Binding of heparin to human high molecular weight kininogen

The binding of heparin to high molecular weight kininogen (H-kininogen) was analyzed by the effect of kininogen in decreasing the heparin-induced enhancement of the rate of inactivation of thrombin by antithrombin. The conditions were arranged so that the heparin-catalyzed antithrombin-thrombin reaction, monitored in the presence of the reversible thrombin inhibitor p-aminobenzamidine, followed pseudo-first-order kinetics and the observed rate constant (kappa obsd) varied linearly with the heparin concentration. In the absence of metal ions, H-kininogen minimally affected kappa obsd, measured at a constant concentration of heparin with high affinity for antithrombin (30 nM), at I = 0.15, pH 7.4 and 25 degrees C. However, at a saturating concentration of Zn2+ (10 microM), kappa obsd was reduced to 50% at approximately 20 nM H-kininogen and to that of the uncatalyzed reaction at greater than or equal to approximately 0.2 microM H-kininogen. Conversely, at a saturating concentration of H-kininogen (0.5 microM), kappa obsd was decreased to 50% at approximately 0.6 microM Zn2+ and to the kappa obsd of the uncatalyzed reaction at greater than or equal to 10 microM Zn2+. Other metal ions were effective in the order Zn2+ approximately Ni2+ greater than Cu2+ approximately Co2+ approximately Cd2+. The single-chain and two-chain forms of H-kininogen and the H-kininogen light chain reduced the heparin enhancement in the presence of Zn2+ to the same extent, whereas low molecular weight kininogen had no influence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling

Stereospecific 1H and 13C NMR assignments were made for the two diastereotopic methyl groups of the 14 valyl and leucyl residues in the DNA-binding domain 1-69 of the 434 repressor. These results were obtained with a novel method, biosynthetically directed fractional 13C labeling, which should be quite widely applicable for peptides and proteins. The method is based on the use of a mixture of fully 13C-labeled and unlabeled glucose as the sole carbon source for the biosynthetic production of the protein studied, knowledge of the independently established stereoselectivity of the pathways for valine and leucine biosynthesis, and analysis of the distribution of 13C labels in the valyl and leucyl residues of the product by two-dimensional heteronuclear NMR correlation experiments. Experience gained with the present project and a previous application of the same principles with the cyclic polypeptide cyclosporin A provides a basis for the selection of the optimal NMR experiments to be used in conjunction with biosynthetic fractional 13C labeling of proteins and peptides.     

Proton-metal distance determination in cobalt(II) stellacyanin by 1H nuclear magnetic resonance relaxation measurements including Curie-spin effects: a proposed structure of the metal-binding region

1H nuclear magnetic resonance (1H NMR) experiments on Co(II)-substituted stellacyanin have been performed. Large paramagnetic hyperfine shifts are observed, the whole spectrum covering a range of 190 ppm. Experiments were mainly performed at 270 MHz from which temperature and pH* dependencies of the out-shifted resonances were reported, as well as determinations of the longitudinal (T1) and transverse (T2) relaxation times. These relaxation times are among other things, dependent on the individual proton-metal distance, and the aim of this work has been to determine these distances, by use of the Solomon-Bloembergen equations modified to include the so-called "Curie spin". The application of this method to a protein has not been reported earlier. Experiments were also performed at 100, 400, and 500 MHz in order to estimate the size of the Curie spin from the field dependence of the line widths. Furthermore, determination of the values for the rotational correlation time, tau r, and the effective magnetic moment, mu eff, was necessary for the present approach. With apostellacyanin, tau r was found to be (6.0 +/- 0.4) X 10-8 s. From the paramagnetic susceptibility of Co(II) stellacyanin, the value (4.53 +/- 0.03)beta was determined for mu eff. The proposed assignments of several paramagnetically out-shifted resonances. the proton-metal distances obtained, and the known peptide sequence of stellacyanin have allowed us to build a three-dimensional model of the metal site and its surrounding structure consistent with all the experimental data. It is revealed that both histidine ligands bind the metal with their 3-nitrogens. Also we find strong indications that a second sulfur atom is actually binding the metal, this being the long-sought-after fourth ligand. The model suggests that this sulfur belongs to Cys-59, which together with Cys-93 constitutes the disulfide bridge known to be present in the structure. A potential fifth ligand, an amide oxygen from Asn-47, is also found.     

Membrane solubilization by detergent: use of brominated phospholipids to evaluate the detergent-induced changes in Ca2+-ATPase/lipid interaction

The solubilization and delipidation of sarcoplasmic reticulum Ca2+-ATPase by different nonionic detergents were measured from changes in turbidity and recovery of intrinsic fluorescence of reconstituted ATPase in which tryptophan residues had been quenched by replacement of endogenous phospholipids with brominated phospholipids. It was found that incorporation of C12E8 or dodecyl maltoside (DM) at low concentrations in the membrane, resulting in membrane "perturbation" without solubilization, displaced a few of the phospholipids in contact with the protein; perturbation was evidenced by a parallel drop in ATPase activity. As a result of further detergent addition leading to solubilization, the tendency toward delipidation of the immediate environment of the protein was stopped, and recovery of enzyme activity was observed, suggesting reorganization of phospholipid and detergent molecules in the solubilized ternary complex, as compared to the perturbed membrane. After further additions of C12E8 or DM to the already solubilized membrane, the protein again experienced progressive delipidation which was only completed at a detergent concentration about 100-fold higher than that necessary for solubilization. Delipidation was correlated with a decrease in enzyme activity toward a level similar to that observed during perturbation. On the other hand, Tween 80, Tween 20, and Lubrol WX failed to solubilize SR membranes and to induce further ATPase delipidation when added after preliminary SR solubilization by C12E8 or dodecyl maltoside. For Tween 80, this can be related to an inability to solubilize pure lipid membrane; in contrast, Tween 20 and Lubrol WX were able to solubilize liposomes but not efficiently to solubilize SR membranes. In all three cases, insertion of the detergent in SR membranes is, however, demonstrated by perturbation of enzyme activity. Correlation between detergent structure and ability to solubilize and delipidate the ATPase suggests that one parameter impeding ATPase solubilization might be the presence of a bulky detergent polar headgroup, which could not fit close to the protein surface. We also conclude that in the active protein/detergent/lipid ternary complexes, solubilized by C12E8 or dodecyl maltoside, most phospholipids remain closely associated with the ATPase hydrophobic surface as in the membranous form. Binding of only a few detergent molecules on this hydrophobic surface may be sufficient for inhibition of ATPase activity observed at high ATP concentration, both during perturbation and in the completely delipidated, solubilized protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Construction of an artificial bifunctional enzyme, beta-galactosidase/galactose dehydrogenase, exhibiting efficient galactose channeling

The in-frame fusion between two oligomeric enzymes, beta-galactosidase and galactose dehydrogenase, is described. The lacZ gene was fused to the 3' end of the galdh gene with a linker encoding only three amino acids. The purified artificial bifunctional enzyme displayed the enzymic activity of both gene products. The hybrid protein was found in two major forms, consisting of four and six subunits, but other forms could also be identified. The molecular weight of each subunit was determined to be 145,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bifunctional enzyme shows kinetic advantages over the identical native system in conversion of lactose to galactonolactone. A higher steady-state rate and a reduction of the transient time are observed. This phenomenon is especially pronounced at low initial substrate concentrations and when the pH is adjusted to a level at which the galactose dehydrogenase activity is much higher than that of the beta-galactosidase.     

Interdigitation of phosphatidylcholine and phosphatidylethanolamine mixed with complexes of acidic lipids and polymyxin B or polymyxin B nonapeptide

A fatty acid spin label, 16-doxyl-stearic acid, was used to determine the percent interdigitated lipid in mixtures of a neutral phospholipid and an acidic phospholipid. Interdigitation of the acidic lipid was induced with polymyxin B (PMB) at a mole ratio of PMB to acidic lipid of 1:5. This compound does not bind significantly to neutral lipids or induce interdigitation of the neutral lipids by themselves. The neutral lipids used were dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or dipalmitoylphosphatidylethanolamine (DPPE), and the acidic lipids were dipalmitoylphosphatidylglycerol (DPPG) or dipalmitoylphosphatidic acid (DPPA). The percent interdigitated lipid was determined from the percent of the spin label which is motionally restricted, assuming that the spin label is homogeneously distributed in the lipid. Assuming further that 100% of the acidic lipid is interdigitated at this saturating concentration of PMB, the percentage of the neutral lipid which can become interdigitated along with it was calculated. The results indicate that about 20 mole % DPPC can be incorporated into and become interdigitated in the interdigitated bilayer of PMB/DPPG at 4 degrees C. As the temperature approaches the phase transition temperature, the lipid becomes progressively less interdigitated; this occurs to a greater degree for the mixtures than for the single acidic lipid. Thus the presence of DPPC promotes transformation of the acidic lipid to a non-interdigitated bilayer at higher temperatures. At the temperature of the lipid phase transition little or none of the lipid in the mixture is interdigitated. Thus the lipid phase transition detected by calorimetry is not that of the interdigitated bilayer. The shorter chain length DMPC can be incorporated to a greater extent than DPPC, 30-50 mol%, in the interdigitated bilayer of PMB-DPPG. This may be a result of reduced exposure of the terminal methyl groups of the shorter myristoyl chains at the polar/apolar interface of the interdigitated bilayer. Less than 29% of the total lipid was interdigitated in a DPPC/DPPA/PMB 1:1:0.2 mixture indicating that none of the DPPC in this mixture becomes interdigitated. This is attributed to the lateral interlipid hydrogen bonding interactions of DPPA which inhibits formation of an interdigitated bilayer. DPPE was found to be incorporated into the interdigitated bilayer of PMB-DPPG to a similar extent as DPPC if the amount of PMB added is sufficient to bind to only the DPPG in the mixture. Differential scanning calorimetry showed that the remaining non-interdigitated DPPE-enriched mixture phase separates into its own domain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Association of Tat protein and viral mRNA with nuclear matrix from HIV-1-infected H9 cells

The transactivating protein from human immunodeficiency virus type 1 (HIV-1), Tat, was found to bind to the nuclear matrix from uninfected and HIV-1-infected H9 cells. Addition of the Zn2+, Cd2+ and Cu2+ chelator o-phenanthroline destroyed the matrix fibrils and the binding affinity of Tat to the matrix. A sequential treatment of the matrix, first with o-phenanthroline and then with ZnCl2, partially restored the fibrillar-like matrix structure. Infection of H9 cells with HIV-1 resulted in a displacement of cellular mRNA by viral mRNA from the nuclear matrix. Both the matrix-bound host cell and HIV-1 mRNA were found to dissociate from the matrix in the presence of o-phenanthroline. This could be prevented by coincubation with Zn2+ or Cu2+ (but not Mg2+), which stabilize the mRNA containing nuclear matrix structure.     

Beta-oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

The beta-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fructose-refed rats. The beta-oxidation of lauric acid (12:0) was less inhibited by fructose refeeding and by (+)-decanoyl-carnitine than the oxidation of oleic acid was, suggesting a peroxisomal beta-oxidation of lauric acid. Little lauric acid was esterified in triacylglycerol fraction, except at high substrate concentrations or in the fructose-refed state. With [1-14C]myristic acid (14:0), [1-14C]lauric acid (12:0), [1-14C]octanoic acid (8:0) and [2-14C]adrenic acid (22:4(n - 6] as substrate for hepatocytes from carbohydrate-refed rats, a large fraction of the 14C-labelled esterified fatty acids consisted of newly synthesized palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) while intact [1-14C]oleic acid substrate was esterified directly. With [9,10-3H]myristic acid as the substrate, small amounts of shortened 3H-labelled beta-oxidation intermediates were found. With [U-14C]palmitic acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the carbohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal beta-oxidation followed by synthesis of C16 and C16 fatty acids which can then stored as triacylglycerol.     

Effects of tetrahydrolipstatin, a lipase inhibitor, on absorption of fat from the intestine of the rat

Tetrahydrolipstatin (THL) derived by hydrogenation from lipstatin, a lipase inhibitor produced by Streptomyces toxytricini, has been shown to inhibit in vitro the activity of all three lipases secreted to the gastro-intestinal tract; gastric lipase, pancreatic lipase and carboxylester lipase (cholesterol ester hydrolase). The effects of THL on intestinal absorption of fat (transport to the thoracic duct chyle) has now been investigated after intraduodenal infusion in a rat model. Absorption of label from oleic acid when administered with monoolein in micellar bile salt solution was not affected by THL in concentrations up to 10(-4) M calculated on the volume of the aqueous phase. Absorption of free cholesterol in micellar bile salt solution of the lipolytic products of triolein; oleic acid and monoolein, is not significantly affected at a concentration of THL of 10(-4) M. Absorption of cholesterol from cholesteryl oleate under the same conditions is almost completely inhibited. The results indicate that absorption of free cholesterol is not dependent on the activity of pancreatic cholesterol ester hydrolase. The absorption of emulsified triolein was not significantly affected by 10(-5) M THL but decreased to around 30% of the controls by a concentration 10-times higher. There was no significant decrease of cholesterol absorption when administered in emulsified triolein while absorption of cholesteryl oleate was reduced at both concentrations of THL and almost completely at 10(-4) M. Radioactivity from [2-14C]THL when administered emulsified in triolein was recovered in urine, bile and thoracic duct lymph to 10-14, 8-13 and 1-3%, respectively, largely independent on dose administered. Label from [1"-14C] THL was recovered in the same amounts in lymph but much less in bile and urine indicating that the amino acid moiety has been split off early in the absorption process.     

Structure of the low-density lipoprotein receptor-related protein (LRP) promoter

The low-density Lipoprotein receptor-related protein (LRP) is a 4544-amino-acid membrane protein which closely resembles the LDL receptor in its arrangement of cysteine-rich motifs. Binding studies have suggested that one function of the molecule is as a receptor for ligands containing apolipoprotein E. We present here the sequence and structure of the promoter region of the LRP. These data show that the LRP contains no sterol regulatory element, and is not down-regulated by sterols like the LDL receptor. This lends further support to the identity of the LRP as a chylomicron remnant receptor.