH-resistant mutant of Y-1 cells, called OS-3, is relatively resistant to adenosine. Adenosine and 2-chloroadenosine stimulate adenylate cyclase in membranes from Y-1 and I-10 cells at concentrations slightly greater than are effective for steroidogenesis. Other nucleosides are ineffective. Like the NH2-terminal 24 residues of adrenocorticotropic hormone (1-24 ACTH), the adenosine effect in Y-1 membranes is rapid and is on the Vmax intercept (versus ATP) and not on the Km. In contrast to steroidogenesis, adenosine is only a partial agonist for adenylate cyclase. It effect occurs in the presence of ITP, GTP, or guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Theophylline inhibits adenosine-stimulated steroidogenesis. Inhibition of adenylate cyclase occurs in the same concentration range but is of the mixed type.

Adenosine↗
PubMed1977

Two distinct adenosine-sensitive sites on adenylate cyclase.

The effects of adenosine and adenosine analogs on adenylate cyclases from several tissues have been examined. Two adenosine-reactive sites have been identified: (i) the "R" site, occupancy of which usually leads to activation of cyclase and which requires integrity of the ribose ring for activity, and (ii) the "P" site, which mediates inhibition and requires integrity of the purine ring for activity. Biphasic effects of adenosine are explained by the presence of both sites on a single adenylate cyclase. Comparison of these data with those in the literature indicates that adenosine-reactive "P" and "R" sites are present generally.

Adenosine↗
PubMed1977

Adenyl cyclase in Bordetella pertussis vaccines.

Brodetella pertussis organisms have a large amount of extracytoplasmic adenyl cyclase, part of which is found in the supernatant culture medium during exponential growth. The enzyme differs from previously studied bacterial adenyl cyclases in biochemical characteristics as well as in location. Several commercial pertussis vaccines were found to contain adenyl cyclase activity; this activity is probably due to the extracytoplasmic enzyme associated with and released from the whole cells in the vaccine.

Adenylyl Cyclases↗
PubMed1976

Anion-induced increases in the rate of colchicine binding to tubulin.

The rate of binding of colchicine to tubulin to tubulin is enhanced by certain anions. Among the inorganic anions tested, only sulfate was effective. The organic anions include mostly dicarboxylic acids, among which tartrate was the most effective. This effect occurs onlt at low concentrations of colchicine (less than 0.6 X 10(-5) M). The rate increase dor sulfate and L-(+)-tartrate is ca. 2.5-fold at 1.0 mM and plateaus at a limiting value of ca. 4-fold at 100mM. The overall dissociation rate of the colchicine from the complex, which includes both the true rate of dissociation and the rate of irreversible denaturation of tubulin, is not influenced by 1.0 mM tartrate. The affinity constants for colchicine determined from the rate constants are 8.7 X 10(6) and 2.1 X 10(7) M-1 in the absence and the presence of 1.0 mM L-(+)-tartrate. The limiting value is 3.2 X 10(7) M-1. The affinity constant calculated from steady-state measurements is 3.2 X 10(6) M-1 with or without anions. The binding of other ligands like podophyllotoxin, vinblastine, and 1 -anilino-8-naphthalenesulfonate to tubulin is not affected by tartrate. No major conformational changes resulting from anion treatment could be detected by circular dichroism or intrinsic fluorescence. However, the ability of tubulin to polymerize is inhibited by L-(+)-tartrate at concentrations that increase the rate of colchicine binding. We conclude that anions must have a local effect at or near the binding site which enhances the binding rate of colchicine and which may be related to inhibition of polymerization.

Anilino Naphthalenesulfonates↗
PubMed1976

The treatment of Wilms' tumor: Results of the national Wilms' tumor study.

The National Wilms' Tumor Study, initiated in 1969, tested competing treatment strategems for patients with tumors ranging from Group (Gp) I (tumors confined to the kidney and totally removed) to Gp IV (remote metastases present at diagnosis). Three hundred and fifty-nine of 606 registered patients were randomized in the trial. Gp I patients under 2 years of age fared well whether postoperative radiation therapy (RT) was or was not added to 15 months' maintenance actinomycin D (AMD). Their prognosis was better than that for older cohorts similarly treated, in whom the difference in relapse rates between treatment groups were suggestive of an RT effect. Combined AMD and vincristine (VCR) gave better results than either agent alone in patients with more advanced tumors (Gps II and III) still confined to the abdomen, all of whom received postoperative RT as well. Preoperative VCR given Gp IV patients in addition to postoperative RT, AMD, and VCR did not improve results. The frequency of mesoblastic nephroma (1%), of bilateral tumors (5%), and of incorrect preoperative diagnosis of Wilms' tumor (5%), the toxicities of the various regimens, and other ancillary data are presented and discussed.

Age Factors↗
PubMed1976

Clinically significant cardiac infiltration in acute leukemia, lymphocytic lymphoma, and plasma cell myeloma.

Cardiac infiltration by hematologic neoplasms leading to clinically significant cardiovascular disease is rare. Three such cases are described in this report, and it is suggested that rare manifestations of hematologic neoplasms may become more common in the future since these diseases are more amenable to therapy than heretofore. Cardiac involvement with hematologic neoplasms is of more than academic interest since this complication is likely to respond to radiotherapy.

Adult↗