Biomedical subjects

J E Zull

Publications and source records attributed to J E Zull.

At least 19 recordsLinked to original sources

PubMed1994

Nucleic acid sequences coding for internal antisense peptides: are there implications for protein folding and evolution?

We have asked whether coding segments of nucleic acids generate amino acid sequences which have an antisense relationship to other amino acid sequences in the same chain (i.e. 'Internal Antisense'), and if so, could the internal antisense content be related to the structure of the encoded protein? Computer searches were conducted with the coding sequences for 132 proteins. The result for each search of a specific sequence was compared to the mean result obtained from 1000 randomly assembled nucleic acid chains whose length and base composition were identical to that of the native sequences. The study was conducted in all three reading frames. The normal reading frame (frame one) was found to be contain lower amounts of internal antisense than the randomly assembled chains, whereas the frame two results were much higher. The internal antisense content in frame three was not significantly different from that in the random chains. The amount of internal antisense in frames two and three was correlated with the GC content at the center position of the codons in that frame, but this correlation was absent in frame one. No correlation with chain length was found. Qualitatively similar results were obtained when the random model was limited to retain the same purine/pyrimidine ratio as the native chains at each position in the codons, but in this case the internal antisense in frame three was also significantly greater than the computer-generated sequences. The results suggest that the internal antisense content in the correct reading frame has a qualitatively different origin from that in the other two frames. The high amount in frames two and three is apparently an artifact resulting from the asymmetric distribution of G and C in the codons, while the low amount in frame one may suggest evolutionary selection against internal antisense. Thus, the results do not support a relationship between internal antisense and protein structure.

Amino Acid Sequence↗

PubMed1994

Problems and approaches in covalent attachment of peptides and proteins to inorganic surfaces for biosensor applications.

Some of the fundamental problems in covalent attachment of peptides and proteins to putative biosensor surfaces are reviewed and specific approaches to these problems discussed. In addition, selected aspects of our recent work utilizing self-assembled monolayer (SAM) systems designed to react selectively with the thiol side chain of Cys in proteins are presented. Uniform attachment of a 21-amino acid peptide antigen through a single Cys residue with retention of biological function (antibody binding) has been attained. Further work with this system may lead to solutions for some of the problems which currently prevent the development of reliable biosensors for industrial and medical use.

Amino Acid Sequence↗

PubMed1990

Effect of methionine oxidation and deletion of amino-terminal residues on the conformation of parathyroid hormone. Circular dichroism studies.

Circular dichroism (CD) studies of parathyroid hormone (PTH), its oxidized forms, and some fragments of the hormone are described. The CD spectrum of native PTH (84 amino acids) and the active fragment, 1-34 PTH, suggests that most of the secondary structure resides in the amino-terminal segment of this hormone. Oxidation of the methionine residue at position 18 has a small impact on secondary structure, whereas oxidation of the methionine at position 8 produces substantial changes. Oxidation of both methionines produces secondary structure changes that are greater than the sum of those seen upon oxidation of the individual methionines. The CD spectrum for the 3-34 fragment of PTH is identical to that of the 1-34 fragment, and that of the 7-34 fragment is only slightly different. The spectra of the 13-34 and 19-34 fragments are markedly altered from that of the 1-34 peptide, and those of the 9-84 and 19-84 fragments of native PTH are significantly different from the intact hormone. Computer-assisted estimates of secondary structure content, and difference spectra, were utilized to evaluate the secondary structure content of the peptides. These results suggest that residues 6-12 are important in formation of helical secondary structure and that a reverse turn may be important for the folding of PTH into a conformation with high affinity for receptors. Residues 1 and 2 appear to make no contribution to the secondary structure and may be directly involved in activation of receptors.

Animals↗

PubMed1990

Characterization of the interaction of parathyroid hormone with the mitochondrial ATPase.

Parathyroid hormone (PTH) has been shown to bind specifically to the beta subunit of the mitochondrial ATPase on nitrocellulose blots. We have now examined this interaction further, using intact mitochondria, submitochondrial particles, and the purified F1 ATPase. With intact mitochondria, 1 microM concentrations of PTH and its biologically active 1-34 fragment activate the ATPase about 3-fold. This effect was reduced to a 1.4-fold activation with 3-34 and 7-34 fragments of the hormone, and oxidized PTH gave no detectable activity. Activation could only be observed below pH 7. PTH had no significant effect on the activity of the purified enzyme or on submitochondrial particles. However, specific binding of an iodinated PTH analog, [Nle 8,18-Tyr 34] bPTH (1-34) amide, was found with submitochondrial particles and the purified ATPase. Binding affinity with the purified enzyme was about 10(-3) that of the plasma membrane receptor, and the molar stoichiometry was close to 1:1 (PTH:intact enzyme). With submitochondrial particles the affinity was about 10-fold higher than with the purified enzyme. This binding was further examined with PTH derivatives and fragments, and compared to that seen in the plasma membrane receptor. Oxidation of methionine 18 in PTH reduced the affinity about 50%, oxidation of methionine 8 reduced the affinity 95%, and oxidation of both methionines further decreased affinity in both membranes and submitochondrial particles. However, when compared to the native hormone, the 3-34 and 7-34 PTH fragments had much higher affinity for the submitochondrial particles than for the plasma membranes. PTH also reduced chemical crosslinking of the ATP analog, p-fluorosulfonyl benzoyl 5'-adenosine, to the alpha subunit of this enzyme, but did not alter labeling of the enzyme with 3'-O-(4'-benzoyl) benzoyl ATP, suggesting that the hormone binds near a regulatory nucleotide binding site. Direct chemical crosslinking of PTH to the beta-subunit of the enzyme was attained with a cleavable, photoactivate crosslinker, sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl-1,3-dithiopropionate. The crosslinked protein was cleaved with cyanogen bromide and the labeled fragments were sequenced. The labeled fragments were found to be segments of the protein which have previously been implicated as being close to the noncatalytic ATP binding sites.

Animals↗

PubMed1990

Is genetic code redundancy related to retention of structural information in both DNA strands?

We have noted that the sense-antisense relationships inherent in the genetic code divide the amino acids into three separate groups. The nature of the amino acids in each group may allow the polypeptides coded by the antisense strand to retain the secondary structure patterns of the translated strand. Also, this relationship requires all but eight of the codons in the eukaryotic code and all but four in the mitochondrial code. Thus, genetic code redundancy could be related to evolutionary pressure toward retention of protein structural information in both strands of DNA.

Amino Acids↗

PubMed1989

Examination of parathyroid hormone antisera for the presence of receptor antibodies.

Proteins from bovine kidney membranes were separated by denaturating polyacrylamide gel electrophoresis and blotted onto nitrocellulose paper. The blots were immunostained with parathyroid hormone (PTH) antisera, and the effect of the presence of PTH on immunostaining was determined. Immunostaining of membrane proteins by two specific antisera was altered by PTH. With one antiserum, the immunostaining of two specific proteins (apparent mass 90 and 105 kDa) was prevented by PTH. With the second antiserum the immunostaining of a 150 kDa protein was prevented by the hormone. These effects were strongest with the 90 and 150 kDa proteins and these were investigated further. Antibody binding was prevented either by co-incubation or by preincubation of the blots with PTH, followed by washing and subsequent exposure to the antisera. Concentrations of PTH as low as 1 nM prevented antibody binding to the 90 kDa species, but somewhat higher PTH concentrations were required with the 150 kDa protein. Oxidation of the PTH methionine residues in the amino terminal segment of PTH, and deletion of the first nine residues in the hormone greatly reduced the competition with the 90 kDa protein, but had no effect on immunostaining of the 150 kDa species. The 35-84 fragment of PTH was not a competitor for the 90 kDa species, while the 1-34 fragment was ineffective with the 150 kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Effects of selective oxidation of 1-34 bovine parathyroid hormone on its renal actions in the rabbit.

Oxidation of both of the methionine residues (positions 8 and 18) in parathyroid hormone (PTH) eliminates many of its biological effects. The present studies were performed to examine the actions of 1,34 bovine PTH and 1-34 bovine PTH oxidized selectively at Met 8, at Met 18, and at both sites on renal electrolyte handling and on adenylate cyclase (AC) stimulation. In clearance studies in anesthetized rabbits, PTH caused a phosphaturia and an anticalciuria. PTH also stimulated renal proximal tubular AC in vitro and increased renal cortical cAMP content in vivo. PTH oxidized at Met 18 was anticalciuric, but not phosphaturic, stimulated renal AC and increased cortical cAMP content. PTH oxidized at Met 8 also produced an anticalciuria without a phosphaturia, but only weakly stimulated AC and did not alter cortical cAMP content. PTH oxidized at both Met 8 and Met 18 was phosphaturic but not anticalciuric, was a weak agonist for AC and decreased cortical cAMP content. In the isolated perfused rabbit proximal straight tubule, PTH inhibited fluid and phosphate transport, whereas the doubly oxidized peptide was inactive. The data are consistent with the possibility that the effects of PTH on renal tubular phosphorus transport are mediated by more than one mechanism and are, in part, independent of the cAMP messenger system.

Adenylyl Cyclases↗

PubMed1988

The beta-subunit of the bovine mitochondrial F1 ATPase specifically binds the amino terminal domain of parathyroid hormone.

A protein which specifically binds the amino terminal domain of parathyroid hormone (PTH) on nitrocellulose blots of polyacrylamide gels was fragmented with cyanogen bromide (CNBr), and two fragments were sequenced through 20 residues. The sequence obtained was 100% homologous with the beta-subunit of bovine F1 mitochondrial ATPase. Purified F1 ATPase from bovine heart and Escherichia coli were obtained and the binding of PTH examined on the blots. The beta-subunit of the bovine enzyme bound PTH specifically through its amino terminal domain. However, both the alpha- and beta-subunit of the E. coli enzyme were found to bind the hormone. This binding was also specific for the amino terminal domain of the hormone. The subcellular distribution of the PTH-binding protein from bovine kidney was also examined further. While the mitochondria and plasma membrane appear to possess similar PTH-binding capability, submitochondrial particles enriched in F1 ATPase were also enriched in PTH-binding activity.

Amino Acid Sequence↗

PubMed1987

Identification and purification of a kidney membrane protein which specifically binds the amino-terminal domain of native parathyroid hormone.

Nitrocellulose blots of bovine kidney membrane proteins were prepared from denaturing polyacrylamide gels. Strips of the blots were incubated with parathyroid hormone (PTH), washed, and then incubated with antisera against the hormone. Exposure to horseradish peroxidase-linked second antibody led to staining of a 51-kDa protein. No staining was observed in blots not incubated with PTH. Fragments 35-84 and 19-84 of PTH reacted strongly with the antisera, but did not lead to staining of the 51-kDa protein on the blots. Staining was visible, but greatly reduced, when fragment 9-84 was used. Oxidation of the native hormone at positions 8 and 18 led to reductions in staining of the band which were quantitatively similar to the reductions in biological activity induced by such oxidations. These properties suggested that the 51-kDa protein recognizes the amino-terminal portions of PTH, which is the segment of the molecule required for its biological activities. Several micrograms of the 51-kDa protein were purified to homogeneity by selective extraction from the membranes with detergent and by elution from multiple two-dimensional gels. The purified protein retained its PTH-dependent staining and specificity. This protein may be a PTH receptor or a fragment of a PTH receptor from kidney.

Animals↗

PubMed1987

Proton NMR studies of the biologically active 1-34 fragment of bovine parathyroid hormone: examination of a structural model.

Proton NMR spectra of the biologically active 1-34 fragment of bovine parathyroid hormone (bPTH) were studied as a function of pH over the range of pH 4 to 10, in buffer and in 6 M guanidine DC1. One of the histidine C-2 peaks titrated normally, with a pKa value of 6.8, but the other two histidines in this peptide had pKa values of 6.3. Denatured PTH showed only one histidine C-2 peak with a pKa of 6.7. An aliphatic peak identified as due to either a methionine or a glutamine residue also shifted with pH, and the pKa for this shift was 6.3. Finally, small but significant upfield shifts in the methyl and methylene resonances were observed as a function of pH, and when compared to the denatured peptide. These results indicate that the N-terminal domain of native PTH has considerable structure in solution, and are consistent with a theoretical model for the folding of this peptide.

Animals↗

PubMed1987

Deletion of lysine 13 alters the structure and function of parathyroid hormone.

A peptide of unknown structure was found as a side product in a commercial preparation of the 1-34 fragment of bovine parathyroid hormone (PTH). CNBr cleavage and amino acid analysis showed that this peptide is the des-lys-13 form of 1-34 bovine PTH. The peptide thus represents a deletion mutant of PTH and structure-function studies are of interest. This peptide was a full agonist in the adenylyl cyclase bioassay for PTH, but its potency was about 5% of that found for the complete 1-34 peptide. Proton NMR studies showed that the pK values for the histidine residues in the des-lys-13 form were essentially identical to those of the intact peptide. However, pH-dependent changes in the chemical shifts for the tryptophan protons (residue 23) and several unidentified methyl group resonances were observed in the des-lys peptide. The latter are major shifts and probably represent ring-current effects; these were not seen in the intact 1-34 peptide. The results show that Lys-13 is important in the folding of the active domain of PTH, and are interpreted in the context of a previously published model for the folding of this hormone.

Amino Acid Sequence↗

PubMed1986

Production of biologically active fragments of parathyroid hormone by isolated Kupffer cells.

Cleavage of parathyroid hormone (PTH) by isolated Kupffer cells from rat liver was examined. Iodinated PTH labeled at position 43 was converted into two radioactive fragments which were shown by Edman degradation to have residues 35 and 38 as their NH2 termini. Cleavage at these positions is characteristic of cathepsin D. Amino-terminal fragments were detected by bioassay of fractions obtained by high performance liquid chromatography. These fragments eluted in positions characteristic of the 1-34 and 1-37 peptides also previously shown to be produced by purified cathepsin D. The putative 1-37 fragment was rapidly converted to 1-34 upon digestion with cathepsin D, whereas the putative 1-34 fragment was not further digested by this enzyme, behavior previously shown to be characteristic of 1-37 and 1-34 bovine PTH. Fragmentation of PTH as measured by generation of fragments soluble in trichloroacetic acid was inhibited by methylamine, monensin, and ammonium chloride. In addition, monensin significantly inhibited production of both carboxyl- and amino-terminal fragments. Finally, active PTH fragments were also produced by elicited peritoneal macrophages. It is concluded that Kupffer cells, and other macrophages, can produce active fragments of PTH which appear in the medium. These fragments may be generated by cathepsin D within the cells.

Adenylyl Cyclases↗

PubMed1986

The role of the methionine residues in the structure and function of parathyroid hormone.

Forms of the biologically active N-terminal fragment of bovine parathyroid hormone oxidized at methionine 8, methionine 18, and both positions were prepared, separated from one another, and characterized as described earlier for the native hormone (A. L. Frelinger and J. E. Zull, (1984) J. Biol. Chem. 259, 5507). The biological properties of the oxidized forms were compared to those of the native hormone, using the renal membrane adenylyl cyclase assay. Oxidation at position 18 produced full agonists of the hormone with slightly reduced potency. Oxidation at position 8 produced partial agonists of greatly reduced potency. Oxidation at both positions produced partial agonists of even lower potency. Thus, methionine 8 is implicated both in binding and in activation of adenylyl cyclase, but methionine 18 is implicated only in binding. Further study showed that oxidation of both residues is dependent on the pH, ionic strength, and polarity of the solvent. However, methionine 8 is less easily oxidized than methionine 18. This difference is eliminated in 3 M guanidine-HCl with 1-34 and in 6 M guanidine-HCl with 1-84. On the other hand the difference in reactivity is greatly increased in high ionic strength, with methionine 8 becoming much less reactive. These results suggest that the methionine residues are important in the biologically active conformation of parathyroid hormone and that methionine 8 is less accessible than methionine 18 under certain conditions. These conclusions are discussed in the context of a specific model for the folding of parathyroid hormone.

Adenylyl Cyclases↗

PubMed1985

ATP activation of protein degradation by extracts of crude and purified lysosomal preparations.

Activation of proteolysis by ATP was studied in lysates of crude and purified lysosomal preparations from liver and kidney at acid pH. In the crude system, from kidney, it was found that ATP activates proteolysis over a concentration range of 0.1-2 mM. Up to 4-fold activation was observed. GTP and CTP also activated proteolysis, but to a lesser extent. Proteolysis was inhibited by vanadate and molybdate. Fractionation of the kidney lysosomes on Percoll gradients produced two fractions containing lysosomal marker enzymes. Most of the acid phosphatase and the acid pyrophosphatase were found in the lighter band, while most of the beta-galactosidase and cathepsin activity was found in a more dense band. Proteolysis by lysates of both fractions was activated by ATP and inhibited by vanadate and molybdate. In the dense band proteolysis was also nearly totally blocked by pepstatin, and was enhanced by an inhibitor of pyrophosphatases, sodium fluoride. ATP also activates proteolysis in crude lysosomes from liver, but upon fractionation of this tissue it was found that all the lysosomal enzyme markers are present in the dense fraction obtained from the Percoll gradient. Again, proteolysis by lysates of the purified fractions was activated by ATP and inhibited by vanadate and molybdate. These data indicate that ATP can activate proteolysis at acid pH in a lysosomal milieu containing enzymes which also catalyze its breakdown. In the kidney there may be two lysosomal compartments which separate the enzymes catalyzing ATP breakdown from the proteolytic enzymes, but this is not essential for ATP activation as shown by the data from the liver and the crude lysosomal fractions.

Adenosine Diphosphate↗

PubMed1985

Effects of ATP, vanadate, and molybdate on cathepsin D-catalyzed proteolysis.

The effects of ATP, vanadate, and molybdate on cathepsin D-catalyzed hydrolysis of proteins and peptides were examined. Hydrolysis of bovine serum albumin, hemoglobin, parathyroid hormone, and a synthetic octapeptide was activated by ATP. Degradation of the protein substrates all had similar ATP concentration dependence, but the magnitude of the activation varied. Kinetic constants for ATP activation were obtained with a synthetic substrate. ATP increased kcat from 0.4 to 2 s-1 but did not change KM. Kact for ATP was 800 microM. Studies with pepstatin-Sepharose confirm that ATP does not alter the substrate binding site on cathepsin D. Pepsin, a homologous aspartate protease, was not activated by ATP. It was also found that vanadate and molybdate inhibit cathepsin D-catalyzed proteolysis. However, this inhibition was dramatically dependent on substrate concentration and was eliminated at high substrate. Hydrolysis of the synthetic peptide was not inhibited at concentrations of molybdate below 50 microM, and above this concentration the peptide precipitated. Protein substrates were also found to precipitate in the presence of molybdate. The ATP dependence of the enzyme was not altered by molybdate or vanadate. These results suggest that inhibition by vanadate and molybdate is related to interactions with the substrate rather than with cathepsin D. It is concluded that ATP activation of cathepsin D may play a physiological role in regulation of proteolysis in lysosomes, but that vanadate and molybdate inhibition of lysosomal proteolysis does not establish ATP dependence.

Adenosine Triphosphate↗

PubMed1985

Characterization of parathyroid hormone fragments produced by cathepsin D.

Cleavage of parathyroid hormone by cathepsin D was studied. Four primary products were detected and separated by high performance liquid chromatography. Two of the fragments are fluorescent and therefore contain residue 23 (tryptophan). These fragments are NH2-terminal in origin. The other two cross-react with antisera directed against COOH-terminal portions of the hormone; they are the complementary COOH-terminal fragments. Microsequencing and amino acid analysis showed that the two COOH-terminal fragments are 35-84 and 38-84 bovine parathyroid hormone. By CNBr cleavage and amino acid analysis, the two NH2-terminal fragments were shown to be the complementary 1-37 and 1-34 fragments. The 1-37 fragment is transitory and is rapidly hydrolyzed to 1-34, so that only relatively small amounts are detected at any one time. However, 34-84 was not converted to 38-84, although cleavage at other sites in the COOH-terminal fragments was observed with more exhaustive digestion. The 1-34 fragment appears to be the final product of the action of cathepsin D on parathyroid hormone. Both enzymatically produced NH2-terminal fragments were fully active in the renal membrane adenylyl cyclase assay system.

Adenylyl Cyclases↗

PubMed1985

The effects of vanadate and molybdate on cathepsin D; relationship to ATP activation of lysosomal proteolysis.

The effects of the phosphate analogues, vanadate and molybdate, on the ATP-activated enzyme, cathepsin D, were investigated. Both were found to inhibit proteolysis but this appeared to be the result of non-specific interactions with the protein substrates which result in precipitation, rather than interactions with the enzyme. Inhibition of proteolysis was induced by the same concentration of inhibitors as that which induced precipitation (measured by turbidity), and was dependent on the concentration of substrate. Precipitation did not occur at neutral pH but was maximal below pH 5. High concentrations of salt (greater than 1M KC1) prevented precipitation of proteins by vanadate and molybdate and under these conditions little inhibition of proteolysis was observed even at high inhibitor concentrations. Nonetheless, ATP was found to activate proteolysis catalyzed directly by lysosomal enzymes at acid pH, while vanadate and molybdate inhibited proteolysis in this system and induced precipitation of substrate. These results indicate that inhibition of proteolysis at acid pH by vanadate (or molybdate) has no relationship to inhibition of proteases and/or ATP dependence of such enzymes. However, direct activation of cathepsin D in lysosomes by ATP remains a viable hypothesis.

Adenosine Triphosphate↗

PubMed1984

Oxidized forms of parathyroid hormone with biological activity. Separation and characterization of hormone forms oxidized at methionine 8 and methionine 18.

Bovine parathyroid hormone (PTH) was oxidized with hydrogen peroxide, and the oxidation products were separated by reverse phase high performance liquid chromatography. Using a shallow gradient, four major peaks (peaks I-IV in order of elution) were identified and completely separated from one another. Peak IV co-eluted with fully reduced PTH. The earliest eluting peak (peak I) could be reduced with mercaptoethylamine to produce all three of the later eluting ones. The second peak could be reduced back to peak IV but not to peak III, while peak III could be oxidized to peak I but not to peak II. These data plus the kinetics of oxidation showed that peaks II and III are intermediate in the generation of I from IV or IV from I, but were not generated from one another. Amino acid analysis showed that peak I contains no methionine and two residues of methionine sulfoxide, peaks II and III one methionine sulfoxide each, and peak IV two residues of methionine and no sulfoxide. Study of the peptides produced from each form of PTH by cleavage with cyanogen bromide showed that peak II is oxidized at methionine 8 and peak III at methionine 18 while peak I is oxidized at both methionines. The biological activity of each peak was determined in the kidney membrane adenylyl cyclase assay. All forms were active but with widely varying potencies (peak IV greater than peak III greater than peak II greater than peak I).

Adenylyl Cyclases↗