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PubMed1984

Cathepsin B-related proteases in the insulin secretory granule.

The distribution of proteases potentially reactive with peptide sequences containing pairs of basic amino acids or single basic amino acids was studied in subcellular fractions of a transplantable rat insulinoma using the affinity probes 125I-Tyr-Ala-Lys- ArgCH2Cl and 125I-Tyr-Ala-norleucine- ArgCH2Cl . Both probes labeled predominantly proteins of Mr = 39,000, 31,500, and 25,000. The Mr = 25,000 component appeared to be of lysosomal origin, while the Mr = 39,000 and 31,500 proteins were present in both the lysosomes and insulin granules. The Mr = 39,000 and 31,500 proteins were identified as precursor/product forms of the cysteine protease cathepsin B, while assays performed with fluorigenic peptide substrates suggested that the Mr = 25,000 protein was probably cathepsin L and/or H. The greater reactivity of the Mr = 39,000 form with the dibasic probe suggests that the relative proportions of the Mr = 39,000 and 31,500 forms of cathepsin B in different organelles may determine the extent to which the enzyme expresses activity as a specific (prohormone processing) endopeptidase or a more general (degradative) peptidase.

Adenoma, Islet Cell↗
PubMed1995

Proferrioxamine synthesis in Erwinia amylovora in response to precursor or hydroxylysine feeding: metabolic profiling with liquid chromatography-electrospray mass spectrometry.

Metabolic profiling by capillary liquid chromatography-electrospray mass spectrometry was used to monitor shifts in the proferrioxamine profiles of Erwinia amylovora in response to externally supplied potential proferrioxamine precursors, selected stable-isotope-labeled precursors and atypical precursors. Based on the qualitative and quantitative shifts in the proferrioxamine profiles, lysine and arginine are unambiguous, and agmatine, ornithine, diaminobutyric acid and the corresponding C3-5 diamines are highly likely precursors for proferrioxamine biosynthesis in E. amylovora. 5-Hydroxylysine (Hyl), a recently discovered growth inhibitor for E. amylovora, suppresses proferrioxamine production. The Hyl-induced growth inhibition can be reversed by basic amino acids. The basic amino acids also partly restore proferrioxamine synthesis.

Chromatography, Liquid↗
PubMed1998

High-resolution solution NMR structure of the minimal active domain of the human immunodeficiency virus type-2 nucleocapsid protein.

The retroviral nucleocapsid (NC) protein is a multifunctional protein essential for RNA genome packaging and viral infectivity. The NC protein, NCp8, of the human immunodeficiency virus type-II (HIV-2) is a 49 amino acid peptide containing two zinc fingers, of the type C-X2-C-X4-H-X4-C, connected by seven amino acid residues, called the "basic amino acid cluster." It has been shown that the N-terminal zinc finger flanked by the basic amino acid cluster is the minimal active domain for the specific binding to viral RNA and other functions. However, the structure-activity relationships of NCp8 have not been investigated in detail. In the present study, the three-dimensional structure of a 29 amino acid peptide, including the minimal active domain (NCp8-fl), was determined by two-dimensional 1H NMR spectroscopy with simulated annealing calculations. A total of 15 converged structures of NCp8-fl were obtained on the basis of 355 experimental constraints, including 343 distance constraints obtained from nuclear Overhauser effect connectivities, 12 torsion angle (phi, chi1) constraints, and four constraints for zinc binding. The root-mean-square deviation of the 15 converged structures was 0.29 +/- 0.04 A for the backbone atoms (N, C(alpha), C) and 1.27 +/- 0.13 A for all heavy atoms. Interestingly, the basic amino acid cluster itself was defined well, with a loop-like conformation in which three arginine residues in the cluster and one arginine residue in the zinc finger are located approximately in the same plane of the molecule and are exposed to the solvent. The structure-activity relationships are discussed on the basis of the comparison of this well-defined structure with those of other NC proteins.

Amino Acid Sequence↗
PubMed1972

Interactions between amino acid transport systems in Neurospora crassa.

Mutants of Neurospora crassa, selected as resistant to l-canavanine and l-thialysine, are partially deficient in the uptake of basic amino acids. Neutral amino acids completely inhibit uptake of basic amino acids, and this inhibition is dependent on the activity of a neutral amino acid permease. In contradistinction, mutants resistant to 4-methyl-dl-tryptophan are partially deficient in the uptake of neutral amino acids. Basic amino acids completely inhibit neutral amino acid uptake, and this inhibition is dependent on the activity of a basic amino acid permease. It is proposed that these specific transport systems compete with a general amino acid permease for some common element. The general permease is also regulated by ammonia, the amino acid pool, or both. The activity of the general permease can be eliminated phenotypically by a high concentration of glycerol or a high temperature. It is also shown that l-citrulline is transported by the neutral amino acid permease and by the general amino acid permease.

Amino Acids↗
PubMedDate not supplied

Synthetic peptide analogs to barnacle settlement pheromone.

Barnacle pheromone enhances the rate of settlement and metamorphosis of larvae of Balanus amphitrite Darwin. Analogs to the heterogenous pheromone peptides were sought. Settlement assays were used to assess both the pheromone and the potential analogs. The pheromone has a lower threshold of activity at a concentration of 0.2 micrograms BSA protein equivalence l-1. Treatment with carboxypeptidase eliminates biological activity. Series of dipeptides were tested to determine if dipeptides could promote settlement. Combinations of acidic, neutral, and basic amino acids in dipeptides were examined. Specific small peptides can mimic barnacle pheromone. Only peptides with a basic carboxy-terminal amino acid and either a neutral or a basic amino-terminal amino acid enhance settlement. Six peptides were shown to mimic pheromone activity at concentrations comparable to the native molecule. Some peptides were more potent than others. The most effective peptides were L-leucyl-L-arginine and L-histidyl-L-lysine which had a lower threshold of settlement enhancement of 2.0 x 10(-10) M and caused a 130% increase in settlement rate at 2.0 x 10(-8) M. Glycyl-glycyl-L-arginine, glycyl-L-histidyl-L-lysine, L-leucyl-glycyl-L-arginine and L-tyrosyl-L-arginine had thresholds between 2.0 x 10(-8) M and 2.0 x 10(-9) M. Peptide pheromone analogs should be useful in determining the nature and mechanism of barnacle pheromone receptor interactions.

Animals↗
PubMed1984

High-mobility group chromosomal proteins of wheat.

Four proteins have been extracted from purified chromatin of wheat embryos with 0.35 M NaCl. These proteins are soluble in 2% (w/v) trichloroacetic acid and thus meet the original operational requirements to be classified as "high-mobility group" (HMG) chromosomal proteins. The proteins have been characterized by one- and two-dimensional electrophoresis, amino acid analysis, and peptide mapping. Three of the proteins (HMGb, c, and d) share the mammalian HMG characteristic of being rich in both acidic and basic amino acid residues. Unlike their putative mammalian counterparts, these plant HMG proteins contain less than 7 mol % proline. The fourth wheat protein (HMGa) is rich in both proline and in basic amino acid residues. This wheat protein, however, contains only about half the proportion of acidic residues found in mammalian HMG proteins--a characteristic also found in the trout testis HMG protein, H6. Comparative peptide maps show that none of the wheat HMG proteins are degradation products of other HMG proteins or the H1 histones. The peptide maps have not, however, been useful in establishing homologies with mammalian HMG proteins. Wheat HMG proteins are released from DNase I-treated nuclei and co-isolate with micrococcal nuclease-sensitive chromatin fractions. Similar observations concerning the HMG proteins of vertebrate animals have been considered consistent with a role for these proteins as structural components of actively transcribed chromatin.

Amino Acids↗
PubMed1984

Electrophysiological evidence for acidic, basic, and neutral amino acid olfactory receptor sites in the catfish.

Electrophysiological experiments indicate that olfactory receptors of the channel catfish, Ictalurus punctatus, contain different receptor sites for the acidic (A), basic (B), and neutral amino acids; further, at least two partially interacting neutral sites exist, one for the hydrophilic neutral amino acids containing short side chains (SCN), and the second for the hydrophobic amino acids containing long side chains (LCN). The extent of cross-adaptation was determined by comparing the electro-olfactogram (EOG) responses to 20 "test" amino acids during continuous bathing of the olfactory mucosa with water only (control) to those during each of the eight "adapting" amino acid regimes. Both the adapting and test amino acids were adjusted in concentrations to provide approximately equal response magnitudes in the unadapted state. Under all eight adapting regimes, the test EOG responses were reduced from those obtained in the unadapted state, but substantial quantitative differences resulted, depending upon the molecular structure of the adapting stimulus. Analyses of the patterns of EOG responses to the test stimuli identified and characterized the respective "transduction processes," a term used to describe membrane events initiated by a particular subset of amino acid stimuli that are intricately linked to the origin of the olfactory receptor potential. Only when the stimulus compounds interact with different transduction processes are the stimuli assumed to bind to different membrane "sites." Four relatively independent L-alpha-amino acid transduction processes (and thus at least four binding sites) identified in this report include: (a) the A process for aspartic and glutamic acids; (b) the B process for arginine and lysine; (c) the SCN process for glycine, alanine, serine, glutamine, and possibly cysteine; (d) the LCN process for methionine, ethionine, valine, norvaline, leucine, norleucine, glutamic acid-gamma-methyl ester, histidine, phenylalanine, and also possibly cysteine. The specificities of these olfactory transduction processes in the catfish are similar to those for the biochemically determined receptor sites for amino acids in other species of fishes and to amino acid transport specificities in tissues of a variety of organisms.

Adaptation, Physiological↗
PubMed1988

Purification and characterization of a lethal factor in venom from the crown-of-thorns starfish (Acanthaster planci).

A lethal factor in venom of the crown-of-thorns starfish (Acanthaster planci) was obtained in an electrophoretically pure state by chromatography on CM-cellulose and Sephadex G-100. The purified lethal factor is a basic (pI 10.6) glycoprotein (carbohydrate content 3.5%). The mol. wt was estimated to be 20,000 by gel filtration or 25,000 by SDS-disc electrophoresis, suggesting that the lethal factor has no subunit structure. Despite its basicity, the lethal factor was richer in acidic amino acids than in basic amino acids. The lethal factor had an LD50 of 0.43 mg/kg (i.p. injection into mice). Hemolytic, edema-forming and capillary permeability-increasing activities, though very weak, were also exhibited by the lethal factor, while hemorrhagic and phospholipase A activities were not present.

Amino Acids↗
PubMed1994

Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein.

We have previously identified two hyaluronan (HA) binding domains in the HA receptor, RHAMM, that occur near the carboxyl-terminus of this protein. We show here that these two HA binding domains are the only HA binding regions in RHAMM, and that they contribute approximately equally to the HA binding ability of this receptor. Mutation of domain II using recombinant polypeptides of RHAMM demonstrates that K423 and R431, spaced seven amino acids apart, are critical for HA binding activity. Domain I contains two sets of two basic amino acids, each spaced seven residues apart, and mutation of these basic amino acids reduced their binding to HA--Sepharose. These results predict that two basic amino acids flanking a seven amino acid stretch [hereafter called B(X7)B] are minimally required for HA binding activity. To assess whether this motif predicts HA binding in the intact RHAMM protein, we mutated all basic amino acids in domains I and II that form part of these motifs using site-directed mutagenesis and prepared fusion protein from the mutated cDNA. The altered RHAMM protein did not bind HA, confirming that the basic amino acids and their spacing are critical for binding. A specific requirement for arginine or lysine residues was identified since mutation of K430, R431 and K432 to histidine residues abolished binding. Clustering of basic amino acids either within or at either end of the motif enhanced HA binding activity while the occurrence of acidic residues between the basic amino acids reduced binding. The B(X7)B motif, in which B is either R or K and X7 contains no acidic residues and at least one basic amino acid, was found in all HA binding proteins molecularly characterized to date. Recombinant techniques were used to generate chimeric proteins containing either the B(X7)B motifs present in CD44 or link protein, with the amino-terminus of RHAMM (amino acids 1-238) that does not bind HA. All chimeric proteins containing the motif bound HA in transblot analyses. Site-directed mutations of these motifs in CD44 sequences abolished HA binding. Collectively, these results predict that the motif of B(X7)B as a minimal binding requirement for HA in RHAMM, CD44 and link protein, and occurs in all HA binding proteins described to date.

Amino Acid Sequence↗
PubMed1999

Transport of N(G)-nitro-L-arginine across intestinal brush border membranes by Na+ -dependent and Na+-independent amino acid transporters.

PURPOSE: To clarify the transport mechanism of NG-nitro-L-arginine (L-NNA), a potent NO-synthase inhibitor, across intestinal brush border membranes (BBM). METHODS: Dog intestinal BBM vesicles were used. RESULTS: The time course of L-NNA uptake showed a Na+ -dependent overshoot phenomenon. Concentration-dependence curves of L-NNA initial uptake were saturable in the presence and absence of Na+, indicating participation of Na+ -dependent and Na+ -independent carrier-mediated transport systems. The calculated kinetic parameters of L-NNA initial uptake indicate that the former is a low-affinity high-capacity system and the latter is a high-affinity low-capacity one, similar to those in neutral amino acid transport. Neutral and basic amino acids showed cis-inhibitory and trans-stimulatory effects on L-NNA uptake in the presence or absence of Na+. N(G)-Nitro-L-arginine methyl ester, another potent NO-synthase inhibitor, also had both effects, which were smaller than with amino acids. CONCLUSIONS: The present study clearly indicates that transport of L-NNA across the intestinal BBM occurs in the same manner as neutral amino acid transport. However, it is affected by both neutral and basic amino acids in the presence or absence of Na+ differently from that across plasma membranes of nonepithelial cells, because B0,+ and b0,+ amino acid transporters function partly in L-NNA transport across intestinal BBM.

Amino Acid Transport Systems↗
PubMed2000

Effect of IGFBP-derived peptides on incorporation of(35)SO(4)into proteoglycans.

18 amino acid peptides from the C-terminal region of IGFBP-3, -5 (P3, P5), increased the incorporation of(35)SO(4)into proteoglycans in endothelial cells with greater stimulation in large vessel than microvessel cells. The homologous region of IGFBP-6 (P6) also stimulated sulfate uptake, but less potently than P3 and P5. P6 variants were synthesized with one or two amino acids changed to the basic amino acid in the equivalent position of P3. The P6 variants with one additional basic amino acid behaved similarly to P6. The P6 mutant with two altered amino acids was equipotent to P3. P3F, a scrambled version of P3 was less effective than P3. P3, P5, P6, P3F and all P6 variants all stimulated glucose uptake, which occurred only in microvessel cells. P1, P2, P4, and equimolar intact IGFBP-3 stimulated neither glucose uptake nor sulfate incorporation. Thus, C-terminal basic portions of IGFBP-3, -5 and -6 alter two specific functions of endothelial cells with sufficient differences to suggest mediation by distinct mechanisms.

Amino Acid Sequence↗
PubMed1992

Binding of DNA to large fragment of DNA polymerase I: identification of strong and weak electrostatic forces and their biological implications.

Examination of the electrostatic potential of a modeled complex, consisting of the Klenow fragment of E. coli DNA polymerase I and DNA template-primer, suggested the presence of two distinct interacting regions. The one displaying a strong electropositive potential field is generated by side chains of basic amino acid pairs and is directed towards the major groove site in DNA. The second electrostatic potential field around DNA is somewhat weaker and appears to be exerted by a pair of vicinal side chains of acidic and basic amino acids. The distribution of charges in this manner appears well suited for the binding of enzyme to the template-primer required in the enzymatic synthesis of DNA.

Binding Sites↗
PubMed1980

Characterization of rat calcitonin mRNA.

A chimeric plasmic containing cDNA complementary to rat calcitonin mRNA has been constructed. Partial sequence analysis shows that the insert contains a nucleotide sequence encoding the complete amino acid sequence of calcitonin. Two basic amino acids precede and three basic amino acids follow the hormone sequence, suggesting that calcitonin is generated by the proteolytic cleavage of a larger precursor in a manner analogous to that of other small polypeptide hormones. The COOH-terminal proline, known to be amidated in the secreted hormone, is followed by a glycine in the precursor. The cloned calcitonin DNA was used to characterize the expression of calcitonin mRNA. Cytoplasmic mRNAs from calcitonin-producing rat medullary thyroid carcinoma lines and from normal rat thyroid glands contain a single species, 1050 nucleotides long, whch hybridizes to the cloned calcitonin cDNA. The concentration of calcitonin mRNA sequences is greater in those tumors that produce larger amounts of immunoreactive calcitonin. RNAs from other endocrine tissues, including anterior and neurointermediate lobes of rat pituitary, contain no detectable calcitonin mRNA.

Amino Acid Sequence↗
PubMed1996

Viral RNA binding properties of human immunodeficiency virus type-2 (HIV-2) nucleocapsid protein-derived synthetic peptides.

The nucleocapsid (NC) protein of HIV-2 (NCp8) contains two Cys-His arrays which function as zinc finger motifs (ZFMs). In this study, we analyzed the viral RNA-binding properties of NCp8-derived synthetic peptides using ultraviolet (UV) cross-linking assay. Several synthetic peptides containing ZFM(s) interacted pH-dependently with in vitro-synthesized HIV-2 RNA. Although the peptides corresponding to the 1st and 2nd ZFMs, respectively, failed to interact with the viral RNA, the corresponding peptides flanked by basic amino acid clusters interacted tightly. Furthermore, basic amino acid residues within a cluster adjacent to ZFMs contributed to the RNA-binding of NCp8 more than Cys and His residues within the ZFM in vitro. In competitive UV cross-linking assay using non-specific RNA as a competitor, the peptides corresponding to the 1st and 2nd ZFMs flanked by basic amino acid clusters interacted specifically with viral RNA. These findings suggest that both ZFM regions of HIV-2 may be concerned with the specificity of packaging of genomic viral RNA into the virion.

Amino Acid Sequence↗
PubMed2000

Roles of the P1, P2, and P3 residues in determining inhibitory specificity of kallistatin toward human tissue kallikrein.

Kallistatin is a serpin with a unique P1 Phe, which confers an excellent inhibitory specificity toward tissue kallikrein. In this study, we investigated the P3-P2-P1 residues (residues 386-388) of human kallistatin in determining inhibitory specificity toward human tissue kallikrein by site-directed mutagenesis and molecular modeling. Human kallistatin mutants with 19 different amino acid substitutions at each P1, P2, or P3 residue were created and purified to compare their kallikrein binding activity. Complex formation assay showed that P1 Arg, P1 Phe (wild type), P1 Lys, P1 Tyr, P1 Met, and P1 Leu display significant binding activity with tissue kallikrein among the P1 variants. Kinetic analysis showed the inhibitory activities of the P1 mutants toward tissue kallikrein in the order of P1 Arg > P1 Phe > P1 Lys >/= P1 Tyr > P1 Leu >/= P1 Met. P1 Phe displays a better selectivity for human tissue kallikrein than P1 Arg, since P1 Arg also inhibits several other serine proteinases. Heparin distinguishes the inhibitory specificity of kallistatin toward kallikrein versus chymotrypsin. For the P2 and P3 variants, the mutants with hydrophobic and bulky amino acids at P2 and basic amino acids at P3 display better binding activity with tissue kallikrein. The inhibitory activities of these mutants toward tissue kallikrein are in the order of P2 Phe (wild type) > P2 Leu > P2 Trp > P2 Met and P3 Arg > P3 Lys (wild type). Molecular modeling of the reactive center loop of kallistatin bound to the reactive crevice of tissue kallikrein indicated that the P2 residue required a long and bulky hydrophobic side chain to reach and fill the hydrophobic S2 cleft generated by Tyr(99) and Trp(219) of tissue kallikrein. Basic amino acids at P3 could stabilize complex formation by forming electrostatic interaction with Asp(98J) and hydrogen bond with Gln(174) of tissue kallikrein. Our results indicate that tissue kallikrein is a specific target proteinase for kallistatin.

Amino Acid Substitution↗
PubMed1987

Opioid peptide biosynthesis: enzymatic selectivity and regulatory mechanisms.

Certain general principles determine the biosynthesis of most biologically active peptides, including the opioid peptides, from large protein precursors. In almost all instances, the active peptide is embedded in the precursor flanked on both sides by pairs of basic amino acids. The first step in processing involves a trypsinlike enzyme, cleaving to the carboxyl terminus of basic amino acids, and leaving the active peptide with a basic amino acid on the carboxyl terminus. A carboxy-peptidase peptidase B-like enzyme then removes the remaining basic amino acid. It has been unclear whether any endopeptidases with trypsinlike activity are selective for one or another basic amino acid. Recently a soluble endopeptidase has been identified that can cleave to both the carboxyl and amino termini of basic amino acids. Enkephalin convertase (carboxypeptidase E, H) (EC 3.4.17.10) has considerable selectivity, and appears to be physiologically associated with the biosynthesis of enkephalin as well as a limited number of other neuropeptides. The turnover of opioid peptides and other neuropeptides is most effectively ascertained by measuring levels of mRNA either biochemically or by in situ hybridization. Striking dynamic alterations include a pronounced increase in levels of proenkephalin mRNA in the corpus striatum after blockade of dopamine receptors, but changes in opioid peptide mRNA after opiate addiction are less clear.

Animals↗
PubMed1988

Acetylcholinesterase converts Met5-enkephalin-containing peptides to Met5-enkephalin.

Acetylcholinesterase (AChE; E.C. 3.1.1.7) was incubated with a number of enkephalin-containing neuropeptides found in the bovine adrenal medulla. Met5-enkephalin and Leu5-enkephalin were the most stable of the peptides studied, while precursors of Met5-enkephalin were converted to Met5-enkephalin. AChE is therefore capable of limited peptidase activity on Met5-enkephalin precursors. The enzyme hydrolysed the Met5-enkephalin precursor BAM-12P on the C-terminal side of the pair of basic amino acid residues, and cleaved basic amino acids from the carboxy-terminal of Met5-enkephalin-Arg6 and Met5-enkephalin-Arg6-Arg7. These results indicate that AChE, acting alone, is capable of the same pattern of enkephalin processing as that observed in the adrenal medulla.

Acetylcholinesterase↗
PubMed2002

The L-3,4-dihydroxyphenylalanine transporter in human and rat epithelial intestinal cells is a type 2 hetero amino acid exchanger.

Information on the intestinal transport of L-3,4-dihydroxyphenylalanine (L-DOPA) is scarce. We present here the functional characteristics and regulation of the apical inward L-DOPA transport in two intestinal epithelial cell lines (human Caco-2 and rat IEC-6). The inward transfer of L-DOPA and L-leucine was promoted through an energy-driven system but with different sensitivity to extracellular Na(+) concentration: a minor component of L-leucine uptake (approximately 25%) was found to require extracellular Na(+) in comparison with L-DOPA transport which was Na(+)-independent. L-DOPA and L-leucine uptake was insensitive to N-(methylamino)-isobutyric acid, but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH). L- and D-neutral amino acids, but not acidic and basic amino acids, markedly inhibited L-DOPA and [(14)C]L-leucine accumulation in both cell lines. The [(14)C]L-DOPA and [14C]L-leucine outward were markedly increased by L-leucine and BCH present in extracellular medium, but not by L-arginine. In both cell lines, L-DOPA transport was stimulated by acidic pH in comparison with [(14)C]L-leucine inward which was pH-independent. In conclusion, it is likely that system B(0) might be responsible for the Na(+)-dependent uptake of L-leucine in Caco-2 and IEC-6 cells, whereas sodium-independent uptake of L-leucine and L-DOPA may include system type 1 and type 2 L-amino acid transporter (LAT1 and LAT2), the activation of which results in trans-stimulation of substrates outward transfer.

Amino Acid Transport Systems↗