Homocyst(e)ine and risk of cardiovascular disease in the multiple risk factor intervention trial.

A nested case-control study was undertaken involving men participating in the Multiple Risk Factor Intervention Trial (MRFIT). Serum samples from 712 men, stored for upto 20 years, were analysed for homocyst(e)ine. Cases involved non-fatal myocardial infractions, identified through the active phase of the study, which ended on February 28, 1982, and deaths due to coronary heart disease, monitored through 1990. The non-fatal myocardial infarction occurred within 7 years of sample collection, whereas the majority of coronary heart disease deaths occurred more than 11 years after sample collection. Mean homocyst(e)ine concentrations were in the expected range and did not differ significantly between case patients and control subjects: myocardial infarction cases, 12.6 micromol/L; myocardial infarction controls, 13.1 micromol/L; coronary heart disease death cases, 12.8 micromol/L; and coronary heart disease controls, 12.7 micromol/L. Odds ratios versus quartile 1 for coronary heart disease deaths and myocardial infarctions combined were as follows: quartile 2, 1.03; quartile 3, 0.84; and quartile 4, 0.92. Thus, in this prospective study, no association of homocyst(e)ine concentration with heart disease was detected. Homocyst(e)ine levels were weakly associated with the acute-phase (C-reactive) protein. These results are discussed with respect to the suggestion that homocyst(e)ine is an independent risk factor for heart disease.

Homocyst(e)ine and risk of cardiovascular disease in the Multiple Risk Factor Intervention Trial.

A nested case-control study was undertaken involving men participating in the Multiple Risk Factor Intervention Trial (MRFIT). Serum samples from 712 men, stored for up to 20 years, were analyzed for homocyst(e)ine. Cases involved nonfatal myocardial infarctions (MIs), identified through the active phase of the study, which ended on February 28, 1982, and deaths due to coronary heart disease (CHD), monitored through 1990. The nonfatal MIs occurred within 7 years of sample collection, whereas the majority of CHD deaths occurred more than 11 years after sample collection. Mean homocyst(e)ine concentrations were in the expected range and did not differ significantly between case patients and control subjects: MI cases, 12.6 mumol/L; MI controls, 13.1 mumol/L; CHD death cases, 12.8 mumol/L; and CHD controls, 12.7 mumol/L. Odds ratios versus quartile 1 for CHD deaths and MIs combined were as follows: quartile 2, 1.03; quartile 3, 0.84; and quartile 4, 0.92. Thus, in this prospective study, no association of homocyst(e)ine concentration with heart disease was detected. Homocyst(e)ine levels were weakly associated with the acute-phase protein (C-reactive protein). These results are discussed with respect to the suggestion that homocyst(e)ine is an independent risk factor for heart disease.

Identification of zinc proteins in rat parotid saliva.

The presence of unique zinc-binding proteins in human saliva is well documented. These observations have not, however, been extended to other species. The rat has been used extensively to study the salivary gland and its secretion, and it is therefore important to determine if the spectrum of zinc-binding proteins in this experimental model resembles that found in humans. To begin the analysis of zinc-binding proteins in stimulated rat parotid saliva, the saliva was fractionated by DEAE Sephadex and Sepharose 6B chaelate chromatography and the protein patterns analysed by electrophoresis. Zinc-binding proteins from the parotid saliva were identified by incubating Western blots with 65Zn and identifying any bound zinc by autoradiography. Comparison of the autoradiograms with the Coomassie blue-stained filter revealed several proteins with zinc-binding capacity. Isolation of the major zinc-binding proteins revealed an amino acid composition of proline 28%, glutamine 19% and glycine 15%, which is consistent with the amino acid composition of rat salivary acidic proline-rich protein. In addition to the proline-rich proteins, one other zinc-binding protein was analysed. The N-terminal sequence of this protein was found to bear a striking similarity (16 out of 20 amino acids) to secreted carbonic anhydrase VI of the mouse, a known zinc-binding protein. These data demonstrate that rat acidic proline-rich proteins, having an amino acid composition similar to that in humans, have zinc-binding potential. The data also confirm previous reports suggesting secreted carbonic anhydrase in rat parotid saliva.

Human aldehyde dehydrogenase: improved purification procedure and comparison of homogeneous isoenzymes E1 and E2.

An improved purification procedure of human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1 and E2 is presented. This procedure employs only three chromatographic steps to produce homogeneous E1 and E2 isoenzymes at 60% overall yield. The isoenzymes have been tested for homogeneity by electrophoresis of native and denatured species, specific activity determinations following rechromatography, as well as mapping of tryptic and CNBr fragments. Total SH group analysis has also been done on each isoenzyme. The results show that both isoenzymes are homogeneous. Similarities between E1 and E2 isoenzymes are noted in the mobility of about 40% of tryptic fragments, total SH content, and the mobility of two CNBr fragments. The results also show considerable structural differences between the isoenzymes in that CNBr maps show fragments from E1 and E2 of different molecular weight and about 60% of tryptic fragments migrate to distinct locations. Only one of SH-containing tryptic fragments migrates to the same location in both isoenzymes. E1 and E2 each consist of subunits which migrate as single bands in both sodium dodecyl sulfate (SDS) and urea electrophoresis. While the mobility of E1 and E2 subunits in SDS gels is similar, it is different in urea, showing that subunits of E1 are distinct from those of E2 and that the isoenzymes do not share subunits. Structural similarity between isoenzymes must, therefore, result from sequence similarity within regions of distinct polypeptide chains composing E1 and E2 molecules. The results presented offer a simplified procedure for preparation of the homogeneous isoenzymes; they also suggest that E1 and E2 are products of distinct genes which probably diverged from a common genetic ancestor through gene duplication and compartmentation of the cell.

On the interaction of human liver aldehyde dehydrogenase E1 isoenzyme with disulfiram and iodoacetamide.

The E1 isoenzyme of human liver aldehyde dehydrogenase (Km acetaldehyde = 30uM, pH 7.0), when incubated with disulfiram at a stoichiometry of four moles disulfiram/tetrameric E1, is immediately inhibited to within 10% of control activity. The inhibition is reversed by 0.1% (v/v) mercaptoethanol, indicating disulfide bridge formation. An indirect attempt to locate, on maps, a peptide binding disulfiram has yielded inconsistent results. Iodoacetamide inhibits E1 slowly; inhibition is facilitated in the presence of NAD, resulting in loss of ca. 90% of control activity. Incubation with 14C iodoacetamide labels a 16,000 dalton CNBr peptide, and a ca. 4,000 dalton tryptic cleavage product. These fragments can be equated with those which have been suggested by disulfiram.

Human stomach alcohol dehydrogenase: isoenzyme composition and catalytic properties.

Two isoenzymes of alcohol dehydrogenase have been purified from human stomach and characterized with regard to electrophoretic mobility and kinetic properties with ethanol, hexanol, and acetaldehyde. Both undergo a time-dependent formation of multiple electrophoretic bands; the total amount of alcohol dehydrogenase activity in an average human stomach is only about 0.2% of that of the liver.

Rapid modulation of gill Na+ + K+-dependent ATPase activity during acclimation of the killifish Fundulus heteroclitus to salinity change.

The enzymatic properties of membrane-bound Na+ + K+-ATPase from gills of killifish acclimated to fresh water, to 16% sea water, or to 30% sea water appear to be identical, indicating that the same enzyme may function to absorb Na+ in low salinities and excrete Na+ at the gills in high salinities. Ammonium ion is an effective substitute for K+: in the ATPase reaction itself, in blocking phosphorylation of the ATPase protein, and in inhibiting the binding of ouabain to the enzyme. The specific activities of the Na+ + K+-ATPase in the three different salinities are consistent with the expected Na+ pumping rates: higher in fresh water and 30% sea water than in 16% sea water. Within one-half hour after transfer of killifish from one salinity to another, gill Na+ + K+-ATPase activities reach equilibrium levels. The rapid increase in Na+ + K+-ATPase activity in gill microsomes of fish acclimating from fresh water to 30% sea water is accompanied by a slow decrease in the number of binding sites for ouabain, supporting the idea that acclimation to short-term salinity changes may involve modifications in the catalytic rate rather than the number of Na+ + K+-ATPase molecules.