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J E Mace

Publications and source records attributed to J E Mace.

11 recordsLinked to original sources
PubMed1991

Computational method for the design of enzymes with altered substrate specificity.

A combination of enzyme kinetics and X-ray crystallographic analysis of site-specific mutants has been used to probe the determinants of substrate specificity for the enzyme alpha-lytic protease. We now present a generalized model for understanding the effects of mutagenesis on enzyme substrate specificity. This algorithm uses a library of side-chain rotamers to sample conformation space within the binding site for the enzyme-substrate complex. The free energy of each conformation is evaluated with a standard molecular mechanics force field, modified to include a solvation energy term. This rapid energy calculation based on coarse conformation sampling quite accurately predicts the relative catalytic efficiency of over 40 different alpha-lytic protease-substrate combinations. Unlike other computational approaches, with this method it is feasible to evaluate all possible mutations within the binding site. Using this algorithm, we have successfully designed a protease that is both highly active and selective for a non-natural substrate. These encouraging results indicate that it is possible to design altered enzymes solely on the basis of empirical energy calculations.

Algorithms↗
PubMed1991

Roles of the beta 146 histidyl residue in the molecular basis of the Bohr effect of hemoglobin: a proton nuclear magnetic resonance study.

Assessment of the roles of the carboxyl-terminal beta 146 histidyl residues in the alkaline Bohr effect in human normal adult hemoglobin by high-resolution proton nuclear magnetic resonance spectroscopy requires assignment of the resonances corresponding to these residues. Previous resonance assignments in low ionic strength buffers for the beta 146 histidyl residue in the carbonmonoxy form of hemoglobin have been controversial [see Ho and Russu (1987) Biochemistry 26, 6299-6305; and references therein]. By a careful spectroscopic study of human normal adult hemoglobin, enzymatically prepared des(His146 beta)-hemoglobin, and the mutant hemoglobins Cowtown (beta 146His----Leu) and York (beta 146His----Pro), we have resolved some of these conflicting results. By a close incremental variation of pH over a wide range in chloride-free 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer, a single resonance has been found to be consistently missing in the proton nuclear magnetic resonance spectra of these hemoglobin variants. The spectra of each of these variants show additional perturbations; therefore, the assignment has been confirmed by an incremental titration of buffer conditions to benchmark conditions, i.e., 0.2 M phosphate, where the assignment of this resonance is unambiguous. The strategy of incremental titration of buffer conditions also allows extension of this resonance assignment to spectra taken in 0.1 M [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane buffer. Participation of the beta 146 histidyl residues in the Bohr effect has been calculated from the pK values determined for the assigned resonances in chloride-free 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer. Our results indicate that the contribution of the beta 146 histidyl residues is 0.52 H+/hemoglobin tetramer at pH 7.6, markedly less than the 0.8 H+/hemoglobin tetramer estimated by study of the mutant hemoglobin Cowtown (beta 146His----Leu) by Shih and Perutz [(1987) J. Mol. Biol. 195, 419-422]. We have found that at least two histidyl residues in the carbonmonoxy form of this mutant have pK values that are perturbed, and we suggest that these pK differences may in part account for this discrepancy. Furthermore, summation of the positive contribution of the beta 146 histidyl residues and the negative contribution of the beta 2 histidyl residues to the maximum Bohr effect measured in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer suggests that additional sites in the hemoglobin molecule account for proton release upon ligation greater than the contribution of the beta 146 histidyl residues.(ABSTRACT TRUNCATED AT 400 WORDS)

Hemoglobin A↗
PubMed1990

Pectoralis muscle uptake of thallium-201 after arm exercise ergometry. Possible confusion with lung thallium-201 activity.

Pectoralis muscle uptake of thallium-201 was noted in 8 (73%) out of 11 patients after exercise arm ergometry. Uptake varied from mild to marked and potentially could be confused with pulmonary Tl-201 activity with a resulting false-positive diagnosis of exercise-induced left ventricular dysfunction. The three patients exhibiting negative or trace Tl-201 uptake had suboptimal exercise efforts. The characteristics of pectoralis muscle Tl-201 uptake are illustrated, and differentiation from true lung Tl-201 activity is discussed.

Arm↗
PubMed1983

Rubella-specific IgM reactivity in sera from cases of infectious mononucleosis.

Eight sera from 125 cases of infectious mononucleosis (IM) were reactive for rubella-specific IgM in an M-antibody capture radioimmunoassay. The reactivity of individual sera varied depending upon the source of the rubella antigen used in the assay. One serum gave strongly positive results with some rubella haemagglutinating antigens but negative results with others and may have contained an IgM antibody which was capable of distinguishing between strains of rubella virus. If the diagnosis of rubella is based solely on detection in solid-phase immunoassay of rubella-specific IgM, IM should be excluded.

Adult↗
PubMed1981

Are many women immunized against rubella unnecessarily?

Radial haemolysis (RH) was used to test sera for immunity to rubella from 1317 patients attending a general practice. One hundred and forty-one (10.7%) were treated as susceptible and offered an attenuated virus vaccine (RA 27/3). Pre-immunization sera from 43% of these patients were reactive at low levels in RH (less than 15 international units rubella antibody per ml). Pre- (S1) and post- (S2) immunization sera from 66 vaccinees were studied in detail. Antibody was detected by RH, haemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA), and the specific IgM response was measured by a solid-phase M-antibody capture radioimmunoassay (MACRIA). The vaccine-induced IgM response was only detected if the S1 serum was non-reactive by all tests for rubella antibody. It was weaker than that seen following wild virus infection. It could be detected reliably for six weeks, and in most cases for nine weeks, after immunization. In contrast, patients with S1 specimens reactive by RH, HI or ELISA never showed an IgM response in the S2 specimen despite "significant' antibody rises often being present. It was considered that an IgM response to RA 27/3 was the best indicator of pre-immunization susceptibility to rubella. The failure of many vaccinees to make an IgM response implied that a significant proportion were already immune. It is suggested that the threshold for a report of immunity to rubella could be lowered from 15 i.u. antibody per ml and so fewer women immunized without vaccine being withheld from those who need it.

Adolescent↗
PubMed1975

The detection of specific IgM antibodies following infection with rubella virus.

A gel filtration technique using Sephadex G-200 has been used for the detection of specific IgM in sera from (a) 45 cases of clinical rubella in which diagnostic rises of rubella haemagglutination-inhibiting (HAI) antibody could be demonstrated; (b) 70 cases with clinical evidence of rubella in which a rising titre could not be demonstrated because the first serum sample already had high titre HAI antibodies; and (c) 100 patients in whom rubella was not suspected. The results indicate that the high specificity and sensitivity of the method described make it an appropriate technique for use in the routine diagnosis of acquired rubella.

Antibodies, Viral↗
PubMed1975

Elution patterns of rubella IgM, IgA, and IgG antibodies from a dextran and an agarose gel.

The elution pattern of serum proteins and the distribution of rubella HAI activity in 95 sera from 65 cases were determined after gel filtration with (a) Sephadex G-200 and (b) Bio-Gel A-5M. Rubella HAI antibody in peak 1 after Sephadex G-200 fractionation of early convalescent sera consists of both IgM and high molecular weight IgA. However, these two classes of antibody can be distinguished by gel filtration with Bio-Gel A-5M. Bearing these differences in mind, the results of fractionation with the two gels correlate very well although the use of Bio-Gel A-5M gives a slightly less sensitive technique for the diagnosis of recent infection.

Chromatography, Gel↗