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Biomedical subjects

R Byrne

Publications and source records attributed to R Byrne.

At least 37 records · Page 2Linked to original sources
PubMed1984

In vitro effect of Triton WR-1339 on canine plasma high density lipoproteins.

We studied the effect in vitro of various concentrations of Triton WR-1339 on normolipidemic canine plasma and on the high density lipoproteins (HDL) isolated from this plasma by ultracentrifugation. As a preamble to this study, we established that Triton WR-1339 has a unimer molecular weight of 4,500, a micellar molecular weight of 180,000, and a critical micellar concentration (CMC) of 0.018 mM or 0.008 g/dl. Above its CMC, Triton WR-1339 in concentrations between 2 and 10 mg/ml induced concentration-dependent structural changes in HDL which were characterized by a progressive displacement of apoA-I from the HDL surface without loss of lipids. The addition of Triton WR-1339 to the HDL particles modified their electrophoresis mobility and caused an increase in size (95 +/- 5 A to 114 +/- 7 A). At the extreme Triton WR-1339 concentrations utilized in these studies (10 mg/ml) disruption of the HDL particles occurred; at this stage, the original, relatively homogeneous, spherical HDL particles were replaced by a heterogeneous population ranging in size between 50 and 250 A, representing complexes of Triton WR-1339 with lipids essentially free of apoA-I which could be sedimented by ultracentrifugation. The effects of Triton WR-1339 on whole plasma or isolated HDL were comparable. These studies indicate that Triton WR-1339 in vitro alters HDL in a concentration-dependent manner and that these changes vary from a displacement of apoA-I from the HDL surface to a state where all lipids are solubilized into the Triton WR-1339 micellar phase and are driven away from the protein moiety.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Organization of unesterified cholesterol in high density lipoproteins probed by filipin.

The initial rate of filipin association with unesterified cholesterol in high density lipoproteins (HDL) was measured by stopped-flow spectrophotometry to assess the roles played by apolipoproteins and phospholipids in modulating the surface exposure of cholesterol. The initial rate of filipin-unesterified cholesterol association was enhanced upon hydrolysis of the glycerophospholipids of human HDL3 by phospholipase A2. Rate enhancements were also observed following trypsin-catalyzed hydrolysis of apolipoprotein A-I in canine HDL and of apolipoproteins A-I and A-II in human HDL3. However, the initial rate of filipin-unesterified cholesterol association was not altered upon incubation of HDL3 with polymorphonuclear cells, which causes hydrolysis of apolipoprotein A-II but leaves apolipoprotein A-I intact. These results are consistent with the general structural model of HDL in which unesterified cholesterol, apolipoproteins and glycerophospholipids are presumed to be localized at the surface of the HDL particle. From these studies and from results indicating that the initial rate of filipin-unesterified association was enhanced in canine HDL hybrids in which 50% of the apolipoprotein A-I had been replaced by apolipoprotein A-II, we also conclude that apolipoprotein A-I in HDL is in closer proximity to unesterified cholesterol than apolipoprotein A-II. Thus, it appears that rapid kinetic measurements of filipin-cholesterol association may be useful in assessing the organization of unesterified cholesterol in serum lipoproteins.

Animals↗
PubMed1982

Cation binding properties of the multiple subforms of RVV-X, the coagulant protein from Vipera russelli.

The factor X activating enzyme from Russell's viper venom (RVV-X) has been shown to exist in multiple subforms, distinguished from each other by their isoelectric points. The differences in isoelectric points were due, as least in part, to dissimilarities in the respective sialic acid contents of the subforms. No functional difference was, however, discovered between any of the subforms. All of the subforms were found, by equilibrium ultrafiltration, to bind Ca2+ reversibly. At least two equivalent Ca2+ binding sites were observed on each protein molecule (Mr 79 000), with a KD of 50 +/- 15 microM at pH 7.4 and 25 degrees C. A new substrate for RVV-X, which does not bind Ca2+, apoprotein AI from human high-density lipoprotein, was used to show that this reversibly bound Ca2+ was not essential for enzymic activity. All subforms have also been shown, by atomic absorption analysis, to contain nonexchangeable metal ions, to the extent of 1 mol of Ca2+ and 0.7 mol of Zn2+ per mol of protein. No Mn2+ or Mg2+ was detected. This nonexchangeable Ca2+ and Zn2+ could only be removed from the protein by incubation at pH 3.0 or by treatment with 6 M guanidine hydrochloride, conditions under which the protein lost activity irreversibly.

Animals↗
PubMed1982

In vitro mass: activity distribution of lecithin--cholesterol acyltransferase among human plasma lipoproteins.

In order to compare the mass-activity distribution of lecithin-cholesterol acyltransferase (LCAT) among plasma lipoproteins separated by various ultracentrifugal or chromatographic procedures, we have quantified the enzyme by an electroimmunoassay technique using a specific antibody raised in the rabbit. This antibody, when added to whole serum, inhibited all of the enzyme activity present in it. The percent mass distribution of the enzyme among the lipoproteins isolated by rate-zonal ultracentrifugation (d 1.00-1.36 g/ml, SW 40 rotor, 37 000 rpm, 16 h) was as follows: very low density lipoproteins (VLDL), 0; low density lipoproteins (LDL), 6.2; HDL2, 6.5; HDL3, 12 and d greater than 1.21 g/ml fraction, 75. Measurement of LCAT activity of each lipoprotein fraction against mixed single bilayer lecithin-cholesterol vesicles (molar ratio, 4:1) containing apo A-I, indicated that VLDL, LDL and HDL2 were inactive or minimally active under the experimental conditions used, whereas HDL3 and the d greater than 1.21 g/ml fraction contained 17.5 and 79.9% of the total enzyme activity. Prolonged ultracentrifugation of the LCAT-containing lipoproteins resulted in the recovery of activity in the lipoprotein-free infranatant. In studies with lipoproteins linked to Sepharose 4B, LCAT was found to bind LDL, HDL2, and HDL3. It is concluded that LCAT is present in all the major lipoproteins except for VLDL. The activity appears to be dependent, at least in part, on the type of lipoproteins to which the enzyme is associated with.

Centrifugation, Isopycnic↗
PubMed1980

Studies on the mechanism of capacitation: albumin-mediated changes in plasma membrane lipids during in vitro incubation of rat sperm cells.

Plasma membrane isolated from rat sperm cells after incubation in vitro had a significantly lower cholesterol/phospholipid mole ratio when the medium contained serum albumin. Transfer of albumin-bound phospholipids to the membrane can largely account for this effect. The result is broadly consistent with a previously proposed model for albumin-induced destabilization of sperm membrane (capacitation) and its reversal by seminal plasma membrane vesicles. Albumin also decreased sialic acid and, more specifically, ganglioside levels, presumably by promoting release of sperm neuraminidase. Cholesteryl ester comprised up to 0.5 mol/mol of cholesterol in these plasma membrane preparations.

Adenosine Triphosphatases↗
PubMed1980

Interaction of lipids with the plasma membrane of sperm cells. II. Evidence of a membrane thermotropic transition.

Cholesterol and phosphatidylcholine uptake from dipalmitoyl-phosphatidylcholine liposomes by rabbit spermatozoa showed a complex dependence on temperature in these experiments. At 5 degrees and 20 degrees C, the rate of lipid uptake correlated with temperature. However, from 20 degrees to 37 degrees C uptake did not evidently increased. The results in interpreted as evidence of a thermotropic transition in the sperm plasma membrane. Data are presented showing incorporation of these lipids, especially that of cholesterol, into sperm plasma membrane.

Animals↗
PubMed1979

Studies on the mechanism of capacitation. II. Evidence for lipid transfer between plasma membrane of rat sperm and serum albumin during capacitation in vitro.

1. Evidence has been provided for the transfer of phosphatidyl[14C]choline and [3H]cholesterol between bovine serum albumin and cauda epididymal rat spermatozoa in Krebs-Ringer bicarbonate medium, which can promote sperm capacitation. 2. An analysis of the lipid composition in both albumin and spermatozoa revealed that phospholipid levels decreased in the protein and increased by roughly comparable amounts in sperm cells during incubation in vitro. 3. Cholesterol (free + ester) increased in albumin and decreased in spermatozoa. Changes in the amount of esterified cholesterol were solely responsible for the increase associated with albumin, whereas whole sperm cell extracts showed a significant decline in free cholesterol. 4. The composition of albumin-bound fatty acids did not alter appreciably as a result of incubation with spermatozoa. 5. Rates of [14C]palmitic acid utilization by spermatozoa suggest that lipid synthesis accounted for less than 5% of the changes observed under the conditions of this study. 6. These results are interpreted as broadly supporting our previous proposal that lipid exchange between albumin and sperm cells is implicated in sperm capacitation in vitro. Specifically, the results are compatible with the idea that a decreased cholesterol/phospholipid ratio in the sperm plasma membrane facilitates this transformation.

Animals↗
PubMed1979

The binding of calcium to the activation products of bovine factor IX.

Binding isotherms of Ca2+ to the bovine Factor IX activation intermediates and products, i.e. Factor IXalpha, Factor IXa alpha, and Factor IXa beta have been examined. At pH 7.4, Factor IX alpha possesses at least two strong Ca2+ sites, with an average KD of 0.1 mM, and an additional 11 weaker sites, with an average KD of 3.7 mM. Bovine Factor IXa alpha also contains at least two Ca2+ binding sites, with an average KD of 0.1 mM, and an additional 11 weaker sites, with an average KD of 1.3 mM. Factor IXa beta, the ultimate activation product of Factor IX, in the intrinsic system, likewise contains at least two strong Ca2+ sites, of average KD 0.1 mM, as well as seven additional weaker sites, possessing an average KD of 1.0 mM. The Ca2+-binding properties of the above proteins are similar to those of their precursor molecule, Factor IX, which we have earlier shown to possess at least two strong Ca2+ sites, with an average KD of 0.1 mM, and 11 weaker sites, of average KD 1.3 mM (Amphlett, G.W., Byrne, R., and Castellino, F.J. (1978) J. Biol. Chem. 253, 6774-6779). Circular dichroism analysis of all of the above proteins was consistent with the molecules possessing a low alpha-helical content, and a high quantity of beta structure and random coil conformations.

Animals↗
PubMed1978

The binding of metal ions to bovine factor IX.

The binding isotherms of Ca2+ and Mn2+ to bovine factor IX have been determined at pH 6.5 and pH 7.4, at 25 degrees C. At pH 7.4, at least 2 strong Ca2+ sites, with an average KDISS of 0.1 +/- 0.02 mM, are found. An additional 10 to 11 weaker Ca2+ binding sites, with an average KDISS of 1.3 +/- 0.2 mM are noted, at high levels of Ca2+. At pH 6.5, again at least 2 strong Ca2+ sites on factor IX are evident, with an average KDISS of 0.11 +/- 0.02 mM; but slightly fewer (7 to 8) weaker sites, with an average KDISS of 1.9 +/- 0.3 mM, are obtained. Qualitatively, the binding of Mn2+ to bovine factor IX appears similar to that of Ca2+. At pH 6.5, approximately 2 strong Mn2+ binding sites, with an average KDISS of 13 +/- 1.5 micrometer, and an additional 5 to 6 weak sites, with an average KDISS of 160 +/- 15 micrometer, are present. Manganese does not completely displace Ca2+; and Ca2+ does not completely displace Mn2+ from their respective binding sites. On the other hand, Tb3+ and Sm3+ readily displace Ca2+, at pH 6.5, from its sites on factor IX. The rate and extent of activation of bovine factor IX, by bovine factor XIa, is dependent on the Ca2+ concentration, up to concentrations of Ca2+ which saturate its effect on the system. Substitution of Sr2+ for Ca2+ leads to approximately 50% of the maximum rate of factor IXa formation, and final yield of factor IXa, in this activation system. Manganese does not substitute for Ca2+ in this activation, but does inhibit the stimulatory effect of Ca2+. Both Tb3+ and Sm3+ are effective inhibitors of Ca2+ in factor IX activation by factor XIa.

Animals↗