An ultrasensitive human immunodeficiency virus type 1 protease radioimmuno rate assay with a potential for monitoring blood levels of protease inhibitors in acquired immunodeficiency disease syndrome patients.

The angiotensin I-based peptide Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Glu-Ser yields angiotensin I (Ang I) and Leu-Glu-Glu-Ser upon hydrolysis by the human immunodeficiency virus type 1 (HIV-1) protease, but not by human renin. N-terminal sequencing of the reaction products showed that the HIV-1 protease cleaved exclusively at the Leu-Leu bond. The rate of Ang I formation can be measured by a radioimmunoassay, since the parent peptide has minimal cross reactivity in this assay. The rate of enzymatic hydrolysis is maximal at pH 4.5-5.0 and at an ionic strength of 1 M. At 37 degrees C, 0.1 M Na acetate buffer, pH 5.0, 1 M NaCl, 10% glycerol, 5% ethylene glycol, 1 mg/ml bovine serum albumin, and 3 mM EDTA, the reaction obeys Michaelis-Menten type kinetics with Km = 17.2 +/- 3.5 microM and kcat = 2.30 +/- 0.33 min-1. The activity assay readily quantitates as little as 0.25 nM of HIV-1 protease. The production of Ang I by the HIV-1 protease is inhibited in the presence of a HIV-1 protease inhibitor. The newly discovered substrate is relatively insensitive to human or monkey serum. Therefore, the effect of sera from 20 patients with advanced acquired immunodeficiency disease syndrome (AIDS) on Ang I production in the above assay system was examined. Results of this study indicate that it may be possible to adapt the above Ang I-based system to determine blood levels of HIV-1 protease inhibitors in AIDS patients during clinical trials.     

Dissociative inhibition of dimeric enzymes. Kinetic characterization of the inhibition of HIV-1 protease by its COOH-terminal tetrapeptide

Human immunodeficiency virus 1 (HIV-1) protease is an aspartyl protease composed of two identical protomers linked by a four-stranded antiparallel beta-sheet consisting of the NH2- and COOH-terminal segments (Weber, I.T. (1990) J. Biol. Chem. 265, 10492-10496). Kinetic analysis of the HIV-1 protease-catalyzed hydrolysis of a fluorogenic substrate demonstrates that the enzyme is an obligatory dimer. At pH = 5.0, 0.1 M sodium acetate, 1 M NaCl, 1 mM EDTA buffer, 37 degrees C, the equilibrium dissociation constant, Kd = 3.6 +/- 1.9 nM. We found that the tetrapeptide Ac-Thr-Leu-Asn-Phe-COOH, corresponding to the COOH-terminal segment of the enzyme, is an excellent inhibitor of the enzyme. Kinetic analysis shows that the inhibitor binds to the inactive protomers and prevents their association into the active dimer (dissociative inhibition). The dissociative nature of this inhibition is consistent with the results obtained from sedimentation equilibrium experiments in which the apparent molecular weight of the enzyme was observed to be 20,800 +/- 1,500 and 12,100 +/- 300, in the absence and presence of the COOH-terminal tetrapeptide, respectively. The dissociation constant of the protomer-inhibitor complex is Ki = 45.1 +/- 1.8 microM. This is the first kinetic analysis and direct experimental demonstration of noncovalent dissociative inhibition.     

Structure-function relationship of mitochondrial malate dehydrogenase at high dilution and in multicomponent systems

The structure-function relationship of mitochondrial malate dehydrogenase was investigated at low enzyme concentration, as well as in the presence of polyethylene glycol (PEG 6000) and structure making ions. Previous reports claimed the dimeric enzyme to undergo dissociation in dilute solution, and PEG-induced pairing of dimers in the crystalline state. Sedimentation analysis and gel filtration in 0.1 M sodium phosphate pH 7.6 plus 1 mM EDTA and 1 mM dithioerythritol prove the enzyme to be a stable dimer at c greater than or equal to 0.2 microgram/ml (5 nM). In the presence of 8-20% (w/v) PEG 6000, association of the dimer to tetramers and higher aggregates is observed. At 20% (w/v) polyethylene glycol, ultracentrifugal analysis yields up to 50% tetramers; chemical cross-linking by glutaraldehyde confirms the association in a qualitative way. The enzymatic properties of mMDH (specific activity, Km for oxaloacetate and NADH) in the absence and in the presence of PEG 6000 are indistinguishable. At high polyethylene glycol concentrations (greater than or equal to 20%), the thermal stability of the enzyme is found to be increased. The fluorescence emission, as well as the far-UV and near-UV circular dichroism remain unaffected. Accumulated evidence from equilibrium experiments at low enzyme concentration and reconstitution kinetics (after dissociation at acid pH) proves the active species of mMDH to be the dimer.     

Effectors of HIV-1 protease peptidolytic activity

High concentrations of salts dramatically affect the interaction of small ligands with HIV-1 protease. For instance, the Km and kcat values for Abz-Thr-Ile-Nle-p-nitro-Phe-Gln-Arg-NH2 (S) increased 120-fold and 3-fold, respectively, as the NaCl concentration in the assay decreased from 4.0 to 0.5 M. The Kd value for the competitive inhibitor amprenavir increased 12-fold over this concentration range of NaCl. The bimolecular rate constant for association of enzyme with amprenavir was independent of NaCl concentration, whereas the dissociation rate constant decreased with increasing NaCl concentration. Polyanionic polymers such as heparin or poly A substituted for NaCl. For example, the value of kcat/Km for S was 0.18 microM(-1) x s(-1) when the enzyme (<10 nM) was assayed in the standard buffer supplemented with 5 mM NaCl. If 0.01% poly A were included, the value of kcat/Km increased to 8.6 microM(-1) x s(-1). A DNA oligomer (23-mer) with an hexachlorofluoresceinyl moiety linked to the 5' end was studied as a model polyanionic polymer. The enzyme bound HF23 (Kd < 1 nM) with concomitant quenching of the hexachlorofluoresceinyl fluorescence. The stoichiometry for binding was 3 mol of enzyme per mol of oligomer. The hydrolytic activity of the enzyme with this oligomer was similar to that observed with poly A or high salt concentration when the molar ratio of oligomer to enzyme was greater than one. The results presented herein demonstrate that polyanionic polymers substitute for salts as effectors of HIV protease.     

The effect of soluble rat liver proteins on the activity of microsomal stearoyl-CoA and linoleoyl-CoA desaturase.

1. The influence of bovine serum albumin and soluble rat liver proteins on the activity of rat liver microsomal delta9 and delta6 desaturases has been studied. 2. In the absence of bovine serum albumin, the delta9 desaturase which converts stearoyl-CoA into oleoyl-CoA, shows a non-linear correlation between enzyme activity and protein concentration. 3. Optimum concentrations of bovine serum albumin have three main effects on the enzyme activity: (i) establishes a linear relationship between enzyme activity and protein concentration, (ii) stimulates the enzyme activity 2--3-fold and (iii) raises the optimum substrate concentration from 10 to 100 muM. 4. A highly purified soluble liver protein of molecular weight 24 000 also stimulated the enzyme activity and brought about a linear relationship between enzyme activity and protein concentration. 5. It was concluded that the non-linear kinetics were due to limiting amounts of substrate binding protein in the microsomal preparations. 6. The delta6 desaturase which converts linoleoyl-CoA into gamma-linolenoyl-CoA was also stimulated by bovine serum albumin and soluble liver proteins. 7. The significance of the fatty acid-binding proteins is discussed.     

Aryl acylamidase activity of human serum albumin with o-nitrotrifluoroacetanilide as the substrate

Albumin is generally regarded as an inert protein with no enzyme activity. However, albumin has esterase activity as well as aryl acylamidase activity. A new acetanilide substrate, o-nitrotrifluoroacetanilide (o-NTFNAC), which is more reactive than the classical o-nitroacetanilide, made it possible to determine the catalytic parameters for hydrolysis by fatty-acid free human serum albumin. Owing to the low enzymatic activity of albumin, kinetic studies were performed at high albumin concentration (0.075 mM). The albumin behavior with this substrate was Michaelis-Menten like. Kinetic analysis was performed according to the formalism used for catalysis at high enzyme concentration. This approach provided values for the turnover and dissociation constant of the albumin-substrate complex: k(cat) = 0.13 +/- 0.02 min(-1) and Ks = 0.67 +/- 0.04 mM. MALDI-TOF experiments showed that unlike the ester substrate p-nitrophenyl acetate, o-NTFNAC does not form a stable adduct (acetylated enzyme). Kinetic analysis and MALDI-TOF experiments demonstrated that hydrolysis of o-NTFNAC by albumin is fully rate-limited by the acylation step (k(cat) = k2). Though the aryl acylamidase activity of albumin is low (k(cat)/Ks = 195 M(-1)min(-1)), because of its high concentration in human plasma (0.6-1 mM), albumin may participate in hydrolysis of aryl acylamides through second-order kinetics. This suggests that albumin may have a role in the metabolism of endogenous and exogenous aromatic amides, including drugs and xenobiotics.     

Effects of macromolecular crowding on the unfolding and the refolding of D-glyceraldehyde-3-phosophospate dehydrogenase

The effects of crowding agents, polyethylene glycol (PEG 20K), Dextran 70, and bovine serum albumin, on the denaturation of homotetrameric D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) in 0.5 M guanidine hydrochloride and the reactivation of the fully denatured enzyme have been examined quantitatively. Increasing the concentration of PEG 20K to 225 mg/ml decreases the rate constant of slow phase of GAPDH inactivation to 5% but with no change for the fast phase. Chaperone GroEL assists GAPDH refolding greatly and shows even higher efficiency under crowding condition. Crowding mainly affects refolding steps after the formation of the dimeric folding intermediate.     

Bovine dihydropteridine reductase

Dihydropteridine reductase has been purified to homogeneity from bovine liver and bovine adrenal medulla by precipitation with polyethylene glycol, ion exchange chromatography, gel filtration, and affinity chromatography on 5-AMP-Sepharose 4B. The enzymes from the two tissues seem identical by the criteria of gel filtration chromatography, affinity chromatography, polyacrylamide gel electrophoresis in the presence and absence of dodecyl sulfate, isoelectric focusing, amino acid analysis, and binding of NADH. Fluorescence studies show two independent binding sites for NADH and a dissociation constant of 10 nM at pH 6.8. Isoelectric focusing of the enzyme as purified in the presence of NADH revealed three different bands, which by removal of this coenzyme were converted into a single band, corresponding to pI 5.7. The enzyme contains no carbohydrate or zinc.     

Activation of Cytosolic Pyruvate Kinase by Polyethylene Glycol

Homogeneous cytosolic pyruvate kinase from endosperm of germinating castor oil (Ricinus communis L. cv Hale) seeds was potently activated by polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the pyruvate kinase reaction mixture caused a 2.6-fold increase in maximal velocity and 12.5- and 2-fold reductions in Km values for phosphoenolpyruvate and ADP, respectively. Glycerol, ethylene glycol, and bovine serum albumin also enhanced pyruvate kinase activity, albeit to a lesser extent than polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the elution buffer during high-performance gel filtration chromatography of purified cytosolic pyruvate kinase helped to stabilize the active heterotetrameric native structure of the enzyme. A higher degree of inhibition by MgATP, but lower sensitivity to the inhibitors 3-phosphoglycerate and fructose- 1,6-bisphosphate, was also observed in the presence of 5% (w/v) polyethylene glycol. It is concluded that (a) plant cytosolic pyruvate kinase activity and regulation, like that of other regulatory pyruvate kinases, is modified by extreme dilution in the assay medium, probably as a result of deaggregation of the native tetrameric enzyme, and (b) ATP is probably the major metabolic effector of germinating castor endosperm cytosolic pyruvate kinase in vivo.     

Decrease in intestinal permeability to polyethylene glycol 1000 during development in the pig

Changes in intestinal permeability during postnatal development in the pig were investigated by using different-sized polyethylene glycols in the Mr 766-1338 range (polyethylene glycol 1000) as permeability probes. Pigs of varying age, newborn (Oh), 36-45 h old and 22-28 days old, were gavage fed polyethylene glycol 1000 together with the macromolecular markers bovine serum albumin, ovalbumin or FITC-labelled dextran 70,000. The 4-h blood serum concentrations of the different markers were determined and taken as an estimate of their intestinal transmission. In the newborn pigs, high serum levels of polyethylene glycols were obtained, concomitant with high serum levels of bovine serum albumin and FITC-dextran. After intestinal macromolecular closure in the 36-45 h-old pigs, lower serum polyethylene glycol levels were found, especially of those with a Mr greater than 1100 Da. In the 22-28 days-old pigs, polyethylene glycol levels were reduced to one-tenth or less of those in the 36-45 h-old pigs, with the levels decreasing markedly with increasing molecular size. These results show that there is a correlation between the intestinal permeability of polyethylene glycols, especially those larger than 1100, and macromolecules in the newborn pig around intestinal closure, suggesting that such polyethylene glycols traverse the gut by the macromolecular route. During later development, further intestinal maturation results in a markedly reduced permeability to polyethylene glycol 1000.     

Preparation and characterization of cross-linked laccase aggregates and their application to the elimination of endocrine disrupting chemicals

Laccase from the white rot fungus Coriolopsis polyzona was immobilized for the first time through the formation of cross-linked enzyme aggregates (CLEAs). Laccase CLEAs were produced by using 1000g of polyethylene glycol per liter of enzyme solution as precipitant and 200muM of glutaraldehyde as a cross-linking agent. These CLEAs had a laccase activity of 148Ug(-1) and an activity recovery of 60.2% when using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrate. CLEAs formed by co-aggregation with bovine serum albumin (BSA) as a stabilizer showed lower laccase activity and affinity for ABTS than those without BSA. The CLEAs co-aggregated with BSA showed higher residual activity against a protease, NaN(3), EDTA, methanol and acetone. The thermoresistance was higher for CLEAs than for free laccase and also higher for CLEAs co-aggregated with BSA than for simple CLEAs when tested at a pH of 3 and a temperature of 40 degrees C. Finally, laccase CLEAs were tested for their capacity to eliminate the known or suspected endocrine disrupting chemicals (EDCs) nonylphenol, bisphenol A and triclosan in a fluidized bed reactor. A 100-ml reactor with 0.5mg of laccase CLEAs operated continuously at a hydraulic retention time of 150min at room temperature and pH 5 could remove all three EDCs from a 5mgl(-1) solution.     

Cooperative binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate to aspartate transcarbamoylase and the heterotropic effects of ATP and CTP

Most investigations of the allosteric properties of the regulatory enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli are based on the sigmoidal dependence of enzyme activity on substrate concentration and the effects of the inhibitor, CTP, and the activator, ATP, on the saturation curves. Interpretations of these effects in terms of molecular models are complicated by the inability to distinguish between changes in substrate binding and catalytic turnover accompanying the allosteric transition. In an effort to eliminate this ambiguity, the binding of the 3H-labeled bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) to aspartate transcarbamoylase in the absence and presence of the allosteric effectors ATP and CTP has been measured directly by equilibrium dialysis at pH 7 in phosphate buffer. PALA binds with marked cooperativity to the holoenzyme with an average dissociation constant of 110 nM. ATP and CTP alter both the average affinity of ATCase for PALA and the degree of cooperativity in the binding process in a manner analogous to their effects on the kinetic properties of the enzyme; the average dissociation constant of PALA decreases to 65 nM in the presence of ATP and increases to 266 nM in the presence of CTP while the Hill coefficient, which is 1.95 in the absence of effectors, becomes 1.35 and 2.27 in the presence of ATP and CTP, respectively. The isolated catalytic subunit of ATCase, which lacks the cooperative kinetic properties of the holoenzyme, exhibits only a very slight degree of cooperativity in binding PALA. The dissociation constant of PALA from the catalytic subunit is 95 nM. Interpretation of these results in terms of a thermodynamic scheme linking PALA binding to the assembly of ATCase from catalytic and regulatory subunits demonstrates that saturation of the enzyme with PALA shifts the equilibrium between holoenzyme and subunits slightly toward dissociation. Ligation of the regulatory subunits by either of the allosteric effectors leads to a change in the effect of PALA on the association-dissociation equilibrium.     

Bovine brain cathepsin D: inhibition by pepstatin and binding to concanavalin A.

1. Cathepsin D from bovine brain has been purified 1100-fold in 46% recovery. Three isozymes are present with pI (+/- 0.05) = 6.10, 6.30 and 6.40. 2. The isozymes are single polypeptide chains with apparent Mr = 42,000 and are similar with respect to substrate binding and cleavage; the pH-optimum is 3.5 with virtually no activity at neutral pH. 3. Pepstatin inhibits the enzyme and kinetic data are consistent with a "tight binding" mechanism. 4. The dissociation constant for the concanavalin A-enzyme complex is Kd = 19 nM at pH 5.0. 5. Under conditions where 90% of the enzyme is bound to soluble concanavalin A, full enzymatic activity is observed.     

Inhibition of HIV-1 protease by short peptides derived from the terminal segments of the protease.

The active HIV-1 protease is a homodimeric enzyme. A beta-sheet consisting of N- and C-terminal segments provides the main driving force for dimerization of the inactive protomers. Several short peptides with sequences derived from the N- and C-termini of the protease were tested for inhibition of protease activity and for inhibition of HIV-1 replication in lymphocytes. Medium inhibitory activity was found with each of the peptides in the enzyme test and no inhibition of the lymphocytes was found up to 200 micrograms/ml. The enzyme tests indicate that HIV-1 protease is the target of the inhibitory action. Synergistic action could not be found with pairs of the peptides derived from the two different termini. Prolonged incubation with one of the peptides increased inhibition indicating a slow dissociation of the protease dimers. No cytotoxic effect of the inhibitors could be found below 200 micrograms/ml.     

Binding of Progesterone to Nerve Cell Membranes of Rat Brain Using Progesterone Conjugated to 125I-Bovine Serum Albumin as a Ligand

Radioiodinated bovine serum albumin conjugated to progesterone was used as a probe to examine binding parameters of steroids to membrane preparations from rat brain tissue. The binding of 11 alpha-hydroxyprogesterone-11-hemisuccinate-125I-bovine serum albumin conjugate reached saturation after 30 min of incubation at 5 degrees C. Several bovine serum albumin-conjugated steroids were then tested for competition displacement studies. Among these steroid conjugates, the bovine serum albumin conjugate at position 3 of progesterone had the highest affinity, with an estimated inhibition constant of 28.5 +/- 2.1 nM (n = 3), whereas bovine serum albumin itself and the 17 beta-estradiol 6-(O-carboxy-methyl)oxime-bovine serum albumin conjugate showed no specific displacement. In addition, the binding sites were localized in an axolemma-enriched fraction of rat brainstem. Specific binding was obtained in tissues from cerebral cortex, brainstem, cerebellum, corpus striatum, and hypothalamus, but little or no binding occurred in uterus, ovary, liver, and spleen. The present data indicate that progesterone-125I-bovine serum albumin conjugate can be used as a ligand to study progesterone-membrane receptor interactions.     

Kinetic analysis of the interaction between HIV-1 protease and inhibitors using optical biosensor technology

The interaction between HIV-1 protease and reversible inhibitors was studied by surface plasmon resonance biosensor technology. The steady-state binding level and the time course of association and dissociation could be observed by measuring the binding of inhibitors injected in a continuous flow of buffer to the immobilized enzyme. Fourteen low molecular weight inhibitors (500-700 Da), including the four clinically used HIV-1 protease inhibitors (indinavir, nelfinavir, ritonavir, and saquinavir), were analyzed. Affinities were estimated as B(50) values from a series of sensorgrams at different concentrations of inhibitors. These values were found to be correlated with inhibition constants (K(i)) determined by an enzyme inhibition assay (r(2) = 0.84, logarithmic values). Dissociation rates were estimated at a single saturating concentration of the inhibitors as t(1/2,obs), but these values did not correlate with K(i) (r(2) = 0.26, logarithmic values). Indinavir had the highest affinity (B(50) = 11 nM) and the fastest dissociation (t(1/2,obs) = 500 s) among the clinically used inhibitors while saquinavir had a lower affinity (B(50) = 25 nM) and the slowest dissociation rate (t(1/2,obs) = 6500 s). Since these two inhibitors have similar K(i) values, the differences in dissociation rates reveal important characteristics in the interaction that cannot be obtained by the inhibition studies. The biosensor data are expected to be of greater in vivo relevance since the experiments were performed in a buffer more similar to physiological conditions.     

Two phase aqueous extraction

Rheological properties have been measured for aqueous solutions of dextran, polyethylene glycol and bovine serum albumin. Mixtures of these materials have also been studied. A rotating concentric cylinder viscometer was used to study the rheological properties of these materials over the temperature range 10 to 40°C. Over the range of concentrations, molecular weights, temperature and shear rates covered in this work, all aqueous solutions exhibited Newtonian behaviour. Correlations have been reported for viscosities of dextran, polyethylene glycol, and bovine serum albumin. The viscosity of mixtures of these materials is not linear with respect to concentration.     

The recBC enzyme of Escherichia coli K12: premature cessation of catalytic activities in vitro and reactivation by potassium ions.

It is shown that in vitro the degradation of native and single-stranded DNA as well as the hydrolysis of ATP by purified recBC enzyme ceases 2-3 min after the start of the reaction. The presence of potassium ions (60-100 mM), bovine serum albumin (1 mg/ml) or protein from cell-free Escherichia coli extract (10 microgram/ml) prevents the cessation of the activity. Once the cessation has occurred, the activity of the enzyme can be completely restored by the addition of potassium ions, but not by bovine serum albumin. Sedimentation studies revealed that, in contrast to the active recBC enzyme, the 'silent' enzyme is no longer associated with substrate DNA of high molecular weight. On the basis of these results and other observations it is hypothesized that during the degradation of DNA in the absence of potassium ions or bovine serum albumin the recBC enzyme is subject to an alteration of its molecular conformation which results in an inactive form.