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Biomedical subjects

B N Jones

Publications and source records attributed to B N Jones.

At least 37 records · Page 2Linked to original sources
PubMed1988

A rapid fluorometric assay for N-terminal glutaminyl cyclase activity using high-performance liquid chromatography.

A rapid, sensitive method for the quantification of glutaminyl cyclase activity has been developed. The assay involves enzymatic conversion of the model peptide Gln-Leu-Tyr-Glu-Asn-Lys-epsilon-(Dns)-OH to less than Glu-Leu-Tyr-Glu-Asn-Lys-epsilon-(Dns)-OH. Both the product and substrate of this reaction are detected in a single assay in quantities as low as 100 fmol using isocratic reverse phase high-performance liquid chromatography with fluorometric detection. The method is highly reproducible and ideally suited for the rapid analysis of large numbers of samples. The applications of the assay to both the detection of glutaminyl cyclase activity during enzyme purification and the more rigorous enzymology studies dependent on the precise measurement of initial reaction velocities are demonstrated.

Acyltransferases↗
PubMed1988

Purification of a peptidylglycine alpha-amidating enzyme from transplantable rat medullary thyroid carcinomas.

A peptidyl glycine alpha-amidating activity has been isolated from total tissue extracts of rat medullary thyroid carcinoma (MTC). Purification of the activity by ammonium sulfate fractionation, Sephacryl S-300 chromatography, and strong anion-exchange chromatography at pH 6.0 has resolved at least four peaks of activity. The activity associated with peak III has been further purified to apparent homogeneity by strong anion-exchange chromatography at pH 8.0. The purified peak III enzyme has an apparent molecular mass of 75,000 Da as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The identity of the 75,000-Da band as the alpha-amidating enzyme has been confirmed by recovery of activity from a nondenaturing polyacrylamide gel. The enzyme is catalytically active as a monomer, exhibits a pH optimum between 5.0 and 5.5, and has a turnover number of 300 min-1 for N-dansyl-Tyr-Val-Gly amidation at pH 5.5. The larger size, more acidic pH optimum, and higher specific activity distinguish the purified peak III rat MTC enzyme from the enzymes isolated from bovine and porcine pituitary or from frog skin.

Animals↗
PubMed1988

A fluorometric assay for peptidyl alpha-amidation activity using high-performance liquid chromatography.

A rapid and sensitive method for the determination of peptidyl alpha-amidation activity has been developed and is based on reverse-phase high-performance liquid chromatographic separation and fluorometric detection. A dansylated tripeptide, N-dansyl-Tyr-Val-Gly-OH, is used as the substrate in the assay and the amount of alpha-amidation activity is determined by quantitating the extent of its conversion to product, N-dansyl-Tyr-Val-NH2. Both product and substrate can be detected in a single assay in quantities as low as 5 fmol by isocratic elution using C-18 reverse-phase columns. The method yields highly reproducible results and requires less than 3 min per sample for separation and quantitation. The assay procedure is applicable to the screening of a large number of samples under different pH conditions and is readily adaptable for use in a variety of studies. For example, the procedure is ideal for detecting alpha-amidation activity in various tissues, monitoring activity at the different stages during purification of a particular alpha-amidation enzyme, determining kinetic parameters of the purified enzyme, and identifying both competitive and noncompetitive inhibitors.

Amides↗
PubMed1988

Structure-activity relationships for glycine-extended peptides and the alpha-amidating enzyme derived from medullary thyroid CA-77 cells.

A peptidyl alpha-amidating enzyme has been partially purified from conditioned medium derived from cultured medullary thyroid CA-77 cells. The interactions of this enzyme with a series of tripeptides, pentapeptides, and mature glycine-extended prohormones has now been studied using a competition assay that features the enzymatic alpha-amidation of N-dansyl-Tyr-Val-Gly. While a peptide C-terminal glycine was obligatory for tight binding to the alpha-amidating enzyme, other peptide structural elements modulated the interaction. Thus, a greater than 1300-fold range in apparent inhibitor constants was observed by substitution at the -1 (penultimate) position in a C-terminal glycine-containing tripeptide with each of the 20 common L-amino acids. Peptide inhibitory potency decreased through the following amino acid groupings: sulfur containing greater than aromatic greater than or equal to histidine greater than nonpolar greater than polar greater than glycine greater than charged. This pattern was qualitatively dissimilar to that observed for a more limited series of substitutions at the -2 position, demonstrating the positional selectivity of these structural requirements. The structure-activity relationships observed with the tripeptides at the -1 position were consistent with the apparent inhibitor constants obtained for a collection of prohormones and their pentapeptide mimics. Finally, selected prohormones and their pentapeptide mimics were equipotent inhibitors, demonstrating that the peptide structural elements important for alpha-amidating enzyme recognition are located entirely within the C-terminal pentapeptide segment.

Animals↗
PubMed1987

Degradation of atrial natriuretic factor by kidney cortex membranes. Isolation and characterization of the primary proteolytic product.

Synthetic rat atrial natriuretic factor (r-ANF, 1-28) was incubated with rat kidney cortex membranes, and a predominant degradation product was identified by reverse-phase high performance liquid chromatography. The degradation product was subjected to amino acid analyses and found to have a composition identical to r-ANF. Amino-terminal sequence analyses identified two distinct amino-terminal residues and suggested that cleavage had occurred between the cysteine-phenylalanine bond (residues 7 and 8) of r-ANF. This degradative process could be inhibited by 1,10-phenanthroline and EDTA, suggesting that the enzyme responsible for proteolysis is a metalloendoprotease. The enzyme exhibits a Michaelis-Menten constant of approximately 10 microM for the metabolism of r-ANF and has a broad pH optimum between 8.5 and 9.5. These findings suggest that ANF may be initially degraded in the kidney at a single cleavage site within the 17-residue ring structure.

Amino Acid Sequence↗
PubMed1986

Biosynthesis of calcitonin gene-related peptide and calcitonin by a human medullary thyroid carcinoma cell line.

cDNA analyses predict that the human calcitonin gene encodes the two precursor proteins of calcitonin and calcitonin gene-related peptide (CGRP). The mRNAs for the putative precursors of these peptides are derived from alternative processing of the primary transcript of this gene. The 75 amino-terminal residues of each preprohormone are predicted to be identical. We have developed the TT human medullary thyroid carcinoma cell line as a model to study human calcitonin gene expression. Mature calcitonin and CGRP are major secretory products of this cell line. Extracts of TT cells radiolabeled with amino acids for 1 h contained only one peak of either immunoprecipitable calcitonin or CGRP on reverse-phase high-pressure liquid chromatography. Each of these species had an Mr = 12,800 as estimated by gel filtration chromatography. The results of partial amino-terminal microsequencing of these two precursors were identical. Residues 2, 11, and 14 of each precursor were proline, and residues 7, 17, 24, 25, 26, and 29 were leucine. The unique alignment of the positions of these amino acids with the cDNA-predicted sequence for the common region of preprocalcitonin and preproCGRP suggests that the site of signal sequence cleavage occurs after residue 25 of both preprophormones. These studies represent the first identification of procalcitonin (116 amino acids) from a human cell line and of proCGRP (103 amino acids) from any tissue.

Amino Acid Sequence↗
PubMed1985

Protease nexin. Properties and a modified purification procedure.

The present paper describes chemical and functional properties of protease nexin, a serine protease inhibitor released from cultured human fibroblasts. It is shown that protease nexin is actually synthesized by fibroblasts and represents about 1% of their secreted protein. Analysis of the amino acid composition of purified protease nexin indicates that it is evolutionarily related to antithrombin III and heparin cofactor II. Protease nexin contains approximately 6% carbohydrate, with 2.3% amino sugar, 1.1% neutral sugar, and 3.0% sialic acid. The Mr calculated from equilibrium sedimentation analysis is 43,000. Protease nexin is a broad specificity inhibitor of trypsin-like serine proteases. It reacts rapidly with trypsin (kassoc = 4.2 +/- 0.4 X 10(6) M-1 s-1), thrombin (kassoc = 6.0 +/- 1.3 X 10(5) M-1 s-1), urokinase (kassoc = 1.5 +/- 0.1 X 10(5) M-1 s-1), and plasmin (kassoc = 1.3 +/- 0.1 X 10(5) M-1 s-1), and slowly inhibits Factor Xa and the gamma subunit of nerve growth factor but does not inhibit chymotrypsin-like proteases or leukocyte elastase. In the presence of heparin, protease nexin inhibits thrombin at a nearly diffusion-controlled rate. Two heparin affinity classes of protease nexin can be detected. The present characterization pertains to the fraction of protease nexin having the higher affinity for heparin. The low affinity material, which is the minor fraction, is lost during purification.

Amino Acids↗
PubMed1983

o-Phthaldialdehyde precolumn derivatization and reversed-phase high-performance liquid chromatography of polypeptide hydrolysates and physiological fluids.

A rapid and ultrasensitive fluorescence amino acid analysis system has been developed which utilizes omicron-phthaldialdehyde as a precolumn derivatizing agent. omicron-Phthaldialdehyde in the presence of mercaptan reacts rapidly with primary amino acids to form intensely fluorescent derivatives. These derivatives are analyzed with good selectivity by high-performance liquid chromatography employing 3-microns particle size reversed-phase columns. Resolution of the amino acid derivatives is accomplished with a methanol gradient in 0.1 M aqueous sodium acetate, pH 7.2. The quantitation of the individual amino acid derivatives is reproducible within an average relative deviation of +/- 1.5% and has a detection limit of less than 100 fmoles. Amino acid mixtures obtained by either enzymatic or acid hydrolysis of polypeptides are efficiently resolved with an analysis time of less than 18 min. Methods for the amino acid analysis of physiological fluids such as serum, urine and cerebrospinal fluid were also developed which employ the above separation procedure for the identification and quantitation of amino acids and other biological amines. Mixtures which contained as many as 48 components were resolved with an analysis time of less than 50 min.

Aldehydes↗
PubMed1983

Is adrenal proenkephalin glycosylated?

Adrenal proenkephalin contains the sequence -Asn-Ser-Ser- that is a typical site for the attachment of asparagine-linked carbohydrate. The 5300- and 18,200-Da bovine adrenal proteins derived from proenkephalin contain this recognition sequence and were therefore analyzed for the presence of both amino and neutral sugars. Les than 0.05 mol of amino sugar and less than 0.1 mol of neutral sugar were found per mole of each protein. No amino sugar was detected in other high-molecular-weight adrenal [Met]enkephalin-containing proteins. Together these findings indicate that bovine adrenal proenkephalin does not contain asparagine-linked carbohydrate.

Adrenal Glands↗
PubMed1982

Glucagon carboxyl-terminal derivatives: preparation, purification, and characterization.

Chemical and enzymatic methods have been used to prepare the following series of seven glucagon derivatives modified in the carboxyl-terminal region important for hormone-receptor binding: [des-Asn28,Thr29](homoserine lactone27)glucagon, [des-Asn28,Thr29](homoserine27)glucagon, (S-methyl-Met27)glucagon, [des-Thr29](S-methyl-Met27)-glucagon, [des-Thr29]glucagon,[des-Asn28,Thr29](S-methyl-Met27)glucagon, and [des-Asn28,Thr29]glucagon. The derivatives were isolated in high yield, extensively purified, and chemically characterized. All were found to be full agonists of native glucagon. Binding affinity was evaluated by displacement of mono[125I]iodoglucagon prepared by new methods. Binding and biological activities closely correlated, indicating that most modifications affected the relative binding affinity and relative biological potency of glucagon to a comparable extent. Circular dichroism measured in dilute acid solution resembled that of native glucagon except for [des-Asn28,Thr29]glucagon which displayed increased alpha helicity (25%). All derivatives formed helical structures in 2-chloro-ethanol, although the amount of helicity induced was not closely correlated with biological activity. Binding and biological activities were not affected by removal of Thr-29, though both were reduced 20-fold when Asn-28 was also removed, irrespective of whether homoserine or native methionine remained at the carboxyl terminus. Lactone formation was associated with a further 5-fold reduction in binding affinity but not in activity. Methylation of Met-27 had essentially the same effect as removing the two carboxyl-terminal residues, although the combined effect of both modifications was greater than 100-fold reduction in binding and activity. These findings provide additional insight concerning glucagon structure-function relationships.

Adenylyl Cyclases↗
PubMed1982

Enkephalin biosynthetic pathway: a 5300-dalton adrenal polypeptide that terminates at its COOH end with the sequence [Met]enkephalin-Arg-Gly-Leu-COOH.

An enkephalin-containing polypeptide of 5300 daltons has been isolated from extracts of bovine adrenal medulla and purified to homogeneity, and its sequence has been determined by a combination of automated Edman degradation and enzymatic time-course hydrolysis. The polypeptide contains a single [Met]enkephalin sequence followed by Arg-Gly-Leu-COOH, which forms the COOH terminus of the molecule. The enkephalin sequence is preceded by a Lys-Arg linkage, typical of prohormone cleavage sites. Posttranslational processing of the polypeptide at this site would lead to release of the octapeptide [Met]enkephalin-Arg6-Gly7-Leu8.

Amino Acid Sequence↗
PubMed1982

Adrenal opioid proteins of 8600 and 12,600 daltons: intermediates in proenkephalin processing.

[Met]Enkephalin-containing proteins of 8600 and 12,600 daltons have been isolated from acid extracts of bovine adrenal medulla and purified to homogeneity, and their sequences have been determined by a combination of automated Edman degradation, tryptic mapping, and enzymatic time-course hydrolysis. The 8600-dalton protein contains one copy of the [Met]enkephalin sequence at the COOH terminus and the 12,600-dalton protein contains three copies of [Met]enkephalin, of which two are internal and the third is at the COOH terminus. They possess identical NH2-terminal amino acid sequences, suggesting that the 8600-dalton protein is derived from the 12,600-dalton protein by intracellular proteolytic processing. This is supported by results from tryptic maps of both proteins. Furthermore, chemical analysis of the tryptic peptides obtained from the 12,600-dalton protein indicates that it also contains the amino acid sequence that corresponds to a previously characterized enkephalin-containing polypeptide of 3800 daltons (peptide F) [Jones et al. (1980) Arch. Biochem. Biophys. 204, 392-395]. All three polypeptides appear to be intermediates in posttranslational processing of a still larger polyenkephalin precursor molecule, proenkephalin, and part of a biosynthetic pathway leading to smaller enkephalin-containing polypeptides and free enkephalins.

Adrenal Medulla↗
PubMed1982

An 18,200-dalton adrenal protein that contains four [Met]enkephalin sequences.

An 18,200-dalton adrenal enkephalin-containing polypeptide was purified to homogeneity and shown to contain four internal [Met]enkephalin sequences. Protein sequence analysis and mapping of trypsin-derived peptides demonstrated that the previously described 12,600-dalton and 5,300-dalton adrenal enkephalin-containing polypeptides compose the amino-terminal and carboxyl-terminal sequences, respectively, of this larger protein. They are linked by two basic amino acid residues to form the entire sequence of the 18,200-dalton protein. This protein is the largest enkephalin-containing polypeptide yet isolated from beef adrenal medulla and serves as the precursor for several other previously characterized adrenal enkephalin-containing proteins and peptides.

Adrenal Medulla↗