Detailed characterization of an anti-factor IX monoclonal antibody that neutralizes the prolonged ox brain prothrombin time of hemophilia B(M) by synthetic peptides

In a previous study, we prepared a monoclonal antibody (MoAb) to coagulation factor IX (FIX), designated 65-10, which interfered with the activation of FIX by the activated factor XI/Ca(2+) and neutralized the prolonged ox brain prothrombin time of hemophilia B(M) [11,12]. The location of the epitope on the FIX for 65-10 MoAb is (168) Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val(182) [21]. In this paper, we studied in more detail an epitope on FIX using the systematic substitution of different amino acids at each residue of the epitope peptides and the influence of the epitope peptide on the prolonged ox brain prothrombin time of the hemophilia B(M) plasma of 65-10 MoAb. In the replacement set of amino acids, peptides showing low or no reactivity to 65-10 were (175)Phe --> Asp, Glu, Gly, Lys, Arg, Thr, Val, (176)Asn --> Asp, Glu, Phe, Ile, Lys, Leu, Pro, Val, Tyr, (177)Asp --> Cys, Glu, Phe, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, and (178) Phe --> Pro. These results imply that a hydrophobic molecule of (175) Phe, a hydrophilic molecule of (176)Asn, and a negative charge molecule of (177)Asp were important to the epitope. The 65-10 MoAb antibody neutralized the prolonged ox brain prothrombin time of hemophilia B(M) Nagoya 2 ((180)Arg -->Trp) and Kashihara ((181)Val --> Phe) as well as B(M) Kiryu ((313)Val --> Asp) and Niigata ((390)Ala --> Val). This reaction was inhibited by preincubation with a (168) Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val(182) peptide conjugated with bovine serum albumin (BSA). 65-10 MoAb that has been useful in detailing epitopes will be useful for qualitative analysis of hemophilia B(M).     

X-ray diffraction studies of enkephalins. Crystal structure of [(4''-bromo) Phe4,Leu5]enkephalin.

In order to investigate the structure-activity relationship of [Leu5]- and [Met5]enkephalins, [(4'-bromo)Phe4, Leu5]-, [(4'-bromo)Phe4, Met5]- and [Met5] enkephalins were synthesized and crystallized. The crystal structure of [(4'-bromo) Phe4, Leu5]- enkephalin was determined by X-ray diffraction method using the heavy atom method and refined to R = 0.092 by the least-squares method. The molecule in this crystal took essentially the same type I' beta-turn conformation found in [Leu5]enkephalin [Smith & Griffin (1978) Science 199, 1214-1216). On the other hand, the preliminary three-dimensional Patterson analyses showed that the most probable conformations of [(4'-bromo)Phe4,Met5]- and [Met5]enkephalins are both the dimeric extended forms. Based on these insights, the biologically active conformation of enkephalin was discussed in relation to the mu- and delta-receptors.     

Subsite specificity of memapsin 2 (beta-secretase): implications for inhibitor design

Memapsin 2 is the protease known as beta-secretase whose action on beta-amyloid precursor protein leads to the production of the beta-amyloid (Abeta) peptide. Since the accumulation of Abeta in the brain is a key event in the pathogenesis of Alzheimer's disease, memapsin 2 is an important target for the design of inhibitory drugs. Here we describe the residue preference for the subsites of memapsin 2. The relative k(cat)/K(M) values of residues in each of the eight subsites were determined by the relative initial cleavage rates of substrate mixtures as quantified by MALDI-TOF mass spectrometry. We found that each subsite can accommodate multiple residues. The S(1) subsite is the most stringent, preferring residues in the order of Leu > Phe > Met > Tyr. The preferences of other subsites are the following: S(2), Asp > Asn > Met; S(3), Ile > Val > Leu; S(4), Glu > Gln > Asp; S(1)', Met > Glu > Gln > Ala; S(2)', Val > Ile > Ala; S(3)', Leu > Trp > Ala; S(4)', Asp > Glu > Trp. In general, S subsites are more specific than the S' subsites. A peptide comprising the eight most favored residues (Glu-Ile-Asp-Leu-Met-Val-Leu-Asp) was found to be hydrolyzed with the highest k(cat)/K(M) value so far observed for memapsin 2. Residue preferences at four subsites were also studied by binding of memapsin 2 to a combinatorial inhibitor library. From 10 tight binding inhibitors, the consensus preferences were as follows: S(2), Asp and Glu; S(3), Leu and Ile; S(2)', Val; and S(3)', Glu and Gln. An inhibitor, OM00-3, Glu-Leu-Asp-LeuAla-Val-Glu-Phe (where the asterisk represents the hydroxyethylene tansition-state isostere), designed from the consensus residues, was found to be the most potent inhibitor of memapsin 2 so far reported (K(i) of 3.1 x 10(-10) M). A molecular model of OM00-3 binding to memapsin 2 revealed critical improvement of the interactions between inhibitor side chains with enzyme over a previous inhibitor, OM99-2 [Ghosh, A. K., et al. (2000) J. Am. Chem. Soc. 14, 3522-3523].     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Purification and amino acid sequence of vasoactive intestinal peptide, peptide histidine isoleucinamide (1-27) and secretin from the small intestine of guinea pig

The neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucinamide (1-27) (PHI) and the hormone secretin were purified from the small intestine of guinea pig, being detected by radioimmunoassay and radioreceptor assay throughout six to seven chromatographic steps. After elution on a reverse-phase C18 column, the three peptides were separated on a Fractogel column. After cation-exchange chromatography of each peptide on Mono S, the final steps were performed using a reverse-phase RP8-e column. Guinea pig PHI differed from porcine PHI in having Tyr and Arg residues instead of Phe and Lys in, respectively, position 10 and 20. We confirmed the original sequence of guinea pig VIP previously documented (with Leu5, Thr9, Met19 and Val26). We also established the similarity of the primary structure of guinea pig secretin with that of porcine and bovine.     

Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph

The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr 30,000-32,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala(2)-Pro-Phe-pNa with a Km-1.36 x 10(-4) M and Kcat-24 s(-1), and N-Suc-Ala(2)-Pro-Leu-pNa with Km-6.65 x 10(-4) M and Kcat-11 s(-1). The specificity index k(cat)/K(m) for the tested substrates was calculated to be 176,340 s(-1) M(-1) and 17,030 s(-1) M(-1), respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide bonds: Phe(1)-Val(2), Leu(15)-Tyr(16), and Gly(23)-Phe(24). The proteinase appeared to be sensitive to the specific synthetic inhibitors of the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by AMCI-1 (with an association constant of 3 x 10(10) M(-1)), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus is a subtilisin-like serine proteinase.     

Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonists with N-terminal D-Trp or D-Tpi.

In an attempt to produce more powerful (effective) bombesin/GRP receptor antagonists, the D forms of Trp or Trp analog (Tpi) were introduced at position 6 in two pseudononapeptides, Leu13 psi (CH2NH)Leu14-bombesin(6-14) and Leu13 psi(CH2NH)Phe14-bombesin (6-14). These antagonists were tested for their ability to inhibit basal and gastrin releasing peptide (GRP) (14-27)-induced amylase release from rat pancreatic acini in a superfusion assay. They were also assessed for the inhibition of 125I-Tyr4-bombesin binding to Swiss 3T3 and small cell lung carcinoma cell line H-345 and the mitogenic response of Swiss 3T3 cells induced by GRP(14-27). The peptides, when given alone, did not stimulate amylase secretion, but were able to inhibit gastrin releasing peptide (14-27)-induced amylase release. All of the antagonists showed strong binding affinities for Swiss 3T3 and H-345 cells and suppressed the GRP(14-27)-induced increase of [3H]thymidine incorporation into DNA of Swiss 3T3 cells at nanomolar concentrations. Antagonist D-Tpi6,Leu13 psi (CH2NH)Leu14-bombesin (6-14)(RC-3095) was slightly more potent in these assays than D-Trp6,Leu13 psi (CH2NH)Leu14-bombesin (6-14)(RC-3125). Nevertheless, D-Trp6,Leu13 psi (CH2NH)Phe14-bombesin (6-14) showed the highest binding affinity for Swiss 3T3 and H345 cells and it was the most potent inhibitor of GRP(14-27)-induced amylase secretion. This antagonist RC-3420 was particularly effective in inhibiting the growth of Swiss 3T3 cells, exhibiting an IC50 value less than 1 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Introduction of a pi-pi interaction at the active site of a cupredoxin: characterization of the Met16Phe Pseudoazurin mutant

The Met16Phe mutant of the type 1 copper protein pseudoazurin (PACu), in which a phenyl ring is introduced close to the imidazole moiety of the His81 ligand, has been characterized. NMR studies indicate that the introduced phenyl ring is parallel to the imidazole group of His81. The mutation has a subtle effect on the position of the two S(Cys)-->Cu(II) ligand-to-metal charge transfer bands in the visible spectrum of PACu(II) and a more significant influence on their intensities resulting in a A(459)/A(598) ratio of 0.31 for Met16Phe as compared to a A(453)/A(594) ratio of 0.43 for wild-type PACu(II) at pH 8. The electron paramagnetic resonance spectrum of the Met16Phe variant is more axial than that of the wild-type protein, and the resonance Raman spectrum of the mutant exhibits subtle differences. A C(gamma)H proton of Met86 exhibits a much smaller hyperfine shift in the paramagnetic (1)H NMR spectrum of Met16Phe PACu(II) as compared to its position in the wild-type protein, which indicates a weaker axial Cu-S(Met86) interaction in the mutant. The Met16Phe mutation results in an approximately 60 mV increase in the reduction potential of PACu. The pK(a) value of the ligand His81 decreases from 4.9 in wild-type PACu(I) to 4.5 in Met16Phe PACu(I) indicating that the pi-pi contact with Phe16 stabilizes the Cu-N(His81) interaction. The Met16Phe variant of PACu has a self-exchange rate constant at pH 7.6 (25 degrees C) of 9.8 x 10(3) M(-)(1) s(-)(1) as compared to the considerably smaller value of 3.7 x 10(3) M(-)(1) s(-)(1) for the wild-type protein under identical conditions. The enhanced electron transfer reactivity of Met16Phe PACu is a consequence of a lower reorganization energy due to additional active site rigidity caused by the pi-pi interaction between His81 and the introduced phenyl ring.     

Four hydrophobic amino acids of the factor VIII C2 domain are constituents of both the membrane-binding and von Willebrand factor-binding motifs.

Factor VIII binds to phospholipid membranes and to von Willebrand factor (vWf) via its second C domain, which has lectin homology. The crystal structure of the C2 domain has prompted a model in which membrane binding is mediated by two hydrophobic spikes, each composed of a pair of residues displayed on a beta-hairpin turn, and also by net positive charge and specific interactions with phospho-l-serine. To test this model, we prepared 16 factor VIII mutants in which single or multiple amino acids were changed to alanine. Mutants at Arg(2215), Arg(2220), Lys(2227), Lys(2249), Gln(2213), Asn(2217), and Phe(2196)/Thr(2197) had specific activities that were >70% of the wild type. Mutants at Arg(2209), Lys(2227), Trp(2313), and Arg(2320) were degraded within the cell. Hydrophobic spike mutants at Met(2199)/Phe(2200), Leu(2251)/Leu(2252), and Met(2199)/Phe(2200)/Leu(2251)/Leu(2252) (4-Ala) exhibited 43, 59, and 91% reduction in specific activity in the activated partial thromboplastin time assay. In a phospholipid-limiting factor Xa activation assay, these mutants had a 65, 85, and 96% reduction in specific activity. Equilibrium binding of fluorescent, sonicated phospholipid vesicles to mutants immobilized on Superose beads was measured by flow cytometry. The affinities for phospholipid were reduced approximately 20-, 30-, and >35-fold for 2199/2200, 2251/2252, and 4-Ala, respectively. A dimeric form of mature vWf bound to immobilized factor VIII and the same mutants, but the affinities of the mutants were reduced approximately 5-, 10-, and >20-fold, respectively. In a competition, solution phase enzyme-linked immunosorbent assay, plasma vWf bound factor VIII and the same mutants with the affinities for the mutants reduced >5-, >5-, and >50-fold, respectively. We conclude that the two hydrophobic spikes are constituents of both the phospholipid-binding and vWf-binding motifs. In plasma, vWf apparently binds the inherently sticky membrane-binding motif, preventing nonspecific interactions.     

A Decrease of the Respiratory Burst in Neutrophils after Exposure to Weak Combined Magnetic Fields of a Certain Duration

Biophysics - Abstract—A significant decrease in the respiratory burst activity of a neutrophil suspension has been recorded in response to N-formyl–Met–Leu–Phe peptide...     

Structural determinants of HscA peptide-binding specificity

The Hsp70-class molecular chaperone HscA interacts specifically with a conserved (99)LPPVK(103) motif of the iron-sulfur cluster scaffold protein IscU. We used a cellulose-bound peptide array to perform single-site saturation substitution of peptide residues corresponding to Glu(98)-Ile(104) of IscU to determine positional amino acid requirements for recognition by HscA. Two mutant chaperone forms, HscA(F426A) with a DnaK-like arch structure and HscA(M433V) with a DnaK-like substrate-binding pocket, were also studied. Wild-type HscA and HscA(F426A) exhibited a strict preference for proline in the central peptide position (ELPPVKI), whereas HscA(M433V) bound a peptide containing a Pro-->Leu substitution at this location (ELPLVKI). Contributions of Phe(426) and Met(433) to HscA peptide specificity were further tested in solution using a fluorescence-based peptide-binding assay. Bimane-labeled HscA and HscA(F426A) bound ELPPVKI peptides with higher affinity than leucine-substituted peptides, whereas HscA(M433V) favored binding of ELPLVKI peptides. Fluorescence-binding studies were also carried out with derivatives of the peptide NRLLLTG, a model substrate for DnaK. HscA and HscA(F426A) bound NRLLLTG peptides weakly, whereas HscA(M433V) bound NRLLLTG peptides with higher affinity than IscU-derived peptides ELPPVKI and ELPLVKI. These results suggest that the specificity of HscA for the LPPVK recognition sequence is determined in part by steric obstruction of the hydrophobic binding pocket by Met(433) and that substitution with the Val(433) sidechain imparts a broader, more DnaK-like, substrate recognition pattern.     

Binding sites for alpha-bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the alpha-subunit of the nicotinic acetylcholine receptor.

The binding of the competitive antagonist alpha-bungarotoxin (alpha-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the alpha-subunit of the Torpedo acetylcholine receptor has been characterized. 125I-alpha-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by alpha-Btx (IC50 = 5.0 x 10(-8) M), d-tubocurarine (IC50 = 5.9 X 10(-5)M), and NaCl (IC50 = 7.9 x 10(-2)M). In the presence of 0.02% sodium dodecyl sulfate, 125I-alpha-Btx bound to the 56-residue peptide with a KD of 3.5 nM, as determined by equilibrium saturation binding studies. Because alpha-Btx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptide comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, [3H]PCP was bound to the 172-227 peptide. [3H]PCP binding was inhibited by chlorpromazine (IC50 = 6.3 x 10(-5)M), tetracaine (IC50 = 4.2 x 10(-6)M), and dibucaine (IC50 = 2.7 x 10(-4)M). Equilibrium saturation binding studies in the presence of 0.02% sodium dodecyl sulfate showed that [3H]PCP bound at two sites, a major site of high affinity with an apparent KD of 0.4 microM and a minor low-affinity site with an apparent KD of 4.6 microM. High -affinity binding occurred at a single site on peptide 205-227 (KD = 0.27 microM) and was competed by chlorpromazine but not by alpha-Btx.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of Bacillus stearothermophilus Leucine Aminopeptidase II by Hydrogen Peroxide and Site-Directed Mutagenesis of Methionine Residues on the Enzyme

Leucine aminopeptidases (LAPs) are exopeptidases that remove the N-terminal L-leucine from peptide substrates. Oxidative stability assay showed that the recombinant Bacillus stearothermophilus LAP II (rLAPII) was sensitive to oxidative damage by hydrogen peroxide at the elevated temperature. The H2O2-treated enzyme experienced obvious changes in the secondary structure when the oxidant concentration increased to 300 mM. To investigate the role of methionine residues on the oxidative inactivation, each of the five methionine residues in the rLAPII was replaced with leucine by site-directed mutagenesis. The mutant enzymes with an apparent Mr of approximately 44.5 kDa were overexpressed in Escherichia coli and were purified to homogeneity by nickel-chelate chromatography. The specific activities for Met82Leu, Met88Leu, Met254Leu, and Met382Leu were similar to that of the wild-type enzyme, whereas a reduced activity was observed in Met136Leu. The 50% decrease in the catalytic efficiency (kcat/Km) for Met136Leu was caused by 47% decrease in kcat value. As compared with the wild-type enzyme, all mutant proteins were more sensitive to the oxidant, implying that the methionine residues of B. stearothermophilus LAP II are important for the protection of the enzyme from oxidative inactivation.     

Primary structure and function of the catecholamine release inhibitory peptide catestatin (chromogranin A(344-364)): identification of amino acid residues crucial for activity

The novel chromogranin A fragment catestatin (bovine chromogranin A(344-364); RSMRLSFRARGYGFRGPGLQL) is a potent inhibitor of catecholamine release (IC50, approximately 0.2-0.3 microM) by acting as a nicotinic cholinergic antagonist. To define the minimal active region within catestatin, we tested the potencies of synthetic serial three-residue deletion (amino-terminal, carboxyl-terminal, or bidirectional) fragments to inhibit nicotine-stimulated catecholamine secretion from PC12 pheochromocytoma cells. The results revealed that a completely active core sequence of catestatin was constituted by chromogranin A(344-364). Nicotinic cationic signal transduction was affected by catestatin fragments in a manner similar to that for secretion (confirming the functional importance of the amino-terminus). To identify crucial residues within the active core, we tested serial single amino acid truncations or single residue substitutions by alanine on nicotine-induced catecholamine secretion and desensitization. Nicotinic inhibition by the active catestatin core was diminished by even single amino acid deletions. Selective alanine substitution mutagenesis of the active core revealed important roles for Met346, Leu348, Phe350, Arg351, Arg353, Gly354, Tyr355, Phe357, and Arg358 on catecholamine secretion, whereas crucial roles to inhibit desensitization of catecholamine release were noted for Arg344, Met346, Leu348, Ser349, Phe350, Arg353, Gly354, Tyr355, Gly356, and Arg358. We conclude that a small, 15-amino acid core of catestatin (chromogranin A(344-364)) is sufficient to exert the peptide's typical inhibitory effects on nicotinic cholinergic-stimulated catecholamine secretion, signal transduction, and desensitization. These studies refine the biologically active domains of catestatin and suggest that the pharmacophores for inhibition of nicotinic secretion and desensitization may not be identical.     

In vitro pituitary hormone releasing activity of 40 residue human pancreatic tumor growth hormone releasing factor

The hypophysiotropic activities of a synthetic human pancreatic growth hormone releasing factor (hpGRF) with 40 residues was examined in vitro using rat pituitary halves. At concentrations from 10(-10) M to 10(-7) M the peptide stimulated GH release in a dose-dependent manner with the ED50 being 1.2 x 10(-9) M. The concentration of 10(-10) M hpGRF is comparable to the basal hypophyseal portal blood levels of other known hypothalamic hypophysiotropic hormones. However, GH release was enhanced three-fold by concentration as low as 10(-12) M, though no dose-response relationship was observed up to 10(-10) M. Thus, this peptide not only stimulates the release of GH in a dose-dependent manner, but at lower concentrations also maintains elevated GH levels. The release of ACTH, beta-endorphin, LH, and FSH was not affected by hpGRF at any of the concentrations tested. At hpGRF concentrations less than 10(-7) M, the release of TSH and PRL were unaffected. However, at 10(-6) M, TSH release was enhanced about 2.5 fold and prolactin release was elevated slightly.     

Designing of substrates and inhibitors of bovine alpha-chymotrypsin with synthetic phenylalanine analogues in position P(1)

The primary specificity residue of a substrate or an inhibitor, called the P(1) residue, is responsible for the proper recognition by the cognate enzyme. This residue enters the S(1) pocket of the enzyme and establishes contacts (up to 50%) inside the proteinase substrate cavity, strongly affecting its specificity. To analyze the influence on bovine alpha-chymotrypsin substrate activity, aromatic non-proteinogenic amino acid residues in position P(1) with the sequence Ac-Phe-Ala-Thr-X-Anb(5,2)-NH(2) were introduced: L-pyridyl alanine (Pal), 4-nitrophenylalanine - Phe(p-NO(2)), 4-aminophenylalanine - Phe(p-NH(2)), 4-carboxyphenylalanine Phe(p-COOH), 4-guanidine phenylalanine - Phe(p-guanidine), 4-methyloxycarbonyl-phenylalanine - Phe(p-COOMe), 4-cyanophenylalanine - Phe(p-CN), Phe, Tyr. The effect of the additional substituent at the phenyl ring of the Phe residue was investigated. All peptides contained an amide of 5-amino-2-nitrobenzoic acid, which served as a chromophore. Kinetic parameters (k(cat), K(M) and k(cat)/K(M)) of the peptides synthesized with bovine alpha-chymotrypsin were determined. The highest value of the specificity constant k(cat)/K(M), reaching 6.0 x 10(5) [M(-1)xs(-1)], was obtained for Ac-Phe-Ala-Thr-Phe(p-NO(2))-Anb(5,2)-NH(2). The replacement of the acetyl group with benzyloxycarbonyl moiety yielded a substrate with the value of k(cat) more than three times higher. Peptide aldehydes were synthesized with selected residues (Phe, Pal, Tyr, Phe(p-NO(2)) in position P(1) and potent chymotrypsin inhibitors were obtained. The dissociation constant (K(i)) with the experimental enzyme determined for the most active peptide, Tos-Phe-Ala-Thr-Phe(p-NO(2))-CHO, amounted to 1.12 x 10(-8) M.     

Electrically-Induced Release of Opioid Peptides from the Guinea-Pig Myenteric Plexus Preparation

Abstract

Preparations of guinea-pig myenteric plexus-longitudinal muscle suspended in Krebs solution were stimulated electrically in the presence of cycloheximide and tetraethylammonium. The amounts of eleven endogenous opioid peptides released into the perifusing Krebs solution were determined and correlated with the decrease in the tissue contents induced by stimulation. For pro-enkephalin fragments the ratio of release to reduction in tissue contents was 29 to 43% for [Met]enkephalin, [Leu]enkephalin, [Met]enkephalyl-RF and [Met]enkephalyl-RGL. With [Met]enkephalyl-RRV-NH₂ (BAM-8) the ratio was higher by 50% or more. However, it is of interest that there was no release of the probable precursor [Met]enkephalyl-RRVGRPEWWMDYQ(BAM-18). In this context it may be important that BAM-8 is the only endogenous opioid peptide having-NH₂ at the C-terminal. The low tissue levels of pro-dynorphin derived peptide have made estimation of release unreliable.     

The alterative effect of isobutylmethylxanthine in hypofunctional human neutrophil chemotaxis

The effects of isobutylmethylxanthine on the locomotion of chemotactically normofunctional and hypofunctional human neutrophils were compared. The hypofunctional cells were obtained from the peripheral blood of selected subjects with juvenile periodontitis. The chemically oriented migration (i.e. chemotaxis) of these cells was less than 50% of normal. Isobutylmethylxanthine caused a doubling of the CHO.Met.Leu.Phe.OH-induced chemotactic responsiveness of hypofunctional cells, while no effect of the drug was detected with respect to random migration. Isobutylmethylxanthine had no effect on chemotaxis or random migration in the normofunctional cell populations. Since isobutylmethylxanthine inhibits cAMP catabolism, these data suggest that the cyclic nucleotide plays a role in intracellular transduction mechanisms which affect chemotaxis.     

Stress Impacts the Regulation Neuropeptides in the Rat Hippocampus and Prefrontal Cortex

Adverse life experiences increase the lifetime risk to several stress‐related psychopathologies, such as anxiety or depressive‐like symptoms following stress in adulthood. However, the neurochemical modulations triggered by stress have not been fully characterized. Neuropeptides play an important role as signaling molecules that contribute to physiological regulation and have been linked to neurological and psychiatric diseases. However, little is known about the influence of stress on neuropeptide regulation in the brain. Here, we have performed an exploratory study of how neuropeptide expression at adulthood is modulated by experiencing a period of multiple stressful experiences. We have targeted hippocampus and prefrontal cortex (PFC) brain areas, which have previously been shown to be modulated by stressors, employing a targeted liquid chromatography‐mass spectrometry (LC‐MS) based approach that permits broad peptide coverage with high sensitivity. We found that in the hippocampus, Met‐enkephalin, Met‐enkephalin‐Arg‐Phe, and Met‐enkephalin‐Arg‐Gly‐Leu were upregulated, while Leu‐enkephalin and Little SAAS were downregulated after stress. In the PFC area, Met‐enkephalin‐Arg‐Phe, Met‐enkephalin‐Arg‐Gly‐Leu, peptide PHI‐27, somatostatin‐28 (AA1‐12), and Little SAAS were all downregulated. This systematic evaluation of neuropeptide alterations in the hippocampus and PFC suggests that stressors impact neuropeptides and that neuropeptide regulation is brain‐area specific. These findings suggest several potential peptide candidates, which warrant further investigations in terms of correlation with depression‐associated behaviors.     

Structure-activity relationship of rubiscolins as delta opioid peptides

To study the structure-activity relationship of rubiscolins (YPLDLF and YPLDL), delta opioid peptides derived from the spinach Rubisco, we substituted the amino acid residues and evaluated their activities by mouse vas deferens (MVD) and guinea pig ileum (GPI) assays as well as receptor affinity. Replacement of Leu(3) with Ile and Met in rubiscolin-6 potentiated the delta opioid activity by about four times in MVD assay. Asp(4) cannot be replaced by Ala, Glu or His. The original Leu(5) was optimal, while substitution of Phe(6) with Val potentiated its delta opioid activity by more than 10 times. The most potent derivative we obtained was YPMDLV, which was nearly 20 times more potent than rubiscolin-6 in MVD assay. The derivatives thus obtained showed higher delta receptor affinity and more potent antinociceptive activity than rubiscolins.