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J C Osborne

Publications and source records attributed to J C Osborne.

At least 37 records · Page 2Linked to original sources
PubMed1985

Studies on inactivation of lipoprotein lipase: role of the dimer to monomer dissociation.

Sedimentation equilibrium analysis demonstrated that preparations of bovine lipoprotein lipase contain a complex mixture of dimers and higher oligomers of enzyme protein. Enzyme activity profiles from sedimentation equilibrium as well as from gel filtration indicated that activity is associated almost exclusively with the dimer fraction. To explore if the enzyme could be dissociated into active monomers, 0.75 M guanidinium chloride was used. Sedimentation velocity measurements demonstrated that this treatment led to dissociation of the lipase protein into monomers. Concomitant with dissociation, there was an irreversible loss of catalytic activity and a moderate change in secondary structure as detected by circular dichroism. The rate of inactivation increased with decreasing concentrations of active lipase, but addition of inactive lipase protein did not slow down the inactivation. This indicates that reversible interactions between active species precede the irreversible loss of activity. The implication is that dissociation initially leads to a monomer form which is in reversible equilibrium with the active dimer, but which decays rapidly into an inactive form, and is therefore not detected as a stable component in the system.

Animals↗
PubMed1985

Kinetic mechanisms of two NAD:arginine ADP-ribosyltransferases: the soluble, salt-stimulated transferase from turkey erythrocytes and choleragen, a toxin from Vibrio cholerae.

A subunit of choleragen and an erythrocyte ADP-ribosyltransferase catalyze the transfer of ADP-ribose from NAD to proteins and low molecular weight guanidino compounds such as arginine. These enzymes also catalyze the hydrolysis of NAD to nicotinamide and ADP-ribose. The kinetic mechanism for both transferases was investigated in the presence and absence of the product inhibitor nicotinamide by using agmatine as the acceptor molecule. To obtain accurate estimates of kinetic parameters, the transferase and glycohydrolase reactions were monitored simultaneously by using [adenine-2,8-3H]NAD and [carbonyl-14C]NAD as tracer compounds. Under optimal conditions for the transferase assay, NAD hydrolysis occurred at less than 5% of the Vmax for ADP-ribosylation; at subsaturating agmatine concentrations, the ratio of NAD hydrolysis to ADP-ribosylation was significantly higher. Binding of either NAD or agmatine resulted in a greater than 70% decrease in affinity for the second substrate. All data were consistent with a rapid equilibrium random sequential mechanism for both enzymes.

ADP Ribose Transferases↗
PubMed1985

Molecular size of bovine lipoprotein lipase as determined by radiation inactivation.

We have determined the size of the functional unit of bovine lipoprotein lipase by radiation inactivation. This was done in five different situations: 1) in a buffer with high salt concentration. In this situation the enzyme is relatively soluble and stable. 2) For an enzyme-heparin complex. This may reflect the physiological state of the enzyme at the vascular endothelium, where it is believed to be bound to a heparin-like molecule. 3) In the presence of lipid substrate and 4) with lipid substrate and activator protein. Here most of the enzyme is adsorbed to the substrate droplets. 5) For an enzyme-detergent complex; another model for enzyme-lipid interaction. In all five situations the enzyme activity decayed as an exponential function of radiation dose, and the target sizes were similar. The target size did not vary with the concentration of lipase protein. The combined data for bovine lipoprotein lipase yield a functional size of 72 kDa which is close to that expected for a dimer, 77 kDa.

Animals↗
PubMed1985

The pH-dependent subunit dissociation and catalytic activity of bovine dopamine beta-hydroxylase.

The soluble form of dopamine beta-hydroxylase from bovine adrenal medulla has previously been shown to exist as a tetrameric species of Mr = 290,000 composed of two disulfide-linked dimers. Here we report that this enzyme can also undergo a reversible tetramerdimer dissociation which is dependent on pH. Gel permeation chromatography of dopamine beta-hydroxylase at pH 5.0 demonstrates a Stokes radius of 5.8 nm. When the pH is shifted to 5.7, the Stokes radius changes to 6.9 nm. Sedimentation equilibrium analysis of the purified enzyme demonstrates that this change in molecular size is due to a change in molecular weight. At low protein concentration, the estimated Mr of the enzyme is 145,000 at pH 5.0 and at high protein concentration approaches 290,000 at pH 5.7. This change in Mr is consistent with the existence of a tetramer-dimer dissociation and a change in the equilibrium constant from 1.8 X 10(-6) M to 1.16 X 10(-9) M when the pH is increased from 5.0 to 5.7. This pH-dependent subunit dissociation is correlated with pH-dependent changes in enzyme activity. Purified bovine-soluble dopamine beta-hydroxylase activity is a hyperbolic function of tyramine concentration at pH 5.0. However, the hydroxylase activity displays non-hyperbolic kinetics at pH 6.0. The kinetic data obtained at pH 6.0 can be accounted for by fitting to a model containing two nonidentical catalytic forms of enzyme generated by the pH-dependent partial dissociation of tetrameric enzyme to dimeric subunits. The two catalytic forms have apparently identical maximal velocities; however, they differ in their Michaelis constants for the substrate; the dimeric form having a low Km and the tetrameric form having a high Km. Since the pH inside bovine adrenal medullary chromaffin granules is approximately 5.5, we conclude that the subunits of dopamine beta-hydroxylase are in dynamic dissociation in a physiologically important pH range.

Adrenal Medulla↗
PubMed1985

Chondronectin: physical and chemical properties.

Chondronectin, the chondrocyte attachment factor, was purified from chicken serum and characterized as to its physical and chemical properties. From sedimentation equilibrium data it was found to have a native molecular weight of 175,800 +/- 800 and a subunit molecular weight of 55,540 +/- 800 in the presence of guanidinium chloride and cysteine, suggesting a trimeric structure linked by disulfide bonds. As visualized by electron microscopy after rotary shadowing, the protein appears compact and globular. The amino acid and carbohydrate compositions of chondronectin are distinct from fibronectin, the fibroblast attachment factor, and laminin, the epithelial cell attachment factor. The activity of chondronectin in promoting attachment of chondrocytes is stable to digestion by collagenase, elastase, and neuraminidase, but is destroyed by trypsin treatment. The data suggest that chondronectin is structurally and chemically distinct from fibronectin and laminin.

Amino Acids↗
PubMed1984

Purification and properties of calmodulin-stimulated phosphodiesterase from mammalian brain.

A new, rapid method for purification of calmodulin-stimulated phosphodiesterase from bovine, ovine, and porcine brain using only DEAE-agarose and calmodulin-Sepharose chromatography is described. Purified enzymes from the three species each exhibited a single polypeptide of Mr approximately 59,000 on gel electrophoresis under denaturing conditions. Proteolysis of ovine and bovine enzymes with alpha-chymotrypsin, however, yielded different peptides, indicating that these proteins differ in primary sequence. Homogeneous preparations of bovine and ovine enzymes (purified approximately 5,000- and 2,000-fold, respectively) had different specific activities, although their substrate affinities and activation by calmodulin (8- to 14-fold activation, Kact approximately 1 nM) were very similar. The total amount in ovine was almost twice that in bovine brain. The hydrodynamic properties of bovine and ovine enzymes were indistinguishable with a Stokes radius of 4.35 nm and s20,w of 5.95 S. The calculated frictional ratios of 1.30 to 1.38 suggest a slightly asymmetric molecule. Equilibrium sedimentation data yielded apparent Mr approximately 57,000 in the presence of 6 M guanidine and 124,000 and 112,000 for the native bovine and ovine enzymes, respectively. In addition to the enzyme that was purified to homogeneity (pI approximately 5.6), a major fraction of calmodulin-activated phosphodiesterase with a lower isoelectric point was found in bovine and ovine brain. Whether these represent isozymes, perhaps localized in different types of cells, or whether one is a post-translationally modified form, remains to be determined. The existence of these two otherwise very similar forms of the enzyme has apparently not been previously recognized.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Purification and characterization of ornithine transcarbamoylase from Saccharomyces cerevisiae.

Ornithine transcarbamoylase (OTCase) has been purified in 100-mg quantities from a plasmid-containing, enzyme-overproducing strain of Saccharomyces cerevisiae. The specific activity of the homogeneous enzyme is 2.5-fold above that previously reported. The molecular weight and partial specific volume of OTCase were determined by sedimentation equilibrium in solutions containing H2O and D2O. Data from two rotor speeds were simultaneously fit using nonlinear least squares analysis with multiple independent variables giving a molecular weight of 110,000 +/- 2,200 and a partial specific volume of 0.732 +/- 0.006 ml g-1. The ultraviolet absorption spectrum of OTCase gives a specific absorbance at 280 nm of 0.36. This low value is consistent with a small number of aromatic residues. Amino acid analysis, fluorescence, and multicomponent analysis yield 1 tryptophan, 4 tyrosine, and 24 phenylalanine/polypeptide chain. From an analysis of the circular dichroic spectrum, it was determined that OTCase contained 22% alpha-helix, 43% beta-sheet, 8% beta-turn, and 27% random structure. The fluorescence of the single tryptophan/polypeptide chain has an emission maximum at 320 nm, indicating a hydrophobic environment.

Amino Acids↗
PubMed1984

Activation of an erythrocyte NAD:arginine ADP-ribosyltransferase by lysolecithin and nonionic and zwitterionic detergents.

The activity of an NAD:arginine ADP-ribosyltransferase was stimulated 4-6-fold by lysolecithin; lysolecithins containing long-chain fatty acids such as stearoyl (C18) and palmitoyl (C16) were more effective than those with shorter chains: C14 greater than C12 greater than C10 congruent to C8. The analogue lacking a fatty acid at C-1, alpha-glycerophosphocholine, was inactive as were choline, lysophosphatidic acid, lysophosphatidylserine, lysophosphatidylglycerol, lysophosphatidylethanolamine, lecithin, phosphatidic acid, phosphatidylserine, and phosphatidylethanolamine. Activation of the transferase was, however, also observed with certain nonionic (e.g., Triton X-100) and zwitterionic [3-[ ( cholamidopropyl ) dimethylammonio ]-1-propanesulfonate] detergents. The transferase was shown previously to be stimulated by chaotropic salts or histones; in the presence of maximally effective concentrations of lysolecithin, salt, and histone, the activity was similar to that observed in the presence of histone or salt alone. Maximal activation by lysolecithin and detergents was less than that observed with either salt or histone. It appears that activation by lysolecithin shows significant differences from that observed previously with histones or salt and can be mimicked by certain nonionic and zwitterionic detergents.

ADP Ribose Transferases↗
PubMed1984

Selective inhibition of two soluble adenosine cyclic 3',5'-phosphate phosphodiesterases partially purified from calf liver.

"Low Km" cAMP phosphodiesterase and cGMP-stimulated cyclic nucleotide phosphodiesterase activities were partially purified from calf liver supernatant by chromatography on DEAE-cellulose and DEAE-Sepharose and ammonium sulfate precipitation. The low Km phosphodiesterase was not retained on N6-H2N(CH2)2-cAMP-agarose and could be separated from the cGMP-stimulated phosphodiesterase which was absorbed by this matrix. From the proteins that did not bind, two distinct low Km cAMP phosphodiesterases were separated on Ultrogel AcA 34. One form (fraction C) hydrolyzed cAMP with an apparent Km of approximately 0.5 microM and was very sensitive to inhibition by cGMP. Lineweaver-Burk plots of cAMP hydrolysis by a second form (fraction B) were nonlinear, with an apparent low Km component of approximately 2 microM. This form was rather insensitive to inhibition by cGMP. With both fractions, hydrolysis of cAMP relative to cGMP was much greater at low (approximately 1 microM) than at high (approximately 100 microM) substrate concentrations. Maximal velocities for cAMP and cGMP were similar. From sedimentation equilibrium, the apparent weight-average molecular weight of fraction B was estimated as 174000, and that of fraction C was 85000. Another fraction (A) of cAMP phosphodiesterase eluted at the void volume of the AcA 34 column. On the basis of the relative affinities for cAMP and cGMP and inhibition by cGMP, fraction A is most likely an aggregated form of fraction B. No apparent interconversion of fractions A, B, or C was observed on high-performance liquid chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Molecular properties of radioiodinated apolipoprotein A-I.

Radioiodinated apolipoprotein A-I was separated into two pools by gel chromatography on Sephadex G-150 superfine resin or by high performance liquid chromatography using a TSK-2000 column. The first pool, pool 1, was indistinguishable from unlabeled apo-A-I as judged by sedimentation equilibrium, circular dichroic measurements, and radial immunodiffusion. This pool self-associated according to a monomer-dimer-tetramer-octamer scheme and underwent the expected conformation changes with dissociation as reported previously for unlabeled apo-A-I. The radioiodinated material in pool 2 had less secondary structure as compared to the unlabeled material and did not self-associate. Protein in this pool was immunochemically less reactive than unlabeled apo-A-I. Kinetic studies, in vivo, demonstrated that these two pools of radioiodinated apo-A-I are also distinguished metabolically. Discussion centers around possible mechanisms of formation of the incompetent protein in pool 2 and factors that should be taken into consideration when using unfractionated radioiodinated apo-A-I for experimental studies.

Adult↗
PubMed1983

Complex effects of inhibitors on cyclic GMP-stimulated cyclic nucleotide phosphodiesterase.

We have investigated the effects of several phosphodiesterase inhibitors on the activity of a cGMP-stimulated cyclic nucleotide phosphodiesterase purified from calf liver supernatant. Theophylline, RO 20-1724, and MY 5445 were not effective inhibitors. With 0.5 microM [3H]cGMP as substrate or with 0.5 microM [3H]cAMP in the presence of 1 microM cGMP, activity was inhibited by papaverine, dipyridamole, isobutylmethylxanthine (IBMX), and cilostamide. With 0.5 microM [3H]cAMP as substrate, however, only cilostamide was inhibitory; papaverine, dipyridamole, and IBMX increased activity. The increase was dependent on both drug and substrate concentration with maximal stimulation (150-180%) at concentrations of cAMP between 0.5 and 2.5 microM. At higher cAMP concentrations, the three drugs were inhibitory; inhibition was maximal at approximately 40 microM and decreased at higher cAMP concentrations. Inhibition of cGMP hydrolysis was maximal at approximately 3 microM and decreased at higher concentrations. Papaverine, IBMX, dipyridamole, and cilostamide inhibited [3H] cGMP hydrolysis competitively with Ki values of 3, 6.5, 7, and 11.5 microM, respectively. Papaverine, IBMX, or dipyridamole reduced the Hill coefficient for cAMP hydrolysis from 1.8 to 1.1-1.2, and Lineweaver-Burk plots were linear or nearly linear. With cilostamide, however, Lineweaver-Burk plots remained curvilinear. Thus, three competitive inhibitors, papaverine, dipyridamole, and IBMX, can mimic substrate and effect allosteric transitions that increase catalytic activity, whereas another, cilostamide, apparently cannot. Differences in the actions of these inhibitors presumably reflect differences in the molecular requirements for effective interaction at catalytic and allosteric sites on phosphodiesterase, i.e. differences in the structure of these sites.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Interaction of the serum amyloid A proteins with phospholipid.

The serum amyloid A proteins (SAA) are transported in plasma in association with the high density lipoproteins. We have studied the solution properties of two of the polymorphic forms of SAA, SAA1 and SAA4, and compared the lipid-binding properties of SAA4 to those of the well characterized apolipoproteins, apo-A-I, apo-A-II, and apo-C-III. SAA4 was monomeric at pH 2.9 but considerable self-association was demonstrated at pH 8.2, even in the presence of 1.0 M guanidine HCl. SAA4 differed from the apolipoproteins in its ability to disrupt multilamellar dimyristoylphosphatidylcholine (DMPC) liposomes and generate bilayer discs. Apo-A-I, apo-A-II, and apo-C-III reduced the turbidity of DMPC dispersions at protein:lipid molar ratios of 1:200. SAA4, however, increased turbidity at molar ratios of 1:250 and 1:100 even when preincubated in guanidine HCl before addition to liposomes. Optical density decreased only at ratios of 1:50 and 1:25. At an SAA4:DMPC ratio of 1:50, discoidal particles (long axis, 28.1 nm; short axis, 4.4 nm) were formed which were similar to those produced by apo-C-III. Lipid binding induced changes in SAA4 conformation similar to those observed in the apolipoproteins. The alpha-helical content and intrinsic tryptophanyl fluorescence were increased and quenching of tryptophanyl fluorescence by acrylamide was reduced in the presence of DMPC. In addition, SAA4 as well as the apolipoproteins broadened the range and increased the temperature of the gel-liquid crystal transition temperature of DMPC.

Amyloid↗
PubMed1983

beta 2-Glycoprotein I. Molecular properties of an unusual apolipoprotein, apolipoprotein H.

beta 2-Glycoprotein I (beta 2GI) has recently been identified as a component of circulating plasma lipoproteins. The metabolic role of this apolipoprotein is not known with certainty; it has been reported that beta 2GI has a high affinity for triglyceride-rich particles, causing their selective precipitation by detergents, and activates lipoprotein lipase in the in vitro hydrolysis of artificial lipid emulsions. In the present report, we have evaluated the secondary, tertiary, and quaternary structure of lipid-free beta 2GI. The weight average molecular weight of beta 2GI, as determined by sedimentation equilibrium measurements, was 43,000 in the presence and absence of denaturing agents. Thus, in contrast to other apolipoproteins, apolipoprotein H (apo-H) does not self-associate in aqueous solution. The circular dichroic spectra of apo-H is unusual in that there are no strong negative bands in the far-ultraviolet region of the spectrum; there is a weak positive maximum at 235 nm and a relatively weak negative maximum at 205 nm. Treatment with guanidinium chloride results in a loss of the positive band with only minor changes in the intensity of the band at 205 nm. Apolipoproteins A-I, A-II, C-I, and E, in contrast, have a secondary structure that contains a high percentage of residues in an alpha-helical configuration and undergo major changes in structure at low concentrations of guanidinium chloride. Highly flexible proteins, such as apolipoproteins A-I, A-II, and C-I, absorb rapidly and reversibly to air-water interfaces, whereas more rigid proteins, such as the classical globular proteins, interact with the interface more slowly and irreversibly. This difference is due to the loosely folded tertiary structure of apolipoproteins and the ease with which they can change structure to accommodate a given environment. The surface activity of beta 2GI at neutral pH resembles that of typical globular proteins. Treatment with acid or base, although causing only minor changes in the circular dichroic spectra, resulted in major increases in the rate of absorption to an air-water interface; under these conditions the rates of absorption were similar to that found for apolipoprotein A-I. These results are consistent with a more flexible structure for beta 2GI in acid or base that resembles other loosely folded apolipoproteins. beta 2GI associates with plasma lipoproteins and satisfies all of the criteria to be classified as an apolipoprotein. The secondary, tertiary, and quaternary structure of beta 2GI is, however, quite different from that of other well characterized apolipoproteins. This difference in structure would be expected to affect protein-lipid interactions; the relationship between apo-H and other apolipoproteins may be similar to that proposed for integral versus peripheral membrane proteins.

Chylomicrons↗