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Biomedical subjects

M G Hamilton

Publications and source records attributed to M G Hamilton.

At least 73 records · Page 4Linked to original sources
PubMed1988

Subunit dissociation and denaturation of Fasciolaria tulipa hemocyanin.

1. The hemocyanin from the marine snail, Fasciolaria tulipa has a molecular weight of 8.6 +/- 0.6 x 10(6) determined by light-scattering and a sedimentation constant of (105.9 +/- 1.1)S. 2. The dissociated subunits at pH 11 and in 8.0 M urea (pH 7.4) had molecular weights of 4.4 x 10(5) and 4.7 x 10(5), close to one-twentieth of the parent didecameric assembly. 3. The pH dependence of the molecular weight profile exhibited bell-shaped transitions in both the presence and absence of Ca2+ and Mg2+ ions. In the physiological pH range of about 7.5-8.2 in divalent ion-containing buffers neither the molecular weight behavior nor the sedimentation patterns suggest any significant dissociation. 4. Both the urea and the Hofmeister salt series were found to dissociate the didecameric hemocyanin assembly. The ureas exhibit increasing effectiveness as dissociating agents with the higher alkyl substituted members of the series, suggesting hydrophobic stabilization of the subunit assembly. 5. Denaturation of the hemocyanin subunits by the urea series follows the same trend in effectiveness as the dissociation reaction; the reagent concentrations required to cause unfolding of the globular domains of the hemocyanin chains were, however, much higher than those needed for dissociation.

Animals↗
PubMed1987

Stabilizing influence of calcium and magnesium ions on the decameric structure of chiton hemocyanins.

The stabilizing effects of Ca2+ and Mg2+ ions on the decameric structure of hemocyanins from two representative chitons, Stenoplax conspicua and Mopalia muscosa were investigated by light-scattering molecular weight measurements, ultracentrifugation, absorbance, and circular dichroism methods. The dissociation profiles at any given pH resulting from the decrease in divalent ion concentration, investigated at a fixed protein concentration of 0.1 g.liter-1, could be fitted by a decamer-to-dimer-to monomer scheme of subunit dissociation. The initial decline in the light-scattering molecular weight curves required one or two apparent binding sites per hemocyanin dimer formed as intermediate dissociation product, with apparent dissociation constants (kD,2) for Ca2+ ions of 0.7 to 7 X 10(-4) M, not very different from the value of 2.5 X 10(-4) M obtained by Makino by equilibrium dialysis for the hemocyanin of the opistobranch, Dolabella auricularia. The binding of Mg2+ ion to S. conspicua and M. muscosa hemocyanins appears to be both weaker than the binding of Ca2+ and more pH dependent, with kD,2 values ranging from the 3 X 10(-4) to 4 X 10(-2) M at pH 8.5 to 9.5. The dissociation the decameric hemocyanin species (sedimentation coefficient ca. 60 S) is also observed in the ultracentrifugation with the initial appearance of 18-20 S dimers, followed by a shift in equilibrium to monomeric species of lower sedimentation rates of 11-12 S as the divalent ion concentration is reduced below 1 X 10(-4) M Ca2+ and Mg2+. The dissociation of dimers to monomers in the second step of the reaction is characterized by one or two binding sites per subunit and a somewhat stronger affinity for divalent ions, indicated by apparent dissociation constants (kD,1) of 0.7 X 10(-4) to 3 X 10(-3) M. Circular dichroism and absorbance measurements at 222 and 346 nm suggest no significant changes in the conformation of the hemocyanin subunits produced by the different stages of subunit dissociation.

Animals↗
PubMed1987

Hydrophobic stabilization of chiton hemocyanins: effects of ureas, Hofmeister salts and pH on their dissociation.

The subunit dissociation of the hemocyanins from five members of the Polyplacophora families, Acanthochitonidae, Callistoplacidae, Chitonidae, Ischnochitonidae and Mopalidae, represented by the chitons Cryptochiton stelleri, Nutallina fluxa, Acanthopleura granulata, Stenoplax conspicua and Mopalia mucosa, respectively, have been investigated by light-scattering molecular-weight and ultracentrifugation methods, using the hydrophobic reagents of the urea series and the Hofmeister salt series as probes of the contact areas of the hemocyanin subunits. The polyplacophoran hemocyanins are decamers with molecular weights of (4.2-4.6) X 10(6). The effectiveness of dissociation by the ureas follows the order of increasing hydrophobicity of the reagent, i.e., urea, methyl-, ethyl-, propyl- and butylurea, as expected of hydrophobically stabilized subunit systems. The urea dissociation is found to be a two-step reaction: the dissociation of parent decamers to dimers followed by dissociation of the dimers to monomers. Analysis of the observed decrease in molecular weight requires the interaction with urea of about 27 to 35 apparent amino-acid groups (Napp) at the contact areas of the dimers, and a much larger number of apparent binding groups ranging from about 100 to 120 per monomer at the contact areas of the monomers. Fitting of the pH dissociation profiles of A. granulata, C. stelleri, M. muscosa and S. conspicua requires the participation of a much smaller number of amino-acid residues in the interaction with the probe-solvent components. The ionization or protonation of one acidic and one basic group per dimer, and five to eight acidic and basic groups per monomer is found to be adequate for the description of the two-step pH dissociation reaction.

Animals↗
PubMed1987

Hemocyanin of the chiton, Stenoplax conspicua (Dall).

1. The hemocyanin of the chiton, Stenoplax conspicua, has a molecular weight determined by light-scattering of 4.2 X 10(6) daltons, (dt) and a sedimentation coefficient of 60 S. 2. The fully dissociated subunits in 6.0 and 8.0 M urea, and at pH 8.9-10 in the absence of divalent ions, have molecular weights of 4.15-4.30 x 10(5) and 4.17-4.75 x 10(5) dt, which is close to one-tenth of the molecular weight of the parent hemocyanin assembly. 3. The pH dependence of the molecular weights from pH 4.5 to 11 exhibit bell-shaped transition profiles, best accounted for by a three-species, decamer to dimer to monomer scheme of subunit dissociation, with one acidic and one basic ionizing group per dimer and 5-8 acidic and basic groups per monomer. 4. In the absence of stabilizing divalent ions S. conspicua hemocyanin is relatively unstable. At pH 7.4 in the presence of 0.01 M EDTA, it is predominantly in the dimeric state, characterized by a sedimentation constant of 18 S. It is also more readily dissociated to monomers at high pHs (8-9 and above) than are the C. stelleri and A. granulata hemocyanins. 5. Urea and GdmCl are effective dissociating agents of S. conspicua hemocyanin. The urea dissociation profile obtained at pH 8.5, 0.01 M Mg2+, 0.01 M Ca2+, and analyzed by means of the decamer-dimer-monomer scheme of subunit dissociation gave estimates of about 30 amino acid groups (Napp) at the dimer contacts within the hemocyanin decamers and about 120 groups per monomer within each dimer, suggesting hydrophobic stabilization of hemocyanin assembly.

Animals↗
PubMed1987

Physical investigations of the hemocyanin of the chiton, Cryptochiton stelleri (Middendorff).

The hemocyanin of the giant Pacific chiton, Cryptochiton stelleri has a molecular weight of 4.2 +/- 0.3 X 10(6), determined by light-scattering, and a sedimentation coefficient of 60S. The fully dissociated subunits in nondenaturing solvents, at pH 10.6, 1 X 10(-2)M EDTA and in 8.0 M urea, pH 7.4 have molecular weights of 4.10 X 10(5) and 4.35 X 10(5), close to one-tenth of the molecular mass of the parent hemocyanin decamers. In the pH region from about 3.5 to 11 the molecular weight (Mw), determined at constant protein concentration of 0.10 g1(-1) exhibits a bell-shaped molecular weight profile centering about the physiological pH of the hemolymph of 7.2. The pH-Mw profile is best accounted for in terms of a three state, decamer-dimer-monomer dissociation scheme. Analysis of the Mg2+ and Ca2+ effects on the molecular weight transitions suggest stabilization of the hemocyanin decamers through one bound divalent ion per hemocyanin monomer or dimer. Urea, GdmCl, and the higher members of the chaotropic salt series are effective dissociating agents for Cryptochiton stelleri hemocyanin. The dissociation profile obtained with urea at pH 8.5, 0.01 M Mg2+, 0.01 M Ca2+ has been analyzed in terms of both the two- and three-species schemes of subunit-dissociation. Hydrophobic stabilization of the subunit contacts is suggested by the large number of apparent amino acid groups (Napp), of the order of 30 between dimers stabilizing the decamers, and 120 apparent amino acid groups between each monomer forming the constituent dimers.

Animals↗
PubMed1987

Coronary artery disease and coronary bypass grafting in young men: experience with 138 subjects 39 years of age and younger.

One hundred thirty-eight men aged less than or equal to 39 years had coronary bypass grafting during a 13 year period. Angina was the presenting symptom in 77% and of these patients, one-third had unstable angina. More than half of the patients had experienced at least one myocardial infarct. There was a high incidence of coronary risk factors, especially smoking. Nineteen patients (13.8%) had left main coronary artery stenosis (it was isolated in two); 13.8, 24.6 and 60.2% had single, double and triple vessel disease, respectively. Left ventriculograms showed serious functional impairment in 42%. A total of 461 coronary bypass grafts, 3.34 per patient, were placed; almost all were vein grafts. There were no operative deaths. Transmural myocardial infarction occurred in 4.3% of patients. All bypass grafts were opacified angiographically early after operation, 95% at 1 year, 56% at 5 years and 26% at 10 years after operation. Some patients also had coronary angiograms, dictated by clinical events, between 1 and 5 and between 5 and 10 years postoperatively. Patency rates for bypass grafts were comparable with those previously reported and were acceptable, although they decreased with time. However, increasing evidence of atherosclerosis of bypass grafts was seen beyond 1 year, particularly beyond 5 years. Of 23 subjects with a coronary bypass reoperation, 2 died and 44% had perioperative transmural myocardial infarction. During follow-up, 13.8% of the patients died, survival being 95, 84 and 76% at 5, 10 and 12 years, respectively. It is considered that the patients were advantageously treated with coronary bypass grafting especially in the short-term. However, bypass graft patency steadily decreased with the passage of time and graft atherosclerosis became an increasingly important problem.

Adult↗
PubMed1986

Hemocyanin of the chiton Acanthopleura granulata.

The subunit structure and solution conformation of the hemocyanin of the chiton Acanthopleura granulata were investigated by light-scattering, ultracentrifugation, viscosity, absorbance, and circular dichroism methods. The molecular weight, determined by light scattering at pH 7.4 in the presence of 0.05 M Mg2+ and 0.01 M Ca2+, was (4.2 +/- 0.3) X 10(6), while those of dissociated subunits in the presence of 8.0 M urea (at pH 7.4) and at pH 10.7 were found to be 4.57 X 10(5) and 4.58 X 10(5), respectively. Circular dichroism and absorbance measurements at 222 and 346 nm indicate only minor changes in the conformation of the folded domains of the hemocyanin subunits in these dissociating solvents. As with the hemocyanins of the snails Busycon canaliculatum, Lunatia heros, and Littorina littorea, exposure to 4.0-6.0 M guanidinium chloride (GdmCl) is found to produce unfolding of the domains, resulting in much more pronounced spectral changes and a further drop in molecular weight. A Mw of 3.2 X 10(5) was obtained with Acanthopleura hemocyanin in 6.0 M GdmCl, suggesting hidden breaks in the polypeptide chains analogous to those observed with the gastropodan hemocyanins. Both urea and pH dissociation showed gradual declines in the molecular weights, consistent with a decamer-dimer-monomer scheme of subunit dissociation. The bell-shaped molecular weight profiles obtained in the pH region from 5 to 11 can be accounted for by assuming two proton-linked groups per dimer, characterized by apparent pK values of 5.5 and 9.5, and the further involvement of five to eight acidic and five to eight basic groups per monomer, having apparent pK values of 5.0 and 10.2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Activation of central muscarinic receptors inhibit Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ transport in synaptic membranes.

Preparations of lysed synaptosomes exhibit a high affinity Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ accumulation activity, with a Km for Ca2+ congruent to 0.5 microM, close to the cytosolic concentration of Ca2+. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1-20 microM), both Ca2+/Mg2+ ATPase and ATP-dependent CA2+ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5-1.0 microM) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca2+ channels or Na+-Ca2+ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca2+ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca2+ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.

Animals↗
PubMed1980

Raman spectra and conformational properties of ribosomes during various stages of disassembly.

Raman spectra have been obtained on aqueous solutions of ribosomes, ribosomal subunits, ribosomal proteins, and ribosomal RNA extracted from both rat liver (RL) and Escherichia coli cells. The species examined from RL ribosomes are total ribosomes (80 S), large subunits (60 S), small subunits (40 S), EDTA-treated ribosomes, total rRNA, 28S RNA, 18S RNA, total protein, RNP particles from 80S ribosomes, and RNP particles from 60S subunits. The species examined from E. coli ribosomes are total ribosomes (70 S), large subunits (50 S), small subunits (30 S), mixtures of 50S and 30S subunits, total rRNA, 23S RNA, and 16S RNA. All rRNA molecules are shown by Raman spectroscopy to contain highly ordered secondary structures in which the backbone conformations are predominantly of the A-helix type. The present Raman spectra do not contain sufficient detail, however, to reach firm conclusions about the conformations of ribosomal proteins or their mutual interactions. RNA molecules within ribosomal particles remain highly ordered during various stages of ribosome disassembly, and their conformations are generally invariant to perturbations of ribosome structure, including dissociation into subunits, EDTA treatment, and partial deproteinization in a CsCl density gradient. However, when total protein extraction is carried out on ribosomes and subunits, small but significant changes in rRNA secondary structure are detected. The kind and magnitude of secondary structural change are different for different ribosomal particles. The Raman spectra of the ribosomes are compared also with spectra of a model ribonucleoprotein, the complex formed by poly(riboadenylic acid) and poly(L-arginine).

Animals↗
PubMed1979

Mechanism of inhibition of hepatic protein synthesis in rats by the carcinogen, methylazoxymethanol acetate.

Treatment of rats with the carcinogen, methylazoxymethanol acetate, results in a rapid, marked inhibition of hepatic protein synthesis and disaggregation of polysomes. Studies were undertaken to learn the mechanism by which this carcinogen induces these effects in rat liver. The data show that the inhibition of endogenous protein synthesis is not due to an effect on the high speed supernatant 'factors' but rather at the level of the polysome, and that both free and membrane-bound polysomes are affected. Poly(U)-directed polyphenylalanine synthesis by native ribosomal subunits is greater in preparations isolated from rats treated with carcinogen than it is in controls. Moreover, the native ribosomal subunit fraction from treated livers in response to added rabbit globin mRNA is able to synthesize a protein similar in molecular weight to globin. These studies show that methylazoxymethanol acetate does not induce significant alterations of ribosomal subunits or of initiation factors and suggest that the inhibition of protein synthesis and disaggregation of polysomes may be the results of an alteration of cytoplasmic mRNA, or its association with ribosomes.

Animals↗