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J E Zull

Publications and source records attributed to J E Zull.

At least 37 records · Page 2Linked to original sources
PubMed1984

Preparation and characterization of radioactive monoiodotyrosine and diiodotyrosine derivatives of parathyroid hormone.

Highly purified native parathyroid hormone was iodinated by the enzymatic method and separated from unlabeled hormone by isocratic HPLC. The separation system used also resolved iodohistidine, monoiodotyrosine, and diiodotyrosine forms of the hormone from one another. A simplified procedure for direct bioassay of the carrier-free, high specific activity, mono- and diiodinated parathyroid hormone (PTH) by the renal membrane adenylyl cyclase method was also developed. Both labeled forms of the hormone are very potent in this assay, but the iodinated forms appeared to give a lower Vmax than the native hormone. The methods for iodination, separation and biological characterization of this PTH tracer are exceptionally facile, inexpensive, and convenient.

Adenylyl Cyclases↗
PubMed1983

ATP activation of parathyroid hormone cleavage catalyzed by cathepsin D from bovine kidney.

The acid protease which is activated by ATP and which catalyzes production of fragments of parathyroid hormone similar to those produced in vivo was shown to be cathepsin D. Purified cathepsin D from bovine kidney and spleen is activated by ATP and other nucleoside triphosphates, and to a much lesser extent by nucleoside diphosphates and pyrophosphate. These findings suggest that cathepsin D may be involved in parathyroid hormone metabolism in vivo and that this lysosomal enzyme may be regulated by the energy status of the cell.

Adenosine Triphosphate↗
PubMed1983

Identification of an ATP-activated endopeptidase from rat kidney which catalyzes cleavage of parathyroid hormone to fragments identical to those produced in the rat kidney in vivo.

An endopeptidase catalyzing cleavage of parathyroid hormone to specific C-terminal and N-terminal fragments was identified in a partially purified membrane fraction from rat kidney. Fractionation on sucrose gradient showed that this activity is present primarily in a light membrane fraction rather than in the basal-lateral membranes, or in the classic lysosomal fraction. The endopeptidase can be extracted from the membranes by freezing and thawing, it has an acid pH optimum, and it catalyzes production of specific fragments of PTH. The major C-terminal fragment produced has its N-terminus at residue 39 of the native hormone. This fragment is identical to the primary PTH fragment found in kidney tissue following injection of iodinated PTH into the systemic circulation of rats (D'Amour et al., 1979). Finally, the cleavage of PTH by this acid endopeptidase is activated by physiological concentration of ATP (10(-4) - 10(-3)M). These results suggest that this enzyme may be involved in PTH catabolism by the kidney, that it may be located in a specialized cell fraction and that hormone catabolism may be regulated by the energy status of the cell.

Adenosine Triphosphate↗
PubMed1981

Specific cleavage of bovine parathyroid hormone catalyzed by an endopeptidase from bovine kidney.

Cleavage of parathyroid hormone (PTH) is catalyzed by an endopeptidase associated with a partially purified membrane preparation from bovine kidney cortex. This enzyme was found to have an acid pH optimum and to be easily extracted from the membranes by a single freeze-thaw cycle. The cleavage is remarkable in that it appears to be restricted to a small region of the PTH peptide chain, generating fragments which are not further degraded. The dominant products are a large fragment, COOH-terminal in origin, and a small fragment from the NH2 terminus. The small fragment is biologically active and its activity establishes that it contains at least the first 29 amino acids in PTH. The large fragment has no biological activity. The cleavage of PTH was demonstrated both with iodinated PTH and with unlabeled hormone by immunoassay and by labeling the large fragment after its production. Microsequencing of the large fragment showed that, in fact, two products are produced: one with its NH2 terminus at position 38 of PTH and one with its NH2 terminus at position 35. These fragments are remarkably similar to those generated in both the liver and the kidney in vivo.

Animals↗
PubMed1980

A theoretical study of the structure of parathyroid hormone.

Theoretical analysis of the tertiary and secondary structure of parathyroid hormone was conducted. By combining interpretations from this analysis with chemical data available in the literature, certain structural features of the hormone are consistently predicted. The proposed model for the hormone contains two domains dominated by hydrophobic clustering of critical residues within each domain and separated by an exposed linker region. In the prediction of two domains with a linker region, the model is similar to that proposed by Fiskin et al. [Fiskin, A.M., Cohn, D.M. & Peterson, G.S. (1977) J. Biol. Chem. 252, 8261-8268], but it differs significantly in other respects. The proposed structural features are apparent in the bovine, human, and porcine species of hormone.

Amino Acid Sequence↗
PubMed1980

Activation of bovine renal cortex membrane adenylyl cyclase by low concentrations of parathyroid hormone.

Under conditions designed to reduce non-specific adsorption of parathyroid hormone (PTH), the sensitivity of the renal membrane adenylyl cyclase to PTH was significantly enhanced. Stimulation of the enzyme could be observed at hormone concentrations as low as 2 x 10-11M. In addition, kinetic analysis of hormone activation revealed that under conditions where non-specific adsorption is great, downward concavity of Eadie-Hofstee plots is observed, whereas when such adsorption is reduced these plots become upwardly concave. These results suggest that in the absence of non-specific adsorption, PTH activation of kidney membrane adenylyl cyclase can occur at hormone concentrations approaching physiological. In addition, at low hormone concentrations PTH activation is probably more complex than previously recognized.

Adenylyl Cyclases↗
PubMed1980

Kidney membrane binding of native parathyroid hormone compared to binding of its synthetic 1 - 34 fragment.

Kidney membrane binding of tritiated native parathyroid hormone (PTH) was compared to that of its active fragment 1 - 34 PTH. Native hormone specific binding is transient and disappears rapidly (integral of 30 minutes) at 37 degrees C but is stable up to 2 hours at 0 degrees C. The rate of binding loss appears relatively independent of the amount of hormone-receptor complex or of the amount of cold PTH in the medium. Loss of specific binding is also seen with iodinated PTH. Loss of specific binding of native PTH does not appear to be the result of enzymatic degradation of the hormone since significant amounts of intact hormone are present, both bound to membranes and in the medium after incubation. Biologically active tritiated 1 - 34 PTH binds specifically to isolated membrane, and both total and specific binding is stable for at least one hour at room temperature. The pH dependence for binding of 1 - 84 PTH and its activation of adenylyl cyclase are very similar, but differ from that reported for specific binding of 1 - 34 PTH. these results suggest that the interaction of native PTH with kidney cells may be more complex than that of its 1 - 34 fragment.

Adenylyl Cyclases↗
PubMed1978

Development of parathyroid hormone- and calcitonin-activated adenylate cyclases in embryonic chicken limb and in cultured cells from embryonic chicken limb.

Activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by parathyroid hormone (PTH) and calcitonin was measured as a function of stage of development in embryonic chicken limb buds. Responsiveness to both hormones develops in the tissue at the time when nascent bone is forming. In addition, a temporal sequence of development of hormone response was observed, with a PTH-activated adenylate cyclase appearing earlier than the calcitonin-activated enzyme. The responsiveness to the two hormones was additive, indicating the presence of two receptor populations. Undifferentiated cells obtained from limb buds prior to appearance of hormonal responsiveness were cultured and were found to develop a PTH-activated adenylate cyclase in vitro. However, a calcitonin-stimulated enzyme did not appear in such cultures. The PTH-activated enzyme was found to be similar to that present in bone in regard to its sensitivity to PTH. The enzyme did not respond to other hormones, and myoblast cultures did not develop a PTH-activated adenylate cyclase, indicating that a true bone adenylate cyclase was being measured.

Adenylyl Cyclases↗
PubMed1977

Binding of tritiated bovine parathyroid hormone to plasma membranes from bovine kidney cortex.

A membrane fraction enriched in parathyroid hormone (PTH)-sensitive adenylate cyclase and sodium and potassium ion-activated (Na+, K+)-ATPase was prepared from bovine kidney. Tritiated PTH binding to this membrane fraction was dependent on both hormone and membrane protein concentration. Both total and specific binding of the hormone decreased significantly after 5 to 10 min of incubation at 22 degrees. PTH binding was highly specific, being sensitive to inhibition only with active forms of unlabeled hormone (native and 1-34 PTH). Specific binding showed a pH optimum of 7.3 to 7.5. Inhibition of binding of tritiated hormone by unlabeled PTH was also highly effective at pH 6.0, but this apparently specific binding was also inhibited by adrenocorticotropic hormone, insulin, glucagon, and vasopressin. Dissociation of bound hormone was demonstrated, and an apparent dissociation constant of 4.6 X 10(-2) min-1 was obtained. Specific binding was eliminated by pretreatment of the membranes with trypsin. The concentration dependence for inhibition of binding with unlabeled PTH was identical to that for activation of adenylate cyclase in this membrane preparation, and binding was also inhibited by concentrations of calcium in the 0.5 to 2 mM range.

Adenylyl Cyclases↗
PubMed1977

Studies of binding of parathyroid hormone to a detergent-dispersed preparation from bovine kidney cortex plasma membranes.

Bovine kidney plasma membranes containing parathyroid hormone-sensitive adenylate cyclase activity were dispersed with 1% Triton X-100 and centrifuged at 150,000 X g for 2 h. Approximately 40% of the total membrane protein was extracted by this procedure. The extraction greatly reduces the fluoride-stimulated and the parathyroid hormone-sensitive adenylate cyclase activity of the membranes and yields a supernatnat which binds biologically active, tritiated parathyroid hormone. Hormone binding is stable for up to 15 h and has a linear dependence on protein concentration in the extract. Binding of the labeled hormone at concentrations of 5 to 10 nM is inhibited by preincubation with unlabeled min, and displays a dependence on temperature, time, and pH. Binding specificity is maximal at physiological pH, being inhibited by only the native hormone or its synthetic 1-34 NH2-terminal, biologically active fragment. Binding increases dramatically at pH 6.0, but is nonspecific in character. Half-maximal inhibition of the binding was achieved at 3.2 X 10(-7) M concentrations of the native hormone and 5.0 X 10(-7) M concentrations of the synthetic 1-34 NH2-terminal fragment. Calcium does not inhibit either total or specific binding. Inhibition, kinetic, and pH dependence data suggest that the extracted component(s) represent the parathyroid hormone binding protein(s) formerly identified in particulate membrane preparations.

Animals↗
PubMed1975

Further studies on acetamidination as a technique for preparation of a biologically valid 3-H-labeled tracer for parathyroid hormone.

The technique of acetamidination of amino groups in parathyroid hormone (PTH) for the purpose of preparation of a tritiated, biologically valid tracer for this hormone, has been more extensively studied. It was found that eight of the ten amino groups in PTH are readily reactive but that two appear unreactive. The kinetics of labeling suggest that preparations of PTH which are labeled to this level of 80% of theoretical consist of a homogeneous population of molecules in which each PTH molecule contains eight tritiated acetamidino groups. There is no question of the presence of unlabeled hormone in such preparations. Eighty per cent labeled acetamidino-PTH is identical, qualitatively and quantitatively, in its biological activity with native PTH as shown by three accepted bioassays: serum calcium elevation, urine phosphate excretion, and activation of kidney cortex plasma membrane adenylate cyclase in vitro. The dose-response relationships are identical for labeled and native hormone in all three systems. Tritiated acetamidino-PTH tends to lose biological activity on storage but full activity can be regained by reduction of the hormone with excess cysteine. However, cysteine reduction conducted at 80 degrees for 2 hours causes some loss of tritium from the hormone. Therefore, dithiothreitol reduction at room temperature was utilized to maintain biological activity. Eighty per cent acetamidino-PTH can be further purified by ion exchange chromatography on carboxymethylcellulose using a continuous gradient of sodium acetate in 8 M urea. Such chromatography reveals the presence of isohormones in both cold and radioactive PTH. Co-chromatography of tritiated acetamidino-PTH with a cold isohormone of PTH shows that while the isohormones can be separated from one another, the acetamidino derivatives of each isohormone elute in a virtually identical position with their parent unlabeled PTH molecule.

Adenylyl Cyclases↗
PubMed1975

Studies of a serum albumin-liposome complex as a model lipoprotein membrane.

Electron microscopy shows that the lipoprotein dispersions formed from the interaction of negatively charged liposomes with bovine serum albumin contain closed, vesicu lar, multilamellar structures. Discontinuous density gradient studies indicate that the lipoprotein suspensions are vesicles in which bovine serum albumin homogenously associates with lipid. Low angle X-ray diffraction results show that all the systems, positively and negatively charged, with and without protein, have the characteristic lamellar structure observed in biological membranes. The lamellar spacing (bilayer plus water layer) of negatively charged liposomes without bovine serum albumin is 55 A. The same lamellar separation in the positively charged system is 108 A. The lamellar spacing corresponding to bilayer, water, and protein for the negatively charged lipoprotein system is 93 A while that for the positively charged lipoprotein system is 91 A. These dimensions suggest that a layer of protein one molecule thick is incorporated between the lamellae bound to the surface of the bilayer. Wide angle X-ray diffraction results indicate no major effect of the protein on the 4.1 A spacing, characteristic of hexagonal packing of the hydrocarbon chains. A classical light scattering technique is used to show that the lipoprotein systems are osmotically active. The solute permeability exhibited by these lipoprotein systems follows the sequence (glucose smaller than arabinose smaller than malonamide smaller than glycerol). K+ diffusion from negatively charged lipoprotein systems is greater than that found for positively charged lipoprotein systems.

Animals↗
PubMed1975

Metabolism of bovine parathyroid hormone. Immunological and biological characteristics of fragments generated by liver perfusion.

The metabolism of bovine parathyroid hormone (PTH) by the perfused rat liver was studied. Labeled hormone, with or without cold hormone, was infused into the circulating perfusion medium containing various calcium concentrations. Pefusate samples at various time periods after the introduction of PTH into the system were chromatographed on Bio-gel P-10; radioactivity and/or immunoreactivity were measured in eluted fractions. Before the perfusion, all immuno- and radioactivity eluted in a single peak, with an apparent mol wt of 9,500 (peak I). After perfusion for 15 min, two other peaks with approximate mol wt of 7,000 (peak II) and 3,500 (peak III) were discernible. Peak I contained both NH2-terminal and COOH-terminal immunoreactivity and was biologically active at all time periods tested. The relative contribution of NH2-terminal and COOH-terminal immunoreactivity to the total immunoreactivity remained constant in this peak throughout the perfusion. In every respect, peak I had the characteristics of intact hormone. At all times, peak II consisted of only COOH-terminal immunoreactivity and was biologically inactive. At early time periods, peak III contained predominantly NH2-terminal immunoreactivity and was biologically active. With time, the relative contribution of NH2-terminal immunoreactivity decreased strikingly while that of COOH-terminal immunoreactivity increased. The three peaks identified in these experiments were analogous in size, biological activity, and immunological characteristics to those we have previously described for fractionated human hyperparathyroid serum. The rate of metabolism of PTH appeared to be regulated by the calcium concentration in the medium. At a high concentration of calcium (greater than 11 mg/100 ml), PTH metabolism was greatly retarded. At a low concentration of calcium (smaller than 5 mg/100 ml), the rate of metabolism was greatly increased. The physiological significance of our observations on the metabolism of PTH by isolated perfused rat liver is not known. However, since such metabolism results in a biologically active fragment, it is suggested that metabolism of intact hormone may be required before full biological expression is possible.

Animals↗