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Biomedical subjects

R L Stein

Publications and source records attributed to R L Stein.

At least 55 records · Page 3Linked to original sources
PubMed1986

Mechanism of inactivation of human leukocyte elastase by a chloromethyl ketone: kinetic and solvent isotope effect studies.

The mechanism of inactivation of human leukocyte elastase (HLE) by the chloromethyl ketone MeOSuc-Ala-Ala-Pro-Val-CH2Cl was investigated. The dependence of the first-order rate constant for inactivation on concentration of chloromethyl ketone is hyperbolic and suggests formation of a reversible "Michaelis complex" prior to covalent interaction between the enzyme and inhibitor. However, the observed Ki value is 10 microM, at least 10-fold lower than dissociation constants for complexes formed from interaction of HLE with structurally related substrates or reversible inhibitors, and suggests that Ki is a complex kinetic constant, reflecting the formation and accumulation of both the Michaelis complex and a second complex. It is proposed that this second complex is a hemiketal formed from attack of the active site serine on the carbonyl carbon of the inhibitor. The accumulation of this intermediate may be a general feature of reactions of serine proteases and chloromethyl ketones derived from specific peptides and accounts for the very low Ki values observed for these reactions. The solvent deuterium isotope effect (SIE) on the inactivation step (ki) is 1.58 +/- 0.07 and is consistent with rate-limiting, general-catalyzed attack of the active site His on the methylene carbon of the inhibitor with displacement of chloride anion. The general catalyst is thought to be the active site Asp. In contrast, the SIE on the second-order rate constant for HLE inactivation, ki/Ki, is inverse and equals 0.64 +/- 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Chloromethyl Ketones↗
PubMed1986

Kinetic mechanism of guinea pig neutrophil 5-lipoxygenase.

The kinetic mechanism of guinea pig neutrophil 5-lipoxygenase was investigated using a continuous spectrophotometric assay that monitors product diene formation at 236 nm due to substrate oxygenation. Progress curves for reactions with both arachidonic acid and eicosapentaenoic acid are characterized by 1-3-min lag phases in the attainment of steady-state velocities and product inhibition, as indicated by the total cessation of the reaction prior to complete depletion of substrate. The dependence of the steady-state velocity on arachidonic acid concentration appears to follow Michaelis-Menten kinetics, with Vmax = 4.2 +/- 0.4 nmol of 5-hydroxy-6,8,11,14-eicosatetraenoic acid/min/mg of protein and Ks = 25 +/- 4 microM. The addition of Ca2+ results in an overall activation: lag phases are shortened to 10-20 s, Vmax increases to 24 +/- 2 nmol/min/mg of protein, and Ks decreases to 7.7 +/- 1.7 microM; and a change in a mechanism to one involving substrate inhibition (Kss = 13 +/- 1 microM). The observed activation by Ca2+ has a half-maximal response at around 30 microM. In the presence of Ca2+, ATP causes an increase in Vmax to 30 +/- 4 nmol/min/mg of protein without changing Ks or Kss and a reduction of the lag to less than 5 s. The half-maximal response for ATP is 31 +/- 7 microM. Oxygenation of eicosapentaenoic acid in the presence of Ca2+ and ATP occurs with similar kinetics, except for significantly less substrate inhibition: Vmax = 31 +/- 6 nmol/min/mg of protein, Ks = 7 +/- 1 microM, and Kss = 33 +/- 2 microM. This is the first report suggesting a kinetic mechanism for 5-lipoxygenase, which accounts for substrate inhibition, regulation by Ca2+, and ATP and substrate specificity.

Animals↗
PubMed1985

Proteolytic inactivation of human leukocyte elastase.

Human leukocyte elastase can be proteolytically inactivated by bovine pancreatic trypsin. Neither porcine pancreatic elastase nor bovine pancreatic chymotrypsin causes inactivation of leukocyte elastase, nor are trypsin, pancreatic elastase, or chymotrypsin themselves susceptible to proteolysis. The trypsin-catalyzed inactivation of leukocyte elastase can be slowed by inhibition of trypsin with benzamidine or by occupation of elastase's active site with elastatinal.

Benzamidines↗
PubMed1985

Catalysis by human leukocyte elastase: III. Steady-state kinetics for the hydrolysis of p-nitrophenyl esters.

Steady-state kinetic parameters were determined at pH 7.4 and 25 degrees C for the human leukocyte elastase-catalyzed hydrolysis of several N-carbobenzoxy-L-amino acid p-nitrophenyl esters. The substrate specificity for these esters was quite broad, and included the Gly, Phe, and Tyr derivatives. Together with reports of a much narrower P-1 specificity for peptide-based substrates, these results suggest that interactions remote from the scissle bond between enzyme and substrate regulate primary specificity. Also, it was found that kc and kc/Km did not exhibit the same dependence on substrate structure. This is interpreted to suggest that there are significant differences in P-1 specificity between acylation and deacylation for leukocyte elastase-catalyzed reactions.

Catalysis↗
PubMed1984

Microcomputer for controlled substance record keeping.

The use of a microcomputer to maintain controlled substance inventory and record keeping is described. The system maintains perpetual inventories of the central narcotic vault and proof-of-use controlled drug records outstanding at nursing stations. The computerized system has eliminated (1) the delay previously encountered in the posting of transactions from the numeric log to perpetual inventory logs and (2) the potential addition and subtraction errors inherent in a manual system. Computerizing the controlled drug record-keeping system has saved approximately 166 minutes of labor per day, a cost savings of approximately $26. The new system also helps prevent diversion of controlled substances. The computer may also be used for other tasks while not running the controlled substance program. A microcomputer is well suited to the task of controlled-substance record-keeping functions, and the cost of the system (less than $4000) can be quickly recouped in labor savings.

California↗
PubMed1983

Purification of human leukocyte elastase and cathepsin G by chromatography on immobilized elastin.

Human leukocyte elastase and cathepsin G were isolated from purulent sputum by a simple procedure involving chromatography on elastin-agarose. Salt extracts of sputum were prepared, treated with DNase, and the precipitate which formed extracted and applied to a column of soluble elastin-Sepharose 4B. Contaminating protein was eluted with 50 mM Tris, 50 mM NaCl, pH 8.0 and then two column volumes of 50 mM acetate, 1.0 M NaCl, pH 5.0. The tightly bound elastase and cathepsin G together with a trypsin-like serine protease could finally be eluted with 50 mM acetate, 1.0 M NaCl, 20% DMSO, pH 5.0. Resolution of the proteases was accomplished by cation-exchange chromatography. Disc gel electrophoresis established the purity of elastase and cathepsin G and confirmed the existence of several isozymes for each.

Cathepsin G↗
PubMed1981

Kinetic alpha-deuterium isotope effects for Escherichia coli purine nucleoside phosphorylase-catalyzed phosphorolysis of adenosine and inosine.

Kinetic alpha-deuterium isotope effects have been measured for the purine nucleoside phosphorylase-catalyzed phosphorolysis of adenosine and inosine by a competitive double label technique at saturating concentrations of the second substrate, phosphate. Under these conditions the observed isotope effect, kH/kD, is on the second order rate constant, Vmax/Km, for reaction of nucleoside with the Michaelis complex of enzyme and phosphate. For adenosine, at neutral pH, the isotope effect is unity. For inosine, kH/kD was determined as a function of pH (the numbers in parentheses are the ratios of Vmax at that pH to Vmax at pH 7.3): 1.10 at pH 5.0 (0.19); 1.10 at pH 6.1 (0.72); 1.01 at pH 7.3 (1.00); 1.16 at pH 8.4 (0.22); and 1.18 at pH 9.4 (0.04). These values suggest a mechanism for purine nucleoside phosphorylase involving a change in rate-limiting step from one at pH values near neutrality for which cleavage of the nucleoside C-N bond is not rate limiting to a step at extremes of pH with a transition state having considerable oxocarbonium ion-like character.

Adenosine↗
PubMed1977

Evaluation of Federal man tests for self-contained breathing apparatus.

The transient pulmonary responses to four short duration man tests used in the Federal approval of breathing apparatus were measured. The pulmonary ventilation dropped 30 percent in the first thirty seconds in the periods assigned for samples and readings which were immediately preceded by work. A physiological deficit was incurred during the short duration man tests which meant that less O2 and ventilation were required when compared to steady state responses shown on approval tests. The scheduled man test activities for short duration breathing apparatus should be revised and made more vigorous to match the intended use of escape apparatus.

Adult↗
PubMed1976

Acylation of subtilisin Carlsberg by phenyl esters.

Approximate Hammett reaction constants rho calculated from k2/K8 values of several phenyl esters of N-acetyl-L-phenylalanine, hippuric acid, and beta-phenylpropionic acid are 0.0, 0.4, and 1.0 respectively. To determine whether the lack of substituent effect of k2/K8 with the N-acetyl-L-phenylalanine esters is a result of substituent-insensitive k2 or rate-limiting association of enzyme and substrate, pH-k2/K8 deependences and solvent deuterium isotope effects were determined for certain of the substrates and compared with those found with the corresponding hippurates and beta-phenylpropionates. In the pH range 5 to 8, k2/K8 of the phenyl and 4-nitrophenyl esters of each series is dependent upon the unprotonated form of an enzymatic base of apparent pKa approximately 7.4, identical with the pKa found for the free enzyme. With the phenyl esters of each substrate class, k2/K8 decreased by 2 to 3 times in deuterium oxide compared with water. The results suggest that a step involving a general base-catalyzed proton transfer, almost certainly k2, is rate-limiting with the N-acetyl-L-phenylalaninates, as well as the hippurates and beta-phenylpropionates. Attack by the protein on the latter substrates is prediminantly nucleophilic, judged by the similarity of rho in the enzymatic and reference hydroxide ion-catalyzed hydrolyses. The power rho values for the N-acetyl-L-phenylalaninates and hippurates could result from an electrophilic component in their hydrolytic mechanisms.

Acylation↗
PubMed1975

Efficiency of dust sampling inlets in calm air.

Measurement of airborne dust concentrations usually involves drawing a sample of the dust-laden air into the measuring instrument through an inlet. Even if the surrounding air is calm, theoretical calculations predict that large particles may not be sampled accurately due to the combined effects of gravity and inertia on the particles near the sampling inlet. Tests were conducted to determine the conditions of particle size, inlet radius, and flow rare necessary for accurate dust sampling. A coal-dust aerosol was sampled simultaneously through inlets of different diameters at the same volume flow-rate and collected on filters. The dust was removed from the filters and the particles were counted and sized with a Coulter counter. Results showed that published criteria for inlet conditions for correct sampling are overly restrictive and that respirable-size particles are sampled correctly in the normal range or operation of most dust sampling instruments.

Aerosols↗