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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.

Biosynthesis of mucin-type O-glycans is initiated by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases, which contain several conserved cysteine residues among the isozymes. We found that a cysteine-specific reagent, p-chloromercuriphenylsulfonic acid (PCMPS), irreversibly inhibited one of the isozymes (GalNAc-T1). Presence of either UDP-GalNAc or UDP during PCMPS treatment protected GalNAc-T1 from inactivation, to the same extent. This suggests that GalNAc-T1 contains free cysteine residues interacting with the UDP moiety of the sugar donor. For the functional analysis of the cysteine residues, several conserved cysteine residues in GalNAc-T1 were mutated individually to alanine. All of the mutations except one resulted in complete inactivation or a drastic decrease in the activity, of the enzyme. We identified only Cys212 and Cys214, among the conserved cysteine residues in GalNAc-T1, as free cysteine residues, by cysteine-specific labeling of GalNAc-T1. To investigate the role of these two cysteine residues, we generated cysteine to serine mutants (C212S and C214S). The serine mutants were more active than the corresponding alanine mutants (C212A and C214A). Kinetic analysis demonstrated that the affinity of the serine-mutants for UDP-GalNAc was decreased, as compared to the wild type enzyme. The affinity for the acceptor apomucin, on the other hand, was essentially unaffected. The functional importance of the introduced serine residues was further demonstrated by the inhibition of all serine mutant enzymes with diisopropyl fluorophosphate. In addition, the serine mutants were more resistant to modification by PCMPS. Our results indicate that Cys212 and Cys214 are sites of PCMPS modification, and that these cysteine residues are involved in the interaction with the UDP moiety of UDP-GalNAc.

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.

This paper reports two studies. The first one investigates how two 10-year-old Taiwanese-Hungarian balanced bilingual twin boys translated sentence-embedded words between their native languages over a six-month period as the physical context around them changed. The second one compares how these two bilinguals and four monolingual children defined words in their native languages. The purpose is twofold: to explore the role of an active language in translation, and to propose a new measurement for bilinguals. The reaction time (RT) and accuracy of their verbal protocols were measured and analyzed. The bilingual children’s word translation reveals an active use of metalinguistic skills. The physical context did affect their RT and accuracy in general and in translating concrete/abstract concepts. The two bilinguals defined words as fast and accurate as their monolingual peers in both languages, but different definition aspects were identified from the answers of the two groups. The translation/definition task appears a comprehensive measurement for bilingual and monolingual children with any combination of languages.

Hysteresis loops of nanocrystalline samples of Fe39Co39Nb6B16 - yCuy (y = 0, 1) HITPERM-type alloys were measured from room temperature up to 773K. The alloy with Cu shows a nearly constant value of coercivity (∼ 135A/m) in all the studied temperature range, whereas for the Cu-free alloy, the coercivity increases from 125A/m at 300K up to 190A/m at 773K. Room temperature Mössbauer spectra can be interpreted on the basis of the presence of three different regions: amorphous, crystalline and interface. The observed nanocrystalline microstructure is similar for the two alloys, although bigger crystallites are found in the alloy without Cu. The very small grain size ensures the averaging out of the magnetocrystalline anisotropy but, as the expected value of magnetostriction constant for the crystalline phase is ∼ 60ppm, the magnetoelastic anisotropy cannot be neglected. Gobierno de España MAT 2004-04618 European Commission. Fondo Europeo de Desarrollo Regional 2004-04618

The crystal structure of d[Cp(CH3)G] has been determined as part of a project to study the mechanism of the B----Z transition in DNA. The asymmetric unit contains two dinucleotides and the equivalent of 7.5 water molecules, partially disordered over 12 definable positions. The two symmetry-independent dinucleotides form a duplex with Watson-Crick base-pairing and a right-handed helical sense. Comparison with previously determined structures of the B and A conformation showed that this duplex is closer to B than to A but significantly different from B. It corresponds to a stretched out helix with a 4 A rise per base pair and a helical twist of 32 degrees. This structure may serve as a model for the bending of DNA in certain situations. The configuration at the methyl phosphonate is RP, and a mechanism, based on this assignment, is presented for the B----Z transition in DNA.

Temperature evolution of the coercivity of nanocrystalline samples of Fe60Co18Nb6B15Cu1 alloy with different crystalline fractions was measured from room temperature up to 690 K. Although room temperature coercivity increases as the nanocrystallization progresses, the thermal stability of the magnetic properties of the system is clearly enhanced as the crystalline volume fraction increases. Microstructure was characterized using room temperature Mössbauer spectra which can be interpreted on the basis of the presence of three different regions: amorphous, crystalline and interface. Gobierno de España MAT 2004-04618 European Commission. Fondo Europeo de Desarrollo Regional MAT 2004-04618

The resistance of Gram − bacteria to the broad-spectrum antibiotic tetracycline (Tc) results from energy-dependent drug efflux mediated by the tet gene product, the cytoplasmic membrane Tet protein. Amino acid (aa) sequences deduced from total tet nucleotide sequences of three different resistance determinants (classes A, B and C) indicate that the protein products [Tet(A), Tet(B), and Tet(C)] share a common ancestor. Hydropathic analysis of Tet sequences predicts twelve transmembrane segments in each protein, with six occuring in each half of the molecule. More importantly, the linear distributions of these segments in the N- and C-terminal halves are nearly identical, suggesting that the two halves of each Tet protein are related by a process of tandem gene duplication and divergence. Indeed, a variable but significant conservation of sequence was detected among the N- and C-terminal halves for all possible comparisons of the three proteins. Such conservation was not observed within other prokaryotic integral membrane proteins or when other prokaryotic proteins were compared to Tet halves. Similarity, both in sequence and in predicted transmembrane structural organization, strongly suggests that a common aancestor of Tet(A), Tet(B), and Tet(C) arose by duplication of a gene reading frame specifying a transmembrane protein of approximately 200 aa residues. The two halves of Tet proteins correspond to the two domains, α and β, which have distinct, complementary roles in Tc efflux. Nevertheless, selective constraints to function in the cytoplasmic membrane have apparently led to maintenance of similar patterns of secondary structural organization in these complementary domains.

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.

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.

C 18 :juvenile hormone is quite soluble in water, yielding a monomeric solution greater than 10 -5 M. In vivo injection or addition of aqueous juvenile hormone to the hemolymph in vitro shows the complexation of juvenile hormone to a protein, as demonstrated by gel permeation chromatography and disc-gel electrophoresis. The protein has an apparent molecular weight of 3.4 × 10 4 and is present in the hemolymph at a concentration in the micromolar range. The binding of the hormone to the protein can be described as a simple thermodynamic equilibrium with a dissociation constant of 3 × 10 -7 M, and the protein has a much higher affinity for the hormone than for the hydrolysis products.

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.

In earlier studies, we prepared a docosapeptide, 1, designed with minimum homology as an amphiphilic alpha-helical model of apolipoprotein A-I (apo A-I) and described its lipid-binding characteristics, surface properties, and enzyme-activating ability. Although the affinity of 1 for egg lecithin unilamellar vesicles was comparable with that for the binding of apo A-I, the affinity of 1 for mixed lecithin/cholesterol (4:1 mol/mol) vesicles was less than that of apo A-I. It appeared possible that the 3-hydroxyl group of cholesterol may have a deleterious interaction with the hydrophobic portion of the amphiphilic helix of 1 that is inserted into the vesicles. Examination of the amphiphilic alpha-helical segments of apo A-I suggested that the preferential interaction of apo A-I with the mixed vesicles might be due to the presence of polar arginine residues in the otherwise hydrophobic regions of two of the helices. Therefore, we synthesized a model docosapeptide, 2, corresponding to the sequence of 1 but containing arginine rather than leucine at position 10 in the hydrophobic region of the alpha helix to assess the role of the alcohol function of cholesterol in protein--cholesterol interactions. The results of studies on the binding of 2 to unilamellar vesicles containing lecithin only, lecithin/cholesterol, lecithin/cholesterol hemisuccinate, or lecithin/cholesterol methyl ether were consistent with the postulate that the major role of cholesterol in the binding of proteins to phospholipid surfaces is the creation of free space between the phospholipid head groups that can accommodate the amphiphilic peptide chains at the interface.

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.

Lipoprotein lipase from bovine milk reacted stoichiometrically with diisopropylphosphorofluoridate (DFP), an inactivator of serine esterases, resulting in the loss of enzymatic activity against triacylglycerols. The reaction obeyed first-order kinetics with a rate constant of 0.69 h-1. In order to isolate the peptide containing the diisopropylphosphoryl moiety (DIP), partially purified lipoprotein lipase was covalently labeled with [3H]DFP, and the labeled protein was reduced, carboxymethylated, and further purified to about 90% homogeneity. Cyanogen bromide cleavage followed by gel filtration yielded a radioactive peptide of 6-8 kDa. This peptide was succinylated and then digested with Staphylococcus aureus V8 proteinase. From this digest, a peptide containing 0.95 mol of [3H] DIP/mol of peptide was isolated by gel-permeation chromatography followed by reverse-phase high performance liquid chromatography. Automated Edman degradation provided the following sequence: Ala-Ile-Gly-Ile-His-Trp-Gly-Gly- (DIP)Ser-Pro-Asn-Gln-Lys-Asn-Gly-Ala-Val-Phe-Ile-Asn-(Ser, Leu)-Glu. Analysis of the sequence for secondary structure suggests that the reactive serine of lipoprotein lipase is in a beta-turn, a structure similar to those of the active sites of most other serine proteinases. Lipoprotein lipase appears to share this secondary structure with other serine hydrolases despite significant differences in the primary structure of this domain.

The enzymatic reaction of porcine pancreatic lipase with insoluble monolayers of either trioctanoin or 1,2-dioctanoin can be followed quantitatively by monitoring the considerable change in surface pressure which occurs during the course of the reaction. The rate of each reaction is proportional to the surface concentration of the substrate. The calculated pseudo-first order rate constants are proportional to the enzyme concentration in the bulk solution. At pH 7.6, the value of the second order rate constant for the hydrolysis of the primary ester function in trioctanoin was found to be 8.46 x 105 m-1 s-1, and that for dioctanoin, 2.65 x 104 m-1 s-1. The pH dependency of the rate constant for dioctanoin hydrolysis reveals an ionizable basic group at the active site of the enzyme with pKa of 6.38. The results are consistent with fully hydrated enzyme molecules acting upon substrate molecules lying within the insoluble mono-layer.

The pH dependence of the α-chymotrypsin-catalyzed hydrolysis of l-tryptophan methyl ester in H2O and D2O and of d-tryptophan methyl ester was determined by spectroscopic means (kcat, Km and kcat/Km). No product inhibition or substrate activation was observed. The kinetic parameters of the α-chymotrypsin-catalyzed hydrolysis of l-tyrosine p-nitrophenyl ester were also determined. Values of kcat for both the tyrosine and tryptophan derivatives depend on the ionization of the amino group. For the tryptophan derivatives which were investigated in most detail, the pH dependence of kcat/Km ( = k2/Ks) yields a bell-shaped curve with a maximum at pH 7.2. The deuterium oxide kinetic solvent isotope effect and the dependency of the reaction on a base of pK ∼ 7 for the l derivative are comparable to that seen with the corresponding acylamino derivative. Thus, the mechanism of hydrolysis of the amino acid esters appear to be the same as that for the acylamino acid esters. However, the ionizability of the α-amino group in the former produces a markedly different pH dependency of the enzyme-catalyzed reaction, which can be attributed to the presence of a protonated and unprotonated substrate. The protonated substrate is about 20-fold faster than the unprotonated substrate in both acylation and deacylation of l-tryptophan methyl ester. Full specificity of the enzyme (l over d) is found for l-tryptophan methyl ester in its protonated form. This may be due to a hydrogen bond between the substrate and the enzyme with very strict orientational requirements.

The reaction of porcine pancreatic lipase B (EC 3.1.1.3) with the soluble triglyceride, tripropionin, shows substantial stimulation in the presence of hydrophobic surfaces. The enhancement of the enzymatic rate can be correlated with the reversible binding of the enzyme to the hydrophobic surface with a dissociation constant K = 1.3 x 10-8 m. The binding of the enzyme to the surface is diffusion controlled with a rate constant of 1.8 x 106 m-1 s-1. At saturation of the surface with enzyme each protein molecule occupies an average area of 6000 A2 per molecule. The hydrolytic reaction on the surface is first order with respect to the amount of adsorbed enzyme and the concentration of tripropionin at the solution-solid interface. The second order rate constant of the reaction is 1.3 x 1013 cm2 mole-1 s-1. The surface reaction requires a basic group on the enzyme with a pK of 5.85. The enhancement of the velocity on the surface as compared to the homogeneous reaction can be ascribed to an increased local concentration of the substrate at the liquid-solid interface.

An acidimetric method is proposed to distinguish between carbon dioxide and carbonic acid as the primary product of enzymic decarboxylation reactions. Application of this method to the thiamine pyrophosphate-requiring Escherichia coli glyoxylate carboligase reaction (the decarboxylating condensation of glyoxylate to yield hydroxymalonic semialdehyde) at pH 8 shows a transient disappearance of protons. Kinetic analysis of this proton uptake shows that the reaction occurs according to the pathway [for equation see PDF] The value of kh measured during the enzyme-catalyzed reaction is identical to the independently determined rate constant of carbon dioxide hydration. Thus, the intermediate is carbon dioxide, and the enzyme does not catalyze its hydration. The identity of the intermediate is further confirmed by the fact that addition of carbonic anhydrase to the reaction mixture suppresses the pH changes.