Structural determinants in substrate recognition by proton-amino acid symports in plasma membrane vesicles isolated from sugar beet leaves.

Amino acids are actively transported across the plasma membrane of plant cells by proton-coupled symports. Previously, we identified four amino acid symports in isolated plasma membrane vesicles, including two porters for the neutral amino acids. Here we investigated the effect of amino acid analogues on neutral amino acid transport to identify structural features that are important in molecular recognition by Neutral System I (isoleucine) and Neutral System II (alanine and leucine). D-Isomers of alanine and isoleucine were not effective transport antagonists of the L-isomers. These data are characteristic of stereospecificity and suggest that the positional relationship between the alpha-amino and carboxyl groups is an important parameter in substrate recognition. This conclusion was supported by the observation that beta-alanine and analogues with methylation at the alpha-carbon, at the carboxyl group, or at the alpha-amino group were not effective transport inhibitors. Specific binding reactions were also implicated in these experiments because substitution of the alpha-amino group with a space filling methyl or hydroxyl group eliminated transport inhibition. In contrast, analogues with various substitutions at the distal end of the amino acid were potent antagonists. Moreover, the relative activity of several analogues was influenced by the location of sidechain branches and Neutral Systems I and II were resolved based on differential sensitivity to branching at the beta-carbon. The kinetics of azaserine and p-nitrophenylalanine inhibition of leucine transport were competitive. We conclude that the binding site for the carboxyl end of the amino acid is a well-defined space that is characterized by compact, asymmetric positional relationships and specific ligand interactions. Although the molecular interactions associated with the distal portion of the amino acid were less restrictive, this component of the enzyme-substrate complex is also important in substrate recognition because the neutral amino acid symports are able to discriminate between specific neutral amino acids and exclude the acidic and basic amino acids.     

Sbustrate specificity of the elastase and the chymotrypsin-like enzyme of the human granulocyte.

Human granulocyte elastase (EC 3.4.21.11) differs from hog pancreatic elastase in its specificity for synthetic substrates. Although hydrolyzing peptide bonds adjacent to the carboxyl group of alanine, the granulocyte enzyme prefers valine at the cleaved bond, in contrast to the pancreatic enzyme which prefers alanine. Peptide bonds involving the carboxyl group of isoleucine can be hydrolyzed by the granulocyte enzyme but are not hydrolyzed to any significant extent extent by pancreatic elastase. This difference in specificty could explain the lower sensitivity of the granulocyte enzyme to inhibitors containing alanine analogs, such as the peptide chloromethyl ketones and elastatinal. The human granulocyte chymotrypsin-like enzyme differs from pancreatic chymotrypsin by being able to cleave substrates containing leucine in addition to those containing the aromatic amino acids.     

The initial binding of Cu(II) to some amino acids and dipeptides: a 13C nuclear-magnetic-resonance study.

The initial binding of Cu2+ ot L-lysine, L-histidine, glycyl-histidine and histidyl-glycine in aqueous solutions was examined by 13C nuclear magnetic resonance spectroscopy. The measurements were carried out in a substantially improved way employing the pulse Fourier transform technique. Spectra of both high quality and resolution were obtained. Cu2+ complex formation with L-lysine occurred with the alpha-amino and carboxyl group attributable to the well expressed broadening effect of the 13C signals of the alpha-carbon atom and the carboxyl atom. The epsilon-amino group was not involved. Measurements of the Cu chelates using L-histidine and glycyl-histidine and histidyl-glycine confirmed the ambidentate nature of the histidine residue. It was concluded that an equilibrium exists between two Cu-complex species designated as histamine-like and histamine-like/glycine-like species. In the homogeneous histamine-like Cu complex, the Cu2+ is exclusively bound with 4 nitrogens, while in the other species one oxygen of the glycyl carboxyl group is involved in the Cu2+ binding. Blocking of this carboxyl groups by peptide bonding as found in histidyl-glycine favoured the formation of a Cu complex where the imidazole carbons of the histidyl residue were the most influenced species.     

Introduction of sulfhydryl groups into proteins at carboxyl sites

A two-step procedure for introduction of sulfhydryl groups at protein carboxyl groups is described. The resultant proteins contain 2-aminoethanethiol residues bound by amide linkages to the protein carboxyl groups. First an amide bond is formed between a carboxyl group of the protein and one of the amino groups of cystamine. Then the disulfide bond is reduced with dithiothreitol, yielding the amide of 2-aminoethanethiol. This procedure was used to incorporate sulfhydryl groups into carbonic anhydrase and adrenocorticotropic hormone. The effect of carbodiimide concentration and pH of the coupling reaction on stoichiometry of sulfhydryl group incorporation was examined. The method was used to prepare bovine carbonic anhydrase containing up to nine sulfhydryl groups per molecule with no loss of enzymatic activity and biologically active adrenocorticotropic hormone containing one sulfhydryl group per molecule.     

Oxidation and esterification of geometrical and positional isomers of octadecenoic acids in perfused rat liver

The metabolic fate of the geometrical isomers of 6-, 9-, and 11-octadecenoic acids was studied in isolated perfused rat livers. Although the ketogenicities of these monounsaturated fatty acids generally decreased as the double bond was moved away from the carboxyl group, a dependence on the geometrical difference was found only for 9-octadecenoate, the rate being significantly lower in the cis-isomer. Very low density lipoprotein lipid secretion was distinctly and specifically decreased when trans-9-octadecenoic acid was perfused, but no such difference was observed with other isomers. Various trans-isomers were actively incorporated into the hepatic lipids and secreted apparently at the expense of preexisting endogenous cis-octadecenoate. The trans-isomers were all incorporated exclusively at the 1-position of hepatic phosphatidylcholine. Concentrations of the cis-octadecenoate in the glycerolipids secreted and remaining in the liver were characteristically modified depending on the location of the ethylenic bond of the cis-octadecenoate. Thus, both the location of the double bond in the acyl-chain and the geometrical configuration specifically influence the rate of oxidation and esterification of octadecenoic acid in perfused rat liver.     

松香改性的研究进展

本文综述了近年来松香改性的研究进展情况,对其进行了分类阐述,即基于羧基的改性、基于双键的改性、基于羧基和双键的改性以及其它方面的改性,并展望了此领域的发展趋势和前景。     

Synthesis of methyl 16-trideuteriohexadecanoate

Methyl 16-trideuteriohexadecanoate has been prepared in high isotopic purity and in 29% overall yield, from methyl 7-oxo-16-heptadecenoate. The oxo group was reduced with sodium cyanoborohydride and the CD₃ group was introduced by reduction with lithium aluminum deuteride, first of the ester group to the alcohol and then of the derived mesylate. The carboxyl group was formed by oxidative cleavage of the double bond.     

Aliphatic amino diacid Asu functions as an effective mimic of Tyr(SO3H) in sulfakinins for myotropic and food intake-inhibition activity in insects

The aliphatic amino diacid alpha-aminosuberic acid can function as an effective, stable mimic of the hydrolysis-susceptible Tyr(SO3H) group in sulfakinin neuropeptide analogs for both hindgut contractile activity in cockroach and food intake-inhibition activity in the desert locust. In the analog, the acidic sulfate group is replaced with an acidic carboxyl group. The degree of activity of sulfakinin analogs is correlated with the carboxyl/alpha-carbon distance in the cockroach hindgut contractile assay. The results represent an important step in the design and synthesis of biostable, sulfakinin analogs that could potentially suppress the feeding behavior of destructive insect pests of agricultural importance.     

Comparison of vasodilatory properties of 14,15-EET analogs: structural requirements for dilation

Epoxyeicosatrienoic acids (EETs) are endothelium-derived eicosanoids that activate potassium channels, hyperpolarize the membrane, and cause relaxation. We tested 19 analogs of 14,15-EET on vascular tone to determine the structural features required for activity. 14,15-EET relaxed bovine coronary arterial rings in a concentration-related manner (ED(50) = 10(-6) M). Changing the carboxyl to an alcohol eliminated dilator activity, whereas 14,15-EET-methyl ester and 14,15-EET-methylsulfonimide retained full activity. Shortening the distance between the carboxyl and epoxy groups reduced the agonist potency and activity. Removal of all three double bonds decreased potency. An analog with a Delta8 double bond had full activity and potency. However, the analogs with only a Delta5 or Delta11 double bond had reduced potency. Conversion of the epoxy oxygen to a sulfur or nitrogen resulted in loss of activity. 14(S),15(R)-EET was more potent than 14(R),15(S)-EET, and 14,15-(cis)-EET was more potent than 14,15-(trans)-EET. These studies indicate that the structural features of 14,15-EET required for relaxation of the bovine coronary artery include a carbon-1 acidic group, a Delta8 double bond, and a 14(S),15(R)-(cis)-epoxy group.     

X-ray structure of Glu 53 human lysozyme.

The three-dimensional structure of a modified human lysozyme (HL), Glu 53 HL, in which Asp 53 was replaced by Glu, has been determined at 1.77 A resolution by X-ray analysis. The backbone structure of Glu 53 HL is essentially the same as the structure of wild-type HL. The root mean square difference for the superposition of equivalent C alpha atoms is 0.141 A. Except for the Glu 53 residue, the structure of the active site region is largely conserved between Glu 53 HL and wild-type HL. However, the hydrogen bond network differs because of the small shift or rotation of side chain groups. The carboxyl group of Glu 53 points to the carboxyl group of Glu 35 with a distance of 4.7 A between the nearest carboxyl oxygen atoms. A water molecule links these carboxyl groups by a hydrogen bond bridge. The active site structure explains well the fact that the binding ability for substrates does not significantly differ between Glu 53 HL and wild-type HL. On the other hand, the positional and orientational change of the carboxyl group of the residue 53 caused by the mutation is considered to be responsible for the low catalytic activity (ca. 1%) of Glu 53 HL. The requirement of precise positioning for the carboxyl group suggests the possibility that the Glu 53 residue contributes more than a simple electrostatic stabilization of the intermediate in the catalysis reaction.     

Endoplasmic oleoyl-PC desaturase references the second double bond

The regiospecificity for the gene product of fad2,(1) the microsomal oleoyl-PC desaturase from higher plants, differs from some previous suggestions. Rather than only referencing the carboxyl group (a Delta(12) desaturase) or the methyl terminus (an omega-6 desaturase), this desaturase locates the second double bond in its substrates by first referencing the existing double bond. This specificity was demonstrated for the oleoyl-PC desaturase cDNA from the developing seeds of peanut (Arachis hypogaea L) expressed in yeast (Saccharomyces cerevisae). The expressed enzyme was capable of desaturating monounsaturated fatty acyl groups in membrane lipids. Endogenous palmitoleate was desaturated to cis, cis 9,12 hexadecadienoate (9(Z)12(Z)C16:2), endogenous oleate to linoleate (9(Z)12(Z) octadecadienoate), and cis 10-nonadecenoate (provided as a supplement in the growth medium) to 10(Z)13(Z)C19:2. The rule, Delta(x+3) where x=9 is the double bond location in the substrate, best describes the consistent placement of the second double bond in the above monounsaturated substrates for the oleoyl-PC desaturase of higher plants.     

Aminopropyl silica gel as a solid support for preparation of glycolipid immunoadsorbent and purification of antibodies.

Aminopropyl silica gel was prepared from porous silica gel and was used as a solid support for immunoadsorbent in the purification of anti-glycolipid antibodies. For neutral glycosphingolipids, a carboxyl function was generated by oxidation of the olefinic double bond of the sphingosine moiety, whereas for gangliosides the carboxyl group of sialic acid was used to couple with aminopropyl silica gel in the presence of a carbodiimide. These compounds were used for purifying anti-glycolipid antibodies from serum of immunized rabbits. The antibodies bound to the su-strate were released by 2 M potassium thiocyanate and their immunological properties were studied. Aminopropyl silica gel may be preferred over conventional organic solid supports for the following reasons: 1) faster flow rate; 2) higher capacity; 3) easier handling; 4) more economical; and 5) lower susceptibility to microbial attack.     

Arachidonic acid-related elicitors of the hypersensitive response in potato and enhancement of their activities by glucans from Phytophthora infestans (Mont.) deBary

The dose response for elicitation of the hypersensitive reaction in potato tuber discs by arachidonic acid (AA) suggested saturation at higher concentrations. Glucans from Phytophthora infestans, inactive themselves as elicitors of the hypersensitive reaction, enhanced sesquiterpene accumulation and hypersensitive browning elicited by AA. Significant activity (seven times control values) was observed with 33 pmol AA/3.0-cm potato disc in the presence of glucans. Glucans did not affect accumulation of steroid glycoalkaloids, influence the timing or relative amounts of sesquiterpenes which accumulate, or affect recovery of AA added to potato discs. Glucans enhanced activity whether added to potato discs 18 h prior to AA, at the same time as AA, or 18 h after AA. Elicitor activity in the presence of glucans was evident with 20-carbon unsaturated fatty acids that had little or no elicitor activity in the absence of glucans. The position of double bonds had considerable influence on the specific activity of unsaturated fatty acids. The most active had a minimum of three double bonds in a methylene-interrupted series beginning with delta 5, e.g., delta 5,8,11. A delta 5 double bond conferred significant activity even if it was not part of a methylene-interrupted series. The 20-carbon chain length appeared optimal for elicitor activity. The 22-carbon chain acids had low activity, and 16- and 18-carbon acids were inactive. A free carboxyl group or easily transesterified group appeared necessary for activity. Arachidonyl alcohol had very low activity and arachidonyl cyanide was inactive. AA-containing phosphatidylcholine, lysophosphatidylcholine and monoacylglycerol were at least as active as free AA, AA-containing diacylglycerols were slightly less active than free AA, and triarachidonyl glycerol was inactive.     

High mutagenicity of N-(alpha-acyloxy)alkyl-N-alkylnitrosamines in S. typhimurium: model compounds for metabolically activated N,N-dialkylnitrosamines.

A series of N,N-dialkylnitrosamines (alkyl means methyl, ethyl, n-propyl, n-butyl or tert-butyl group) mono-substituted at the alpha-carbon with an acetoxy group, were tested for their mutagenic action in Salmonella typhimurium TA1530 in the presence or absence of a rat-liver supernatant from 9000 X g. The presumed released of methyl, ethyl, n-butyl and n-propyl carbonium ions from the corresponding alpha-acetoxy derivatives, either by enzymic cleavage or by non-enzymic hydrolysis of the ester group, caused high mutagenicity in the bacteria. As has been demonstrated for certain alpha-acetoxy compounds, the mutagenicity of these compounds was inversely related to their half-lives in aqueous media. N-(Acetoxy)methyl-N-tert-butylnitrosamine and a beta-acetoxy derivative of N,N-diethylnitrosamine were not mutagenic either in the presence or in the absence of hydrolysing rat-liver enzymes. These results support the hypothesis that alpha-carbon hydroxylation is one mechanism involved in the metabolic activation of N,N-dialkylnitrosamines.     

alpha-Aminoboronic acid derivatives: effective inhibitors of aminopeptidases

alpha-Aminoboronic acids and their derivatives have been synthesized as stable white solids. These compounds are effective inhibitors of human enkephalin degrading aminopeptidase, microsomal leucine aminopeptidase (EC 3.4.11.2), and cytosolic leucine aminopeptidase (EC 3.4.11.1) at micro- to nanomolar concentrations. The inhibition of cytosolic leucine aminopeptidase has been studied in some detail. Kinetic data correspond to the mechanism for biphasic slow-binding inhibition: E + I in equilibrium E.I in equilibrium E.I*, in which a rapid initial binding is followed by a slow transformation to a stable enzyme inhibitor complex. The initial and final binding constants are dependent on the nature of the side chain at the alpha-carbon atom but are independent of the protecting group on the boronic acid moiety and follow the trend for the hydrolysis of the corresponding amino acid amides. The first-order rate constant for the transformation of E.I to E.I* is similar for all four compounds studied. These data suggest that the slow-binding step represents the formation of tetrahedral boronate species from trigonal boronic acid.     

Ionic influences on the phase transition of dipalmitoylphosphatidylserine.

The ionization and phase behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphoserine have been investigated under a variety of condtions by several different methods. As measured by turbidity changes, the temperature of the crystal-liquid crystal phase transition of this lipid is influenced by pH and mono- and divalent cation concentrations. The pH-transition temperature curve is congruent with the curve relating temperature to the degree of ionization of the carboxyl group of the crystalline form. The transition temperature falls from an upper plateau of 72 degrees C at low pH values, where the carboxyl group is fully protonated, to a lower plateau of 55 degrees C at high pH values, where this group is fully ionized. The apparent pK (pH at 50% ionization) of the crystalline form shifts from 6.0 to 4.6 to 3.7 with an increase of NaCl concentration from 10(-3) to 0.1 to l.0 M, respectively. These observations are in accord with a simple theoretical analysis that utilizes diffuse double layer theory and the influence of surface potential on surface concentration of protons. In qualitative terms, an increase in electrolyte concentration reduces the surface potential, the result of which is a diminution of the surface-bulk pH difference and a lowering of the apparent pK. Assuming an area of 50 A2/molecule, the intrinsic pKa (apparent pK corrected for surface pH) of the carboxyl group is 2.7. A 1000-fold change of NaCl concentration produces a very large change in surface potential without influencing the transition temperature of the ionized form of the lipid.     

Three types of stereospecificity and the kinetic deuterium isotope effect in the oxidative deamination of dopamine as catalyzed by different amine oxidases

When the stereospecifically deuterated dopamine enantiomers, (R)- and (S)-[alpha-2H1]dopamine, are incubated with amine oxidases, the deuterium atom may be either retained to form monodeuterated 3,4-dihydroxyphenylacetaldehyde, or eliminated to produce the nondeuterated or protio-aldehyde product. These two aldehydes can be separated from one another and identified by high-performance liquid chromatography with electrochemical detection. Three types of stereospecific abstraction of a hydrogen from the alpha-carbon of dopamine during deamination have been observed. In the first type, the pro-R hydrogen is removed from the alpha-carbon. Enzymes in this category are mitochondrial monoamine oxidases A and B, as isolated from different tissues and species. The second type of deamination involves the abstraction of pro-S hydrogen from the alpha-carbon of dopamine. Soluble enzymes, such as rat aorta benzylamine oxidase or diamine oxidase from hog kidney and pea seedling, have been found to belong to this group. Bovine plasma amine oxidase exhibits the third type of deamination where no absolute stereospecificity is required. This enzyme catalyzes the oxidation of either (S)- or (R)-[alpha-2H1]dopamine, preferably breaking the C-H bond rather than the C-2H bond in both cases. The kinetic deuterium isotope effect during the deamination of dopamine catalyzed by the different amine oxidases varies greatly; VH/VD ranges from 1.5 to 5.5. The high magnitude of the isotope effect suggests that hydrogen abstraction may be the rate-limiting step (i.e., in reactions catalyzed by benzylamine oxidase and monoamine oxidase). When the isotope effect is low (i.e., for diamine oxidases from hog kidney or pea seedling), it is uncertain if the breaking of the bond is rate limiting.     

Specificity of arginine binding by the Tetrahymena intron

L-Arginine competitively inhibits the reaction of GTP with the Tetrahymena ribosomal self-splicing intron. In order to define this RNA binding site for arginine, Ki's have now been measured for numerous arginine-like competitive inhibitors. Detailed consideration of the Ki's suggests a tripartite binding model. The dissociation constants of the inhibitors can be consistently interpreted if the guanidino group of arginine binds in the GTP site by utilizing the H-bonds otherwise made to the N1-H and 2 NH2 of the guanine pyrimidine ring. The positive charge of the arginine guanidino group also enhances binding. A second requirement is for the precise length of the aliphatic arm connecting the guanidino with the alpha-carbon. The positive charge of the alpha-amino group is the third feature essential to effective inhibition. The negative carboxyl charge of arginine inhibits binding, and the substituents on the alpha-carbon are probably oriented, with the alpha-amino group near the phosphate backbone of the RNA. This orientation contributes strongly to the L stereoselectivity of the amino acid site on the RNA. When spaced optimally, net contribution to the free energy of binding is of the same order for the guanidino group and for the arginine alpha-carbon substituents, but the guanidino apparently contributes more to binding free energy. Taken together, these observations extend the previous binding model [Yarus, M. (1988) Science (Washington, D.C.) 240, 1751-1758]. The observed dependence of binding on universal characteristics of amino acids suggests that RNA binding sites with other amino acid specificities could exist.     

Studies on the status of free amino and carboxyl groups in cobrotoxin

The status of free amino groups in cobrotoxin was studied by stepwise modification with trinitrobenzene sulfonate. Lys-27 was selectively modified without altering the activity of cobrotoxin. However, complete loss of activity was observed when Lys-27 and Lys-47 were trinitrophenylated, suggesting that the epsilon-amino group of Lys-47 is essential for the activity of cobrotoxin. The alpha-amino group of N-terminal leucine had no correlation with activity, demonstrated by the guanidination of the lysine residues with O-methylisourea followed by trinitrophenylation of the alpha-amino group. The carboxyl groups in cobrotoxin were modified with glycine methyl ester after activation with water-soluble carbodiimide. Six out of seven free carboxyls reacted in the absence of guanidine.HCl without altering the biological activity. When the remaining carboxyl was modified in the presence of 5 M guanidine.HCl, the resulting toxin was devoid of activity. This "buried" carboxyl is essential for activity and was identified as the gamma-carboxyl group of Glu-21.     

Influence of the carboxyl terminus of luteinizing hormone-releasing hormone and bradykinin on hydrolysis by brain endo-oligopeptidases

A homogeneous preparation of endo-oligopeptidase A from rabbit brain cleaves luteinizing hormone-releasing hormone (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) at the Tyr-Gly bond only after the removal of Gly-NH2 from the COOH-terminal position of the molecule. The influence of the carboxyl terminus on hydrolysis by brain endo-oligopeptidases was studied using bradykinin as a model substrate. The substitution of the carboxyl group of bradykinin by the amide reduces by 2.5-fold the rate of Phe-Ser bond hydrolysis by endo-oligopeptidase A but has no effect on the rate of hydrolysis of the Pro-Phe bond by endo-oligopeptidase B. On the other hand, the deletion of Phe-Arg from the COOH-terminal portion of bradykinin makes the peptide resistant to hydrolysis by endo-oligopeptidase A whereas it increases by 5-fold the rate of hydrolysis of the Pro-Gly bond by endo-oligopeptidase B.