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Mucin type O-glycosylation begins with the transfer of GalNAc to serine and threonine residues on proteins by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminlytransferases. These enzymes all contain a lectin-like (QXW)(3) repeat sequence at the C terminus that consists of three tandem repeats (alpha, beta, and gamma). The putative lectin domain of one of the most ubiquitous isozymes, GalNAc-T1, is reportedly not functional. In this report, we have reevaluated the role of the GalNAc-T1 lectin domain. Deletion of the lectin domain resulted in a complete loss of enzymatic activity. We also found that GalNAc-T1 has two activities distinguished by their sensitivities to inhibition with free GalNAc; one activity is sensitive, and the other is resistant. In our experiments, the former activity is represented by the O-glycosylation of apomucin, an acceptor that contains multiple glycosylation sites, and the latter is represented by synthetic peptides that contain a single glycosylation site. Site-directed mutagenesis of the lectin domain selectively reduced the former activity and identified Asp(444) in the alpha repeat as the most important site for GalNAc recognition. A further reduction of the GalNAc-inhibitable activity was observed when both Asp(444) and the corresponding aspartate residues in the beta and the gamma repeats were mutated. This suggests a cooperative involvement of each repeat unit in the glycosylation of polypeptides with multiple acceptor sites.

The amphiphilic novenamines described in this report have been shown previously to be specific inhibitors of human immunodeficiency virus type 1 reverse transcriptase-associated ribonuclease, which they inhibit when they are in the micellar state but not when they are monomeric. These compounds also inhibit the bacterial enzyme DNA gyrase, which is essential for DNA replication. Hence, the present studies were initiated to determine whether the molecular species inhibiting the gyrase reaction was the monomeric or the micellar form. For this purpose, the rate of DNA replication was measured in a toluenized Escherichia coli cell system in the presence of increasing concentrations of novenamines. The resulting concentration-response curves proved anomalous, suggesting the involvement of micelles or some other, noncovalently aggregated forms of the inhibitors. The results were analyzed in terms of a variety of kinetic schemes and were found to be most consistent with the model where novenamines inhibit replicative DNA synthesis predominantly as cooperative dimers and, to a lesser extent, as monomers, but not as highly aggregated micelles. Based on this analysis and the knowledge that novobiocin and all novenamine-containing analogs are powerful gyrase inhibitors, we conclude that the target of the cooperative, dimeric inhibition is the gyrase, whereas the monomers of the novenamines inhibit another enzyme species involved in the bacterial DNA replication process.

U-31,355, or 4-amino-2-(benzylthio)-6-chloropyrimidine is an inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and possesses anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound acts as a specific inhibitor of the RNA-directed DNA polymerase function of HIV-1 RT and does not impair the functions of the DNA-catalyzed DNA polymerase or the RNase H of the enzyme. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-31,355. The data were analyzed using Briggs-Haldane kinetics, assuming that the reaction is ordered in that the template:primer binds to the enzyme first, followed by the addition of dNTP, and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived that allows the calculation of all the essential forward and backward rate constants for the reactions occurring between the enzyme, its substrates, and the inhibitor. The results obtained indicate that U-31,355 acts as a mixed inhibitor with respect to the templatetprimer and dNTP binding sites associated with the RNA-directed DNA polymerase domain of the enzyme. The inhibitor possessed a significantly higher binding affinity for the enzyme-substrate complexes than for the free enzyme and consequently did not directly affect the functions of the substrate binding sites. Therefore, U-31,355 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either inhibition of the phosphoester bond formation or translocation of the enzyme relative to its template:primer following the formation of the ester bond. Moreover, the potency of U-31,355 depends on the base composition of the template:primer in that the inhibitor showed a much higher binding affinity for the enzyme-poly (rC):(dG) 10 complexes than for the poly (rA):(dT) 10 complexes.

Two inhibitors of cynomolgus monkey cholesteryl ester transfer protein were evaluated. One, a monoclonal antibody made against purified cynomolgus monkey cholesteryl ester transfer protein, was capable of severely inhibiting triglyceride transfer, but had a variable effect on cholesteryl ester transfer. At low antibody to antigen ratios, there was what appeared to be a stoichiometric inhibition of cholesteryl ester transfer, but at high antibody to antigen ratios the inhibition of cholesteryl ester transfer was completely relieved, even though triglyceride transfer remained blocked. Fab fragments of the antibody had no effect whatsoever on cholesteryl ester transfer, but were capable of completely blocking triglyceride transfer. The other inhibitor, 6-chloromecuric cholesterol, severely inhibited cholesteryl ester transfer with minimal inhibition of triglyceride transfer. When both inhibitors were added to the assay, both cholesteryl ester and triglyceride transfer were inhibited; an indication that the inhibitors did not compete for the same binding site on cholesteryl ester transfer protein. When the antibody was given subcutaneously to cynomolgus monkeys at a dose which inhibited triglyceride transfer in the plasma by more than 90%, there was no detectable effect on the high density lipoprotein (HDL) cholesterol level, but the HDL triglyceride levels decreased from 13 ± 2 to 1 ± 0 mol/mol of HDL (mean ± S.D.); an indication that the antibody uncoupled cholesteryl ester and triglyceride transfer in vivo. The 6-chloromecuric cholesterol could not be evaluated in vivo because it is a potent lecithin:cholesterol acyltransferase inhibitor. The fact that cholesteryl ester transfer can be inhibited without effect on triglyceride transfer and, conversely, that triglyceride transfer can be inhibited without effect on cholesteryl ester transfer indicates that these two lipids are not transferred by a single, non-discriminatory process.

A continuous recording fluorescence assay was developed for cholesteryl ester transfer protein (CETP). The assay measures the increase in fluorescence accompanying the relocation of fluorescent lipids, cholesteryl esters and triglycerides, from a donor emulsion to an acceptor emulsion. In the absence of CETP, the quantum yields of the fluorescent lipids is low because their high concentrations in the donor emulsions result in self-quenching. CETP catalyzes the redistribution of the fluorescent lipids from the donor to the acceptor emulsions and fluorescence increases substantially. Efficient sonication and incorporation of apolipoproteins from human HDL into the emulsions significantly increased the transfer rates. Under optimal conditions, the redistribution of fluorescent compounds reaches equilibrium within30 min and the kinetics of this process are consistent with a simple, first-order reaction pathway. The redistribution kinetics support a mechanism of adsorption --exchange --desorption --diffusion.

Three fluorescent, lipophilic, heterocyclic antioxidants were incorporated into lipid bilayers and exposed to depth-dependent nitroxyl fatty acid quenchers. The Stern-Volmer plots curved upward at low quencher concentrations. Quantitative analysis of the results showed that this behavior is consistent with complex formation between quencher and fluorescent antioxidant, where the complex is 2-3 times more fluorescent than the parent fluorophore. At higher quencher concentrations, both free antioxidant and 'bright complex' are quenched dynamically, albeit quenching of the latter is less efficient. The complex probably results from ionic, hydrogen bond and pi-pi interactions. Formation of such a 'bright complex' is also observable in a homogeneous solution of the reactants in cyclohexane. Additional evidence for the complexation of these antioxidants with fatty acids in lipid bilayers is provided by the fact that energy transfer from the antioxidants to anthroyloxy fatty acids occurs at surface concentrations where radiative energy transfer between free molecules should be not be efficient. For directly probing the relative depths of these fluorophores in lipid bilayers we used the aqueous quenchers acrylamide and iodide. They showed that in terms of increasing depth in the bilayer, the order was U-78, 517fU-78,518eU-75,412e. Our results, in toto, demonstrate that the Lazaroid antioxidants are incorporated into the lipid bilayer where they occupy strictly defined positions and orientations. Complexation with fatty acyl chains should be mechanistically relevant, since it may enhance antioxidant activity by hindering free radical chain propagation.

cis - Parinaric acid (PnA), cis - trans - trans - cis-9, 11, 13, 15-octadecatetraenoic acid, is fluorescent (ϵ = 74,000 at 324 nm) when partitioned into a lipid environment and the fluorescence is destroyed upon reaction with free radiacals. It has been used to monitor semiquantitatively free-radical-induced lipid peroxidation in human erythrocyte membranes. We have applied this assay to the quantitative evaluation of potential antioxidants. The kinetics of the reaction of PnA with free radicals were measured in erythrocyte ghosts. After initiation of free radical generation by cumene hydroperoxide and cupric ion, a steady-state rate of fluorescence decay is rapidly established. In the steady state the oxidation of PnA and, hence, the loss of fluorescence is a first-order process. In the presence of antioxidants, such as vitamin E, the rate constant of fluorescence loss decreases, thereby indicating that the antioxidant decreases the steady-state concentration of free radicals. By adding various concentrations of potential antioxidants, pseudo-first-order rate constants [k1] which measure the reactivity of antioxidants with free radicals were determined. Results show that, when incorporated into erythrocyte membranes, U-78,517f, a vitamin E analog, is a potent free radical scavenger, being approximately 50% as effective as vitamin E and 10–15 times more potent than the aminosteroids evaluated (see Table 1).

1. 1. A procedure is described for the preparation of an antibody to arterial FABP using a synthetic peptide as an antigen. In order to locate a highly conserved region located on the outer surface of FABP, computer analysis of primary and secondary structures of several proteins from the FABP family was undertaken and a 24 amino acid sequence beginning at the fifth position from the N -terminus of rat heart FABP was chosen. 2. 2. The synthetic peptide consisted of eight replications of the 24 amino acid sequence individually attached to the α and ϵ amino groups of each terminal lysine on an octalysine branched peptide. 3. 3. Antibody to the synthetic antigen was raised in New Zealand rabbits. Western analysis was conducted and detection was accomplished by using goat-anti-rabbit second antibody conjugated to alkaline phosphatase. 4. 4. The antibody produced from the previously described peptide, recognized purified rat heart FABP and demonstrated a high positive correlation ( r = 0.96) when known concentrations of purified hFABP were plotted against densitometric measurement of the bands. 5. 5. Additionally, the antibody recognized FABP from the 104,000 g supernates of rat atrial and arterial tissue fractionated by a Sephadex G-75 column. 6. 6. Therefore, the antibody produced from this particular protocol employing a synthetic peptide can be utilized qualitatively and quantitatively in the analysis of the heart and arterial FABP content.

Insoluble monolayers of dioctanoyl lecithin show a change in surface state at pressures near 3.5 dynes/cm, as judged by surface potential-surface concentration measurements and from surface-pressure area data. The phospholipase A2 of Crotalus atrox venom and one from porcine pancreas show no surface-pressure rate dependency when acting upon a given state of dioctanoyl lecithin monolayer, but the rate changes at the surface state discontinuity. The ratio of the rate constants in the higher and lower pressure regions is 2.1 for the C. atrox enzyme and 1.4 for the pancreatic enzyme. In contrast to this straightforward kinetic behavior, the rates of hydrolysis catalyzed by the honeybee venom enzyme and a second pancreatic phospholipase increase continuously with the surface pressure. The anomalous surface pressure dependency correlates with the tendency of these enzymes to denature at clean air-water interfaces, and with the accumulation of inactive protein at the surface. The rate of enzyme denaturation is proportional to the area not covered by substrate molecules. Inert substances covering the surface prevent denaturation and eliminate surface pressure dependency of the enzymatic rate. Using a kinetic scheme based upon this free area denaturation model, minimum free areas for denaturation of 500 and 900 A2, and surface denaturation rate constants of 3.9 × 105 sec−1 and 1.1 × 109 sec−1 are calculated for the pancreatic and bee venom enzymes, respectively. The surface denaturation model is consistent with all pertinent data that have been published for enzymes that act upon lipid monolayers.

We have developed a rapid method for probing the affinity of peptides toward an amphiphilic surface. Hydrophobic polystyrene-divinylbenzene beads of 5.7 ± 1.5 μm diameter are coated with a monomolecular film of egg lecithin to achieve the equilibrium spreading density of the phospholipid, 6 × 10 −3 molecule/A 2 . The coated beads are ideally suited for assessing the affinity of peptides for phospholipid surfaces: (i) Large quantities of lipid-coated beads of known surface area can be prepared easily and rapidly. (ii) Within the pH range 2.0 to 9.0, the adsorbed phospholipids are relatively resistant to hydrolysis and remain bound indefinitely. (iii) Following incubation with peptide ligands, beads can be separated from the reaction mixture by centrifugation. (iv) Peptides, such as melittin, which destroy or cause fusion of single bilayer phospholipid vesicles, cannot disrupt lecithin-coated beads in a comparable way, and do not displace lecithin from the surface of beads. After incubating these beads in solutions of peptides and proteins, we have determined the parameters for the binding of several ligands to the phospholipid surface. The binding of many amphiphilic peptides obeys a Langmuir adsorption isotherm, i.e., saturable reversible binding to independent and equivalent sites on the bead. That the binding is a true reversible equilibrium is shown by desorption of the ligand upon dilution. From the isotherm, the surface areas occupied by the ligand molecules were calculated, and were observed to be similar to those observed in monolayers at the air-water interface. In comparing the binding of amphiphilic peptides to that of completely hydrophilic peptides, we observed that only the former bind at levels measurable by our techniques. Thus, this method can serve as a rapid assay for detecting amphiphilicity in peptides of putative amphiphilic character.

Apolipophorin III (apoLp-III) from Manduca sexta has a molecular weight of 18,100. Based on its hydrodynamic properties (sedimentation and diffusion coefficients, frictional ratio, intrinsic viscosity) and its behavior during gel permeation chromatography, we concluded that apoLp-III is a prolate ellipsoid with an axial ratio of about 3. The circular dichroic spectrum of apoLp-III suggests that the protein contains approximately 50% alpha-helix. At the air-water interface, apoLp-III forms a monolayer which is gaseous at surface pressures less than or equal to 1 dyne/cm. The isotherm of this phase yields an excluded molecular area of 3800 A2/molecule (23 A2/amino acid). At a surface pressure of 22.1 dynes/cm, the monolayer undergoes a phase transition reminiscent of a first-order phase transition of pure lipids. The monolayer can be compressed in this surface pressure range to an area per molecule of 480 A2 (2.9 A2/amino acid). Since a globular protein of molecular weight 18,100 could occupy an area of only about 2000 A2 when bound to a surface, it is suggested that in the expanded state, apoLp-III must unfold on the surface, whereas in the compressed state, the molecule is oriented with its minor axis parallel to the water surface. ApoLp-III binds with high affinity (Kd = 1.9 X 10(-7)M) to both phosphatidylcholine- and diacylglycerol-coated polystyrene beads. All of these results are consistent with the proposal that apoLp-III plays a key role in increasing the capacity of the insect lipoprotein, lipophorin, to transport diacylglycerol by stabilizing the increment of lipid-water interface that results from diacylglycerol uptake.

The amino acid sequence has been determined for the insecticyanin from the hemolymph of the fifth instar larvae of the tobacco hornworm, Manduca sexta. The apoprotein is a single polypeptide chain of 189 amino acids, molecular weight 21,378, containing two disulfide bridges, 9-119 and 42-176. The sequence analysis was performed by automated Edman degradation of reduced and carboxymethylated insecticyanin and fragments generated therefrom by cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus proteinase. Most of the peptides were purified by reverse-phase high-performance liquid chromatography. A purification procedure for the isolation of insecticyanin in high yields and a simple method of determining disulfide linkages are also reported.

We have investigated the ionization and surface properties of verapamil (5-[(3,4-dimethoxyphenethyl)methylamino]-2-(3, 4-dimethoxyphenyl)-2-isopropylvaleronitrile, 1) and several verapamil analogues since these properties appear to be involved in the biologic activities of these compounds. Our results show that verapamil and its analogues are surface-active and bind to amphiphilic surfaces. The affinity toward, as well as the capacity of, an amphiphilic surface for verapamil and its ionizable analogues is pH dependent, with the surface having both higher affinity and capacity for the neutral form of the molecules. Thus, verapamil exists as protonated and neutral forms, both of which are free in solution and adsorbed to the interface, and the ionization of verapamil at an interface changes with respect to its ionization in solution. From analyses of the pH dependency of surface binding and of solution and interfacial ionizations, we determined the values of the four equilibrium constants. These equilibrium constants permit correlative studies between the pH-dependent abundance of each species and biologic activity. We discuss preliminary studies which indicate that the negative inotropic effect of verapamil is mediated by the membrane-bound neutral form of the drug.

We report here the discovery of a new class of phospholipases A2 in which Asp-49, a residue considered to be an obligate component of the catalytic apparatus, is replaced by a lysine. Asp-49 is invariant among the more than 30 venom and pancreatic phospholipases A2 sequenced to date, and its beta-carboxylate group has been shown to be a ligand for calcium in a binding site which also involves contributions from the peptide carbonyl oxygens of Tyr-28, Gly-30, and Gly-32, the so-called calcium-binding loop. The change of Asp-49 to a lysine, and other substitutions in regions heretofore thought to be invariant, including the calcium-binding loop, suggested that the new phospholipases might differ functionally with respect to calcium and/or substrate binding. Indeed, although the Lys-49 phospholipases A2 show a dependence on calcium similar to that of the Asp-49 enzymes, they may be distinguished by the fact that, in the absence of phospholipid, they do not bind calcium to any measurable extent under conditions where Asp-49 enzymes bind a stoichiometric amount of calcium. Furthermore, in the absence of calcium, they show binding to single bilayer phospholipid vesicles under conditions where Asp-49 phospholipases do not bind at all. These results suggest a reversed order of addition of calcium and substrate in the formation of the ternary catalytic complex in the Lys-49 phospholipases A2. Although the mechanistic implications of these structural and functional alterations are not defined at present, it is clear that Asp-49 is not essential for phospholipase A2 catalysis and that it does not participate in the enzyme-calcium-phospholipid catalytic complex.

The segment corresponding to residues 121 to 164 of human plasma apolipoprotein A-I (apo-A-I) has been synthesized by the Merrifield solid phase method. The peptide binds to unilamellar phospholipid vesicles and to phospholipid-cholesterol mixed vesicles. The surface affinity of the peptide measured in this way indicated that the mechanism of binding is the same as that of apo A-I (144-165) and apo A-I itself. The peptide appears to be a globular monomer in a aqueous solution, with 17% alpha helix content. The peptide bound to vesicles activates lecithin:cholesterol acyltransferase: compared to apo A-I, the peptide is about 30% as efficient in the activation of cholesterol esterification and of phospholipid hydrolysis when the surface is saturated by the activator. For a variety of amphiphilic peptides and for apo A-I, the lecithin: cholesterol acyltransferase-activating ability correlates well with their alpha helix contents in 50% trifluoroethanol.