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D Mantle

Publications and source records attributed to D Mantle.

At least 91 records · Page 5Linked to original sources
PubMed1992

Effect of clenbuterol on protease activities and protein levels in rat muscle.

To elucidate the biochemical mechanism by which the sympathomimetic agent clenbuterol promotes skeletal muscle growth, we have determined the activity of a range of proteolytic enzyme types (acid, neutral and alkaline proteinases and peptidases), together with the levels of soluble and structural proteins (via SDS-polyacrylamide gel electrophoresis) in 5 innervated and denervated muscle types from control and drug-treated rats. No gross change in activity was found for any enzyme type in any muscle (innervated or denervated) following clenbuterol treatment; however, one enzyme, arginyl aminopeptidase, showed a small but consistent decrease in activity in all of the innervated muscles investigated. Similar fractionation profiles were obtained for structural or soluble proteins from corresponding muscle types (innervated or denervated) in control or clenbuterol-treated animals, with the exception of cardiac muscle, which showed a 50% increase in staining intensity of one band (subunit molecular mass 18 kD). We conclude that the anabolic action of clenbuterol in promoting skeletal muscle growth does not occur via downregulation of protease activity, or increase in levels of individual muscle proteins.

Animals↗
PubMed1991

Purification and characterization of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human skeletal muscle.

The purification and characterization of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human skeletal muscle are described. The characteristics of leucyl aminopeptidase were as follows: optimum activity was at pH 9.5 in the presence of 5 mmol/l Mg2+ or 0.5 mmol Mn2+. No activation of enzyme activity was obtained following addition of other divalent cations or sulphhydryl reagents. Only the leucyl-AMC and methionyl-AMC derivatives were appreciably hydrolysed. The mol mass was estimated as 280 kDa. Approx. 50% inhibition of activity was obtained following addition of p-hydroxymercuriphenyl sulphonate (10 mumol/l), N-ethyl maleimide (2 mmol/l), o-phenanthroline (5 mmol/l), bacitracin (1 mmol/l), amastatin (1 microgram/ml) and bestatin (0.1 mumol/l); no inhibition of activity was obtained in the presence of phenylmethanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 microgram/ml) or pepstatin (100 microgram/ml). The following oligopeptides were hydrolysed by the enzyme: luliberin 7-10, proctolin and [Leu5]enkephalin; oligopeptides not appreciably hydrolysed included neurotensin, angiotensin-I, substance-P and bradykinin. Pyroglutamyl aminopeptidase had the following characteristics: optimum activity was at pH 8.5 in the presence of 1 mmol/l dithiothreitol (an absolute requirement for maintenance of enzyme activity). Maximum activity was obtained in the absence of divalent cations. Only the pyroglutamyl-AMC derivative was appreciably hydrolysed. The mol mass of this enzyme was estimated as 22 kDa. Approximately 50% inhibition of activity was obtained on addition of phenanthroline (4 mmol/l) and antipain (7 microgram/ml); no inhibition of activity was obtained following addition of phenyl methanesulphonyl fluoride (1 mmol/l), limabean trypsin inhibitor (100 microgram/ml) or pepstatin (100 microgram/ml). Only oligopeptides with a pyroglutamyl N-terminal residue (thyroliberin, neurotensin, and luliberin) were hydrolysed by the enzyme.

Cations, Divalent↗
PubMed1991

Characterization of dipeptidyl and tripeptidyl aminopeptidases in human kidney soluble fraction.

In an attempt to identify improved biochemical markers for the diagnosis of early kidney damage via urinary analysis of kidney derived enzymes, we have undertaken the systematic identification, quantification and characterization (following purification via anion exchange and gel filtration chromatography, and preparative electrophoresis) of the dipeptidyl and tripeptidyl aminopeptidases in normal human kidney soluble extract (with which the majority of the cellular activity of these enzymes is associated). Four chromatographically separable enzyme types were identified as follows (% relative activity in parentheses): dipeptidyl aminopeptidase I (EC 3.4.14.1; 24%); dipeptidyl aminopeptidase II (EC 3.4.14.2; 8%); dipeptidyl aminopeptidase IV (EC 3.4.14.5; 61%); tripeptidyl aminopeptidase (unclassified; 7%). Comparison of the levels of activity for the above enzyme types in normal and pathological urine may lead to an improvement upon existing procedures for the early detection of renal damage.

Amino Acid Sequence↗
PubMed1991

Comparison of Ca(2+)-activated proteinase enzyme and endogenous inhibitor activity in brain tissue from normal and Alzheimer's disease cases.

Recent evidence has suggested that Alzheimer's disease may result from an underlying defect of protein catabolism. In an attempt to identify such a defect, we have determined the levels of Ca(2+)-activated proteinase (principally calpain II) and endogenous inhibitor (calpastatin) activity in normal and Alzheimer's disease cases, following fractionation of parietal cortex (grey and white matter) via anion-exchange chromatography. The chromatographic elution profiles and levels of calpain II activity were found to be similar in grey and white matter in both normal and Alzheimer's disease cases. The characteristics of calpain II, including Ca2+ concentration required for optimum activity for enzymes partially purified from normal or Alzheimer's disease cortex were identical. Similarly, the chromatographic elution profiles and levels of total calpastatin activity (approximately equal to that for calpain II activity) were found to be similar in grey and white matter from normal and Alzheimer's disease cases. These data suggest that the characteristic neurodegeneration associated with Alzheimer's disease does not result from alteration in the level of activity or characteristics of the calpain/calpastatin system in the cerebral cortex of patients with this disorder.

Aged↗
PubMed1991

Human brain leucyl aminopeptidase: isolation, characterization and specificity against some neuropeptides.

In order to obtain a greater understanding of the role of aminopeptidases in the degradation of peptides and proteins in the nervous system, we have isolated and characterized leucyl aminopeptidase (EC 3.4.11.1) from human cerebral cortex and studied its action on some physiologically important neuropeptides. The enzyme has a low specificity constant for the hydrolysis of Leu-7-amido-4-methylcoumarin (69s-1M-1) but the peptides Tyr-Gly-Gly and Tyr-Gly-Gly-Phe-Leu (Leu5-enkephalin) were much better substrates (specificity constants 8300 and 18050s -1M-1 respectively). Optimum activity for the degradation of Leu-enkephalin was obtained at pH10.5 in the presence of 5mM-Mn++. A sharp drop in specificity constant occurred with increasing chain length in the series Leu-enkephalin, dynorphin 1-8, 1-10 and 1-13, suggesting that the enzyme functions only as an oligopeptidase. Other neuropeptides were poor substrates (cholecystokinin octapeptide, angiotensin-I) or not hydrolysed at all (somatostatin, Arg8-vasopressin).

Amino Acid Sequence↗
PubMed1990

Characterization of aminopeptidases in human kidney soluble fraction.

Fractionation of human kidney soluble extract (with which the majority of cellular aminopeptidase activity is associated) via anion exchange chromatography resolved four types of separable aminopeptidase (relative activity in parenthesis): alanyl aminopeptidase (EC 3.4.11.14; 50%); arginyl aminopeptidase (EC 3.4.11.6; 30%); leucyl aminopeptidase (EC 3.4.11.1; 18%) and pyroglutamyl aminopeptidase (EC 3.4.19.3; 2%). The further purification (via gel filtration chromatography and preparative electrophoresis) and characterization of each aminopeptidase has been described; the aminopeptidase tissue profile for human kidney was found to be similar to that previously obtained for human skeletal muscle and brain tissue using a similar experimental approach, i.e. the same enzymes, with corresponding similar characteristics, are present in each tissue. These results suggest that soluble aminopeptidases may be of fundamental importance in general cell protein catabolism. Degradation of the following aminoacyl-AMC derivatives via kidney soluble extract has been shown to be due principally to hydrolysis by alanyl aminopeptidase: glutamyl, glycyl, isoleucyl, methionyl, ornithyl, phenylalanyl, prolyl, seryl, tyrosyl and valyl. We would suggest that measurement of the soluble extract-derived aminopeptidases described in this paper in urine may lead to an improvement upon existing assay procedures for early detection of kidney damage.

Aminopeptidases↗
PubMed1990

Comparison of aminopeptidase, dipeptidyl aminopeptidase and tripeptidyl aminopeptidase activities in brain tissue from normal and Alzheimer's disease cases.

Recent evidence has suggested that Alzheimer's disease may result from an underlying defect of protein catabolism. In an attempt to identify such a defect, we have undertaken the systematic identification and characterization of the proteolytic enzymes responsible in human brain for oligopeptide breakdown via hydrolysis of NH2-terminal residues--the aminopeptidases, dipeptidyl aminopeptidases and tripeptidyl aminopeptidases. The activities of these enzymes were determined following anion exchange chromatographic fractionation of cortical soluble extracts, to prevent interference in the respective assay procedures by the major cortical aminopeptidase. Corresponding enzyme types showed similar levels of activity in soluble extracts of frontal cortex from normal and Alzheimer's disease cases. These data suggest that the characteristic neurodegeneration associated with Alzheimer's disease does not result from altered aminopeptidase, dipeptidyl aminopeptidase or tripeptidyl aminopeptidase activity in cortical tissues of patients with this disorder.

Aged↗
PubMed1989

Comparison of major cortical aminopeptidase activity in normal brain and brain from patients with Alzheimer's disease.

In order to develop a greater understanding of the importance of peptide catabolism in the intracellular protein degradation process in normal and pathological human brain, we have undertaken a systematic investigation of the aminopeptidase group of enzymes. Although a wide range of aminoacyl-7-amino-4-methylcoumarin derivatives (which are used to measure aminopeptidase activity) were hydrolysed by normal human cortical soluble extract, fractionation of the latter via anion exchange and gel filtration chromatography resolved only 4 separable aminopeptidase types (activity relative to alanyl aminopeptidase in parenthesis): alanyl (EC 3.4.11.14, 100%); arginyl (2 isoenzymes, EC 3.4.11.6, 15%); pyroglutamyl (EC 3.4.19.3, 4%); and leucyl (EC 3.4.11.1, 1%). Thus approx. 80% of the total soluble aminopeptidase activity in normal human cerebral cortex can be accounted for by a single enzyme, the major cortical aminopeptidase. The activity of this enzyme was measured in the soluble fraction prepared from 4 cortical regions (occipital, frontal, temporal and parietal) in a series of 8 patients with Alzheimer's disease and compared with corresponding data in 8 control normal patients. Although enzyme activity varied in these cortical regions, the activity in corresponding brain areas in the Alzheimer's disease and normal groups was very similar. These findings suggest that the characteristic neurodegeneration associated with Alzheimer's disease does not result from altered activity of the major cortical aminopeptidase in the cortical tissue of patients with this disorder.

Aged↗
PubMed1988

Rationalization of aminopeptidase activities in human skeletal muscle soluble extract.

Although a wide range of aminoacyl-7-amino-4-methylcoumarin derivatives (which are used to measure aminopeptidase activity) were found to be hydrolysed by human skeletal muscle soluble fraction, fractionation of the latter via anion-exchange and gel-filtration chromatography resolved only five types of separable aminopeptidase (with activity relative to alanyl aminopeptidase in parentheses): alanyl aminopeptidase (alpha-aminoacyl-peptide hydrolase, EC 3.4.11.14, 100%), arginyl aminopeptidase (two isoenzymes, L-arginyl-L-lysyl)-peptide hydrolase, EC 3.4.11.6, 15%); pyroglutamyl aminopeptidase (5-oxoprolyl-peptide hydrolase, EC 3.4.19.3, 3%); leucyl aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1, 1.5%) and alpha-glutamyl aminopeptidase (0.2%). Thus over 80% of the total aminopeptidase activity (expressed in relative terms) in human skeletal muscle soluble fraction can be accounted for by a single enzyme, the major aminopeptidase. A single peak of activity, which co-eluted with the major aminopeptidase after anion-exchange and gel-filtration chromatography, was obtained after assay with the following aminoacyl-7-amino-4-methylcoumarin derivatives: glycyl-, isoleucyl-, lysyl-, methionyl-, ornithyl-, phenylalanyl-, prolyl-, seryl-, tyrosyl- and valyl-. Thus, the hydrolysis of these derivatives by skeletal muscle soluble fraction occurs principally via the major aminopeptidase and not by specific enzymes, as previously suggested (Wada and Aoyagi, 1983). These results illustrate the difficulty in measuring individual aminopeptidase activities in muscle homogenate and soluble fraction, and the danger in ascribing apparent aminopeptidase activity to 'specific' enzymes.

Aminopeptidases↗
PubMed1988

Purification and characterization of two soluble Cl(-)-activated arginyl aminopeptidases from human brain and their endopeptidase action on neuropeptides.

Two closely related Cl(-)-activated arginyl aminopeptidases (I and II) were purified from a soluble extract of postmortem human cerebral cortex by anion-exchange chromatography and preparative gel electrophoresis. The electrophoretic mobility of II was approximately 80% that of I; the molecular mass of both enzymes was approximately 70 kilodaltons (kDa) (gel filtration). The aminopeptidase action of I and II on aminoacyl-7-amido-4-methylcoumarin (AMC) substrates was restricted to the Arg and Lys derivatives. Both enzymes had significant endopeptidase activity, hydrolysing several biologically active peptides including neurotensin, bradykinin, angiotensin-I, substance P, luliberin, and somatostatin at internal bonds. Other peptides [Leu-enkephalin, proctolin, thyroliberin, adrenocorticotropin18-39 (ACTH18-39), ACTH11-24, and dynorphin (1-13)] were not appreciably hydrolysed. The amino- and endopeptidase activities had pH optima at 6.5 and 7, respectively, and were both inhibited by metal ion chelators and sulphydryl group blocking agents. The aminopeptidase activity was stimulated 20-fold by Cl- ions, whereas the endopeptidase activity was unaffected by the latter. Km values for neurotensin degradation were 20 microM (I) and 37 microM (II) and for Arg-AMC hydrolysis they were 167 microM (I) and 125 microM (II). The endopeptidase activity was not inhibited by the aminopeptidase inhibitors arphamenine or bestatin (IC50 = 9 nM and 0.1 microM, respectively, with Arg-AMC substrate).

Aged↗
PubMed1987

Absence of biochemical heterogeneity in McArdle's disease. A high resolution SDS-polyacrylamide gel electrophoresis study.

Muscle biopsy extracts from a series of 6 patients with McArdle's disease were investigated by analytical SDS-polyacrylamide gel electrophoresis, to establish the presence or absence of the myophosphorylase protein subunit. In 4 cases, the band corresponding to the myophosphorylase subunit was totally absent from the electrophoretic staining pattern, and in 2 cases was present, but with a greatly reduced staining intensity compared with control normal patients; thus in none of the cases of McArdle's disease investigated was there evidence for a myophosphorylase subunit band of comparable staining intensity to that found in control normal patients. This result contrasts with previously reported findings (Feit and Brooke 1976) which suggested that McArdle's disease exists in biochemically heterogeneous forms; in one form of the disease myophosphorylase being totally absent and in a second form present to a similar extent as normal, but in an inactive form. On the basis of the results reported in this paper, we would suggest that myophosphorylase deficiency is a single gene disorder characterized by the absence or marked reduction of the myophosphorylase protein.

Adolescent↗
PubMed1985

Specificity of neuropeptide degradation by two calcium-activated neutral proteases from human skeletal muscle.

Two calcium-activated neutral proteases (CAPI & II) were purified from human skeletal muscle by anion exchange, gel filtration and affinity (antipain-Sepharose and Blue Ultrogel A4R) chromatography. The enzymes were homogenous as judged by polyacrylamide gel electrophoresis, and have similar properties with the exception of the Ca2+ concentration required for optimum activity (CAP I = 0.1 mM; CAP II = 1 mM). Both enzymes hydrolysed a wide variety of neuropeptides. In six cases, the products were separated and identified by hplc and amino acid analysis. Neurotensin was hydrolysed at Tyr3-Glu4; dynorphin1-13 at Arg8-Arg9; LH-RH at Gly6-Leu7; CCK-8 at Phe8-NH2, substance-P at Met10-NH2; somatostatin at Thr10-Phe11. Although differences in the rates of neuropeptide degradation were noted for the two CAP's the specificity was the same for these six peptides. It is suggested that conformational requirements may be more important than side chains adjacent to the cleavage site in directing the specificity of CAP.

Amino Acids↗