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Biomedical subjects

E Shaw

Publications and source records attributed to E Shaw.

At least 127 records · Page 7Linked to original sources
PubMed1985

Evaluation of inhibitor constants and alkylation rates for a series of thrombin affinity labels.

The kinetics for the inactivation of thrombin (EC 3.4.21.5) by a series of peptides containing C-terminal arginyl chloromethane in the presence of substrate were determined. The inhibitor effectiveness was analysed so as to allow for both the evaluation of the affinity with which the enzyme binds the inhibitor before irreversible modification and also the rate of covalent-bond formation between enzyme and inhibitor. The results obtained show that the observed large range in inhibitor effectiveness can be accounted for almost entirely by marked differences in affinity, with only small variations in rates of formation of covalent complex.

Affinity Labels↗
PubMed1984

Preliminary clinical pharmacological studies of S3341, a new hypotensive agent, and comparison with clonidine in normal males.

S3341, a new hypotensive agent which binds to alpha2-receptors in animal brain preparations, was studied in normal healthy male volunteers. A dose ranging study with 15 and 25 micrograms/kg of S3341 was performed in a double blind, placebo controlled randomised and balanced manner with 3 subjects. A decrease or BP without noticeable sedation (assessed by visual analogue scales) was seen. One, 2 mg of S3341, 0.1 and 0.2 mg of clonidine were then compared in a double blind, placebo controlled, randomised and balanced manner in 10 subjects. BP, heart rate, systolic time intervals (STI), critical flicker frequency, choice reaction time, pursuit rotor, stimulated salivary volume, and dryness of mouth and sedation with visual analogue scales, were measured at 0, 1.5, 3.0, 4.5 and 6.0 h after drug administration. The relationship between decreases in BP and sedation was assessed by linear regression analysis, with the former as the independent (predictor) response and the latter as the dependent (response) variate. Both drugs produced a similar decrease of BP which was significantly different from placebo. Changes in psychomotor function tests were not significant. Both drugs produced dryness of mouth and sedation which were significantly different from placebo but changes were less with S3341. Clonidine showed a significantly steeper slope than S3341 in the relationship between decreases in BP and sedation. This must be interpreted with caution as there was wide variation in the correlation between decreases in BP and sedation, but it may be possible to achieve lesser sedation with S3341.

Adrenergic alpha-Agonists↗
PubMed1984

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Humans↗
PubMed1983

Inactivation of cathepsin B by active site-directed disulfide exchange. Application in covalent affinity chromatography.

The ability of cystamine, bis-N-aminoethyl disulfide, to inactivate cathepsin B by disulfide exchange is considerably enhanced by the addition of hydrophobic residues which apparently occupy secondary binding sites in the extended active center of the protease. For example, a peptidyl cystamine derivative such as symmetrical Gly-Phe-cystamine is substrate-like and promotes the disulfide exchange by affinity labeling. The effectiveness of this type of reagent is dependent on the peptidyl sequence. The inactivation is more rapid with increasing pH in the narrow range studied, pH 5-7, and is completely reversed by added thiols. Immobilization of the peptidyl cystamine derivatives provided an effective and rapid procedure for the covalent chromatography of cathepsin B following ammonium sulfate fractionation of beef spleen or pork liver homogenates, replacing the need for ion exchange chromatography. Pork liver cathepsin B thus obtained was a mixture of single chain and double chain forms as found by other methods.

Animals↗
PubMed1983

Isolation and characterization of a trypsin-like serine proteinase from the membranes of Walker 256 carcino-sarcoma cells.

A serine proteinase was isolated from Walker-256-carcino-sarcoma plasma-membrane-enriched preparations by affinity chromatography employing soya-bean trypsin inhibitor as the ligand. This enzyme was termed 'memsin' owing to its membrane location and trypsin-like substrate specificity. Analysis of this preparation by steric-exclusion high-pressure liquid chromatography (h.p.l.c.) resulted in a single peak of enzyme activity. Calculations of the rates of inactivation of memsin by peptidyl-chloromethanes and comparison with rate constants obtained with other serine proteinases indicated that memsin closely resembled trypsin and acrosin. Digestion of oxidized ribonuclease by memsin and analysis of the resulting peptides by h.p.l.c. yielded a chromatogram that was very similar to one generated by a tryptic digest of oxidized ribonuclease. This enzyme could possibly play a role in tumour-cell invasion.

Animals↗
PubMed1983

An exploration of the primary specificity site of cathepsin B.

Peptidyl diazomethyl ketones inactivate cathepsin B apparently by alkylation of the active center thiol following complex formation as in the case of benzyloxycarbonyl (Cbz)-Phe-AlaCHN2. The phenylalanine contributes considerably to binding in the secondary specificity site. In order to define the topography of the active center region comprising the primary specificity site of beef spleen cathepsin B, a series of peptidyl diazomethyl ketones having the general structure Cbz-Phe-X-CHN2 has now been synthesized. The amino acid, X, has been varied in size to include rather large side chains which might reveal available binding potential or limitations. Some of the reagents, in fact, were not inhibitory even at 10(-4) M. Others, however, that did measurably inactivate cathepsin B provided a range of reactivities that extended over 5 orders of magnitude and correlated with affinity in the reversible phase of inactivation. Some large side chains, for example, that of tryptophan, were very poorly tolerated in this region of the active center, whereas others, such as O-benzyl threonine, provided remarkably active inhibitors. A topographical rationalization of the results is offered.

Amino Acids↗
PubMed1983

The effect of a new antiarrhythmic agent, flecainide acetate, on systolic time intervals.

We investigated the effect of flecainide on systolic time intervals in 6 healthy subjects. A randomized, double blind, cross-over study was designed using flecainide (2 mg/kg), propranolol (0.2 mg/kg) or saline. In comparison with placebo, flecainide increased heart rate, diastolic BP, QT-Index, QS2-Index, PEP, and reduced LVET-Index (p less than 0.05). It caused a significant increase in PR interval, QRS duration, and PEP/LVET (p less than 0.01). Propranolol, compared with placebo, decreased heart rate and systolic BP (p less than 0.05). It increased PR interval, PEP, and PEP/LVET ratio (p less than 0.05). In comparison with propranolol, flecainide increased heart rate and QRS duration (p less than 0.01, p less than 0.001 respectively). It increased diastolic BP, PR interval, QS2-Index, PEP, and PEP/LVET ratio, and decreased LVET-Index (p less than 0.05). These changes in STI induced by flecainide may be produced by depression of myocardial contractility. Intraventricular conduction delay may be an additional contributing factor.

Adult↗
PubMed1983

A prolyl endopeptidase from murine macrophages, its assay and specific inactivation.

The presence of a prolyl endopeptidase in the soluble fraction of murine peritoneal macrophages is reported. The prolyl endopeptidase is apparently highly specific for cleaving peptides after proline residues. A sensitive new fluorogenic assay substrate matching this specificity, benzyloxycarbonyl-Ala-Ala-Pro beta-methoxynaphthylamide, is described. The enzyme is rapidly inactivated by benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone, one of a class of reagents specific for cysteine proteinases, and by diisopropyl fluorophosphate, an inhibitor of serine proteinases. Culture of macrophages with the addition of low levels of benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone to the media allows the selective inhibition of the cytoplasmic enzyme as measured in lysates at the termination of culture. After exposure to inhibitor, macrophages resynthesize the enzyme over a period of days, a process which is inhibited by cycloheximide. Similar amounts of activity were found in both normal peritoneal macrophages and those elicited by prior injection of thioglycollate media. The enzyme from murine macrophages appears similar to that reported in bronchopulmonary lavage fluid and lung tissue and to those isolated from brain and pituitary tissues.

Animals↗
PubMed1983

Intra-acrosomal inhibition of boar acrosin by synthetic proteinase inhibitors.

Thirteen serine proteinase inhibitors of the guanidine, monoamidine and diamidine type were tested for their ability to inhibit the proteinase acrosin present in the acrosome of ejaculated and capacitated boar spermatozoa. All compounds studied proved to be potent in-vitro inhibitors of acrosin. Inhibition constants (Ki) in the range of 1.2 x 10(-7) to 6 x 10(-8) M were found for the reversible inhibitors. The intra-acrosomal inhibition of acrosin was assessed by the gelatin substrate film method: 2 guanidinobenzoates, one monoamidine and one diamidine derivative proved to inhibit acrosin completely in intact spermatozoa. Intravenous injection of 6-amidino-2-(4-amidinophenyl)-indole had no effect on fertilization, but application of 4-nitrophenyl-4-guanidinobenzoate in a vaginal suppository gave a 50% reduction of fertilization.

Acrosin↗
PubMed1983

Cysteine protease characteristics of the proteoglycanase activity from normal and pseudoxanthoma elasticum (PXE) fibroblasts.

Pseudoxanthoma elasticum (PXE) is an inherited disease characterized by calcified degenerative changes of elastin in the skin, eye, and vasculature. Previous studies suggested the abnormal presence of a protease from PXE fibroblasts that degrades sulfated proteoglycans. This study describes the use of a radioassay to quantitate proteoglycan degradation by proteases from normal and PXE fibroblasts. PXE protease had optimal activity at pH 6.0. Inhibition of activity by 5 mM diisopropylfluorophosphate, 5 mM phenylmethylsulfonylfluoride, and 0.1 mM HgCl2 was reversed by 10 mM dithiothreitol. Iodoacetamide (1 mM) irreversibly inhibited activity. Carbobenzyloxy-phenylalanyl-alanyl (0.1 mM) and carbobenzyloxy-lysyl-diazomethyl ketone (10 microM) inhibited the proteoglycanase activity. These data suggest that the PXE proteolytic proteoglycanase activity is a cysteine protease. After blocking activity with 5 mM EDTA, addition of 10 mM Mg++, Mn++, Cu++, or Co++ had little effect (less than 10%) on restoring activity, 10 mM CaCl2 restored approximately 70% recovery of the activity, and 10 mM ZnCl2 stimulated the activity to 500% of the initial level. Similar normal fibroblast samples contained little zinc-dependent activity and a substantial amount of calcium-dependent activity. Thus the distinction between the divalent ion requirements for proteoglycan degradation suggests that the PXE fibroblasts may produce a different cysteine protease than do normal fibroblasts.

Calcium↗
PubMed1982

Kallikrein-like activity of crotalase, a snake venom enzyme that clots fibrinogen.

During the amino acid sequence determination of crotalase (EC 3.4.21.30), the thrombin-like enzyme from the venom of Crotalus adamanteus (eastern diamondback rattlesnake), we found that, in addition to the expected structural homology with bovine thrombin (EC 3.4.21.5), there was even greater homology with porcine pancreatic kallikrein (EC 3.4.21.8). In exploring further the similarities between crotalase and kallikrein, several striking observations were made. First, crotalase was rapidly and specifically inhibited by the tripeptide affinity labeling derivative prolylphenylalanylarginine chloromethyl ketone, which is known to be a specific inhibitor of kallikrein. Second, NaDodSO4/acrylamide gel electrophoresis revealed that crotalase cleaves the plasma kallikrein-susceptible bonds in human high molecular weight kininogen, producing an intermediate with procoagulant activity. Crotalase-catalyzed cleavage of high molecular weight kininogen also liberates kinin as evidenced by rat blood pressure bioassay. Finally, crotalase exhibits substrate specificity not only for the thrombin chromogenic substrate S-2238 but also for the kallikrein substrates S-2302 and S-2266. Interestingly, one of the other reactions catalyzed by plasma kallikrein, the activation of plasminogen, was not one of the activities exhibited by crotalase.

Animals↗