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M T Martin

Publications and source records attributed to M T Martin.

At least 19 recordsLinked to original sources
PubMed1992

Analysis by in situ hybridization of cells expressing mRNA for tumor-necrosis factor in the developing thymus of mice.

We have used in situ hybridization to investigate the expression of TNF-alpha genes by thymic cells during fetal development in mice. In 14-day-old fetal thymuses, very scarce cells produce TNF-alpha mRNA. A second phase of cytokine gene expression starts on day 16. The density of positive cells progressively increases up to day 20. Thymuses at 15 days of gestation and after birth do not express detectable cytokine mRNA. In an attempt to identify the nature of the TNF-alpha mRNA-producing cells, acid phosphatase activity, which is characteristic of the macrophage lineage, was studied in the same thymuses. Acid phosphatase-positive cells only appear on day 15. Their frequency increases up to birth. However, no correlation can be established between acid phosphatase--and TNF alpha mRNA--positive cells. The results indicate that a small subset of thymic cells is responsible for TNF-alpha mRNA production during ontogeny: These cells are not yet identified. The possible role of TNF-alpha in thymic ontogeny is discussed.

Acid Phosphatase↗
PubMed1991

Mechanistic studies of a tyrosine-dependent catalytic antibody.

A pre-steady-state multiple-turnover kinetic burst is observed during hydrolysis of phenyl acetate by the catalytic antibody, 20G9. The burst is caused by partial product inhibition by phenol (Ki,app = 2.5 microM), which lowers both kcat and KM by almost an order of magnitude without affecting kcat/KM. The acid limb of the steady-state kcat pH profile of native 20G9 has a pKa of 9.6, suggesting a catalytic role for tyrosine. Additional evidence for an essential tyrosyl residue is that mild treatment of 20G9 with tetranitromethane nitrates a single tyrosine per equivalent of antigen binding sites and the mononitrated derivative has less than 5% of the native activity. Near-UV absorbance spectroscopy suggests that the alternative substrates N-carbobenzoxyglycine O-phenyl ester (ZG-OPh) and N-acetylglycine O-phenyl ester (AcG-OPh) acylate multiple tyrosines on the antibody. Neither ZG-OPh nor AcG-OPh are measurably catalyzed once appreciable acylation has taken place. Antibody acylated by ZG-OPh is inactive toward phenyl acetate hydrolysis, but can be reactivated by hydroxylamine. The data and derived kinetic rate equations are consistent with an acyl mechanism for phenyl acetate hydrolysis in which phenol inhibits by binding to a covalent O-acetyltyrosyl intermediate, slowing deacylation. Although the data are consistent with such a mechanism, they do not rule out other plausible, yet less unifying mechanisms of phenol inhibition; the observed burst could conceivably result from partial mixed phenol inhibition or from phenol-induced nonproductive substrate binding. Because antibodies often use tyrosines in antigen binding, tyrosyl catalytic antibodies may be commonly encountered in the future.

Antibodies, Monoclonal↗
PubMed1991

Characterization of the mechanism of action of a catalytic antibody.

The time course of phenylacetate hydrolysis by the catalytic antibody 20G9 has a kinetic burst lasting several reaction cycles. The burst is caused by partial mixed inhibition by one product of the hydrolysis, phenol, which binds with an apparent dissociation constant of 4.6 microM. Phenol binding causes kcat to decrease from 9.1 min-1 to 1.0 min-1 and Km to decrease from 300 microM to 36 microM. Because Km decreases but kcat/Km is unaffected, phenol must perturb the ground state structure but not the transition state structure. Structural complementarity to the transition state seems to be an important contributor to catalysis by 20G9 because weak binding in the ground state can be markedly improved by adding phenol, but tight binding of the transition state, which has been optimized by the immune system, cannot be readily improved. Further evidence that the substrate ground and transition states differ greatly in complementarity to the antibody is that the substrate binds more than five orders of magnitude more weakly than the transition state analogue hapten to which the antibody was raised. Two additional phenol molecules bind at higher product concentrations; the first binds over the concentration range of 15 to 86 microM and accelerates hydrolytic activity by 42%; the second is a competitive inhibitor with a Ki of 140 microM. Binding of multiple phenol molecules suggests the presence of abundant hydrophobic amino acids in the complementarity-determining region of 20G9.

Antibodies↗
PubMed1990

Potentiometric biosensor employing catalytic antibodies as the molecular recognition element.

Catalytic antibodies are introduced as an important new class of biomolecules for molecular recognition in biosensors in which the binding sites are continually regenerated by the catalytic reaction of the substrate. Consequently, molecular recognition by catalytic antibodies can yield reversible immunoblosensors. In this example, a prototype potentiometric biosensor is described in which a micro-pH electrode is modified with a catalytic antibody that catalyzes the hydrolysis of phenyl acetate, producing hydrogen ions that can be monitored by the electrode. The reversible response is linear with the log of substrate concentration over a range of 20-500 microM with a detection limit of 5 microM under the conditions of this study. Alternative applications of catalytic antibodies in other biosensor configurations are discussed.

Antibodies, Monoclonal↗
PubMed1990

Beta-lactamases as fully efficient enzymes. Determination of all the rate constants in the acyl-enzyme mechanism.

The rate constants for both acylation and deacylation of beta-lactamase PC1 from Staphylococcus aureus and the RTEM beta-lactamase from Escherichia coli were determined by the acid-quench method [Martin & Waley (1988) Biochem. J. 254, 923-925] with several good substrates, and, for a wider range of substrates, of beta-lactamase I from Bacillus cereus. The values of the acylation and deacylation rate constants for benzylpenicillin were approximately the same (i.e. differing by no more than 2-fold) for each enzyme. The variation of kcat./Km for benzylpenicillin with the viscosity of the medium was used to obtain values for all four rate constants in the acyl-enzyme mechanism for all three enzymes. The reaction is partly diffusion-controlled, and the rate constant for the dissociation of the enzyme-substrate complex has approximately the same value as the rate constants for acylation and deacylation. Thus all three first-order rate constants have comparable values. Here there is no single rate-determining step for beta-lactamase action. This is taken to be a sign of a fully efficient enzyme.

Acylation↗
PubMed1990

Staging equine seminiferous tubules by Nomarski optics in unstained histologic sections and in tubules mounted in toto to reveal the spermatogenic wave.

Nomarski optics were used to identify stages of the spermatogenic cycle of seminiferous tubules in sectioned tissue or in whole dispersed tubules and to characterize the equine spermatogenic wave. Embedded tissues were sectioned at 20 microns. Whole dispersed tubules were obtained by enzymatic digestion of thin slices of fresh testis. Dispersed tubules were fixed, dehydrated in graded levels of alcohol, infiltrated with Epon, and mounted in toto on glass slides. Stages of the spermatogenic cycle could be identified under Nomarski optics in both histologic sections and tubules mounted in toto. Stage dependent nuclear chromatic and cytoplasmic changes in spermatogonia, spermatocytes, and spermatids were evident. Spermatid development included chromatin condensation, nuclear elongation, acrosomal development from the Golgi and proacrosomic granules, migration of the annulus and mitochondrial alignment, and the transient appearance of the chromatoid body and manchette. Both nuclear and cytoplasmic details of Sertoli cells were revealed. In tubules mounted in toto, the spermatogenic wave along the length of the tubules occurred as a consecutive set of stages occupying small regions along the tubular length. The spermatogenic wave in the horse is more similar to that of humans than that of rats. The combination of enzymatic isolation of seminiferous tubules and identification of spermatogenic stages by Nomarski optics facilitates examination of the spermatogenic wave in species whose tubules are tightly bound and not easily teased apart.

Animals↗
PubMed1990

Effects of human alpha interferon on experimentally induced equine herpesvirus-1 infection in horses.

The immunotherapeutic effect of low-dose human alpha interferon on viral shedding and clinical disease was evaluated in horses inoculated with equine herpesvirus-1 (EHV-1). Eighteen clinically healthy weanling horses, 5 to 7 months old, were allotted to 3 equal groups. Two groups were treated orally with human alpha-2a interferon (0.22 or 2.2 U/kg of body weight), on days 2 and 1 before inoculation with EHV-1, the day of inoculation, and again on postinoculation day 1. The horses of the remaining group were given a placebo orally on the same days. The horses were monitored daily for changes in body temperature and for clinical signs of respiratory tract disease. Blood and nasal swab specimens were collected daily for virus isolation. Blood was also collected at intervals throughout the monitoring period for evaluation of CBC, serum IgG and IgM concentrations, and antibody titers to EHV-1. Febrile responses, nasal discharge, viral shedding, changes in CBC, and an increase in antibody titers to EHV-1 were noticed in all horses after inoculation. There was no significant difference (P greater than 0.05) in mean values of the factors measured between treatment and control groups.

Animals↗
PubMed1989

Effects of mechanism-based reversible inhibitors on the metal environment of cobalt(II)carboxypeptidase A: an electronic spectral study.

Electronic absorption, circular dichroic (CD), and magnetic circular dichroic (MCD) spectra have been determined for complexes of cobalt(II)-substituted carboxypeptidase A and five reversible inhibitors. Three of the inhibitors, N-(1-carboxy-5-butyloxycarbonylaminopentyl)-L-phenylalanine, (I); (R,S)-2-benzyl-4-oxobutanoic acid, (III); and 2-benzyl-4-oxo-5,5,5-trifluoropentanoic acid, (IV) are mechanism-based inhibitors. Another, N-(1-carboxy-5-carbobenzoxyaminopentyl)-glycyl-L-phenylalanine, (II), is a tight binding, slowly hydrolyzed substrate. The fifth, phosphoramidon, (V), is a mechanism-based inhibitor of thermolysin, and may also bind to carboxypeptidase in a mechanism-based mode. The absorption and CD spectra of the enzyme-inhibitor complexes all differ from the spectrum of the free enzyme and from each other. The MCD spectra indicate that the tetrahedral coordination geometry of cobalt, which is distorted in the free enzyme, is also distorted in the inhibitor complexes, although to various degrees. The complexes of I and III are spectrally similar despite being structurally dissimilar, and that of IV, whose structure resembles III, is spectrally distinct, indicating that I and III, but not IV, may perturb the metal in nearly the same way. The absorption spectrum of IV is identical to that, at high pH, of Co(II)carboxypeptidase in which Glu-270 has been modified by a carbodiimide reagent, possibly pointing to a common perturbation of this residue. The absorption and CD spectra of II are similar to those of the catalytic intermediate that precedes the rate-limiting step in peptide hydrolysis [D. S. Auld, A. Galdes, K. F. Geoghegan, B. Holmquist, R. Martinelli, and B. L. Vallee, Proc. Natl. Acad. Sci. USA 81, 4675-4681 (1984)]. Since II is a substrate, the steady-state bound species that it generates may therefore be a true productive intermediate rather than a nonproductive mimic of an intermediate. The spectra of the complexes with II and V differ considerably despite structural similarities. The negative CD ellipticity of the free enzyme is reversed in sign in the presence of V, a phenomenon previously observed with complexes of Co(II)carboxypeptidase and dipeptides. This resemblance may result from a similar interaction of cobalt with the phosphoramidate group of phosphoramidon and the N-terminal amine of dipeptides. The spectra of reversible, mechanism-based inhibitors permit general structural predictions about true intermediates but require caution when used for assigning precise conformation and ligands of bound catalytic species.

Binding Sites↗
PubMed1989

An angiotensin converting enzyme inhibitor is a tight-binding slow substrate of carboxypeptidase A.

Carboxypeptidase A-catalyzed hydrolysis of peptides and depsipeptides is competitively inhibited by N-(1-carboxy-5-t-butyloxycarbonylaminopentyl)-L-phenylalanine (Boc-CA-Phe, Ki = 1.3 microM) and the angiotensin converting enzyme inhibitor, N-(1-carboxy-5-carbobenzoxyaminopentyl)-glycyl-L-phenylalanine (Z-CA-Gly-Phe, Ki = 4.5 microM). The latter compound is actually a slow substrate of carboxypeptidase. Indirect observation of inhibitor binding by stopped-flow measurement of radiationless energy transfer between carboxypeptidase tryptophans and dansylated substrates reveals slow binding for both compounds. The visible absorption spectrum of the complex of cobalt(II)-substituted carboxypeptidase and Z-CA-Gly-Phe, which differs from the corresponding spectrum of the Boc-CA-Phe complex, is remarkable in its resemblance to the spectrum of the complex between Co(II)carboxypeptidase and a transient intermediate previously observed during hydrolysis of peptide substrates. The spectrum slowly changes to that of the free enzyme indicating hydrolysis. Chromatographic quantitation of substrate and products confirms that carboxypeptidase converts Z-CA-Gly-Phe to Z-CA-Gly and L-Phe with an apparent kcat of 0.02 s-1. Absorption spectroscopy indicates that the Z-CA-Gly-Phe-Co(II)carboxypeptidase spectrum is not that of bound products. Moreover, spectral titrations indicate that the products (both with spectral Ki values of about 3 mM), as well as D-Phe, compete for the same site on the enzyme.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1988

Kinetic characterization of the acyl-enzyme mechanism for beta-lactamase I.

beta-Lactamase I catalyses the hydrolysis of penicillins by an acyl-enzyme mechanism. A procedure was developed for determining the rate constants for the acylation and deacylation steps for the good substrates benzylpenicillin and phenoxymethylpenicillin; this depends on determining the fraction of enzyme that is present as acyl-enzyme in the steady state.

Acylation↗
PubMed1988

Metal-free chromatographic media.

The three major considerations in reducing metal-gel binding are (1) the type of gel to be used, (2) the ionic strength of the eluent, and (3) alkaline reduction of the gel. Each of these parameters should be considered in advance of an experiment. A method is offered for determining the optimal gel type, gel treatment, and eluent for a critical metal-free chromatography experiment.

Chromatography↗
PubMed1988

Biochemical study of adduct synthesis between fibrin monomers and elastin.

Monomers of fibrin generated by thrombin from fibrinogen reacted with elastin to give a new addition product or adduct. Adduct formation resulted from a covalent bond between fibrin monomers and elastin. The kinetic studies of this reaction confirmed that the adduct was formed before fibrin precipitated to produce the clot. The reaction depended on elastin, fibrinogen and thrombin concentrations. When thrombin-induced and reptilase-induced fibrin were compared, it became obvious that fibrin monomers did intervene more commonly as Des AA-fibrin than as Des AA.BB-fibrin. The adduct synthesis was completely inhibited by 150 microM of the peptide Gly-Pro-Arg-Pro which was previously known to stabilize the fibrin monomers and consequently to inhibit the polymerization completely. It is shown that FXIII could intervene directly in the reaction where homological quality of elastin (human versus bovine) and purity of thrombin were other important factors.

Animals↗