Search

Free fatty acids were determined by high performance liquid chromatography after derivatization with monodansyl Cadaverine (MDC) at room temperature for 15min in the presence of diethyl phosphorocyanidate as a coupling reagent. The fluorescence properties (λex340nm, λem 518nm) of MDC-arachidic acid (C20:0) were identical to those of dansyl alanine. The MDC derivatives of saturated and unsaturated fatty acids were completely separated within 50min by gradient elution on a TSK-gel 80TM ODS column. When fluorometrically detected, the detection limits (S/N=3) of fatty acid were below 100fmol. The calibration curves passed through the origin and gave good linearity in the range from 5 to 200pmol. The method was successfully applied to the determination of fatty acids in the rabbit plasma. This method would be quite useful for the analysis of various biological samples for thermolabile carboxylic acids, since it requires no heating procedure.

Thr6-bradykinin and three kinds of its analogous peptides have been isolated from the methanol extract of the fresh skin of Rana rugosa. The longest bradykinin analogue, Thr6-bradykinyl-Ile-Ala-Pro-Glu-Ile-Val, was named ranakinin-R. Serotonin was also identified in the extract. The new peptide which contracts the rat uterus, guinea pig gall bladder and is hypertensive, was isolated and suggested to be the ranatensin or bombesin analogue by these pharmacological properties and by partial sequence analysis of the peptide.

A revised HPLC condition is presented for the separation of DNTH-aminoacids, together with the enhanced fluorescence detection of DNTH-amino acids. The application of the established method to the sequencing of the human growth factor releasing hormone (GRF) is also described

Goosefish angiotensin produced by incubation of the kidney extract with homologous plasma, was purified. The structure was identified as Asn1-Val5-His9-angiotensin (I) decapeptide by amino acid composition and fluorescent peptide mapping technique.

Crude bullfrog angiotensin was prepared by modified Boucher's procedures. The kidney extract was incubated in vitro with homologous plasma. Crude angiotension, 15 μg equivalent to [Asp 1 , Ile 5 ]angiotensin II in rat vasopressor assay, was purified further by three steps by SEP-PAK C 18 cartridge, SP-Sephadex column chromatography, and high-performance liquid chromatography on Finepak SIL C 18 column. Amino acid analysis was done on 64 ng of the purified material after acid hydrolysis. The fluorescent peptide mapping technique was used to confirm the amino acid sequence. It is proposed that bullfrog angiotensin is [Asp 1 , Val 5 , Asn 9 ]angiotensin I.

Biologically active peptides of the tachykinin and bombesin families occurring in the skin of the Australian leptodectylid frog Uperoleia rugosa were isolated in pure forms and their structures were determined. Uperolein-like peptide 1 was identified, as expected, as authentic uperolein; uperolein-like peptide 2 was found to have the sequence Pyr-Ala-Asp-Pro-Lys-Thr-Phe-Tyr-Gly-Leu-Met-NH and may be considered a Lys, Thr-physalaemin. Bombesin-like peptide 1, in its turn, was identified as Glu(OEt)-litorin, and bombesin-like peptide 2 as authentic litorin. Uperolein and litorin had previously been identified in Uperoleia rugosa and Litoria aurea, respectively; the other two peptides are new. Glu(OEt)-litorin and Lys. Thr-physalaemin appear to have biological activities very similar to those of Glu(OMe)-litorin and physalaemin (or uperolein), respectively.

In addition to wasp kinins, the wasp venom contains a series of hydrophobic peptides, mastoparans and chemotactic peptides as major peptidergic components. The first major component in the venom is mastoparam. The peptides in the mastoparan family are tetradecapeptide amides which cause degranulation of the mast cells to release histamine from the cells, and act on the adrenal chromaffin cells to release catecholamines and adenylic acids. Some mastoparans cause hemolysis and serotonin release from the platelets. The new cytotrophic peptides as the second major components are tridecapeptide amides possessing chemotactic activity for polymorphonuclear leucocytes and monocytes. Some of the peptides in this family also cause histamine release from the mast cells. Mastoparan takes a random coil structure in aqueous solution but changes its conformation to α-helix in methanolic solution or in the presence of lysophosphatidyl choline. This fact is confirmed also by the transferred nuclear overhauser effect by NMR analysis. The similar phenomenon was observed in the family of chemotactic peptides. The helical conformation of these peptides are amphipathic structure in which all of side chains of the hydrophobic amino acids are located on one side of the axis, and those of the basic or the hydrophilic amino acid residues are on an opposite side. Mastoparan enhances the membrane conductivity of the lipid bilayer when the peptide is investigated by the black lipid membrane experiment. This indicates that the peptide may be assembled in the membrane by changing its conformation and. for some reason, enhances the ion transfer through the membrane. These properties of the peptide may reveal various activities on the cell membrane.

Active-site peptides of acetyl transferase, condensing enzyme and acyl carrier protein in the neighborhood of the prosthetic group, 4'-phosphopantetheine, of Cephalosporium caerulens fatty acid synthetase were investigated. The enzyme was reacted with [14C]acetyl-CoA or [14C]iodoacetamide. 14C-Labeled enzyme was digested with pepsin, trypsin or both. 14C-Labeled peptides were isolated by several purification procedures. The amino acid sequence of the active site of condensing enzyme was determined to be Tyr-Gln-Val-Glu-Ser-Cys-Pro-Ile-Leu-Glu-Gly-Lys and that of acetyl transferase was Phe-Ser-Gly-Ala-Thr-Gly-His-Ser-Gln-Gly. The amino acid composition around the 4'-phosphopantetheine-carrying serine was determined to be Asx2, Thr, Ser, Glx3, Gly2, Ala, Ile, Leu3, and Lys. When these active-site peptides were compared with those of Saccharomyces cerevisiae synthetase, a high degree of homology was observed in the active-site peptides of the acetyl transferase and acyl carrier protein domains. However, that of the condensing enzyme domain gave lower homology. These findings may support the assumption that the low reactivity of cerulenin with C. caerulens synthetase is a consequence of the structure of the condensing enzyme domain.

Spermidine synthase was purified to homogeneity from rat and pig liver by a method modified from a previously reported one using DEAE-Sepharose, S-adenosyl(5')-3-thiopropylamine-Sepharose affinity chromatography, Sephacryl S-300 gel filtration and polyacrylamide gel electrophoresis. No apparent difference between the two enzymes was observed in specific activity, molecular weight (74,000), or subunit composition (two subunits). However, significant differences were observed in their pI values, which were 5.16 for the pig enzyme and 5.34 for the rat enzyme, and their peptide maps. Amino acid compositions of the two enzymes were closely related, but differed significantly in some amino acids. In addition, the rat enzyme was more sensitive to inhibition by S-adenosyl-1,8-diamino-3-thiooctane than the pig enzyme.

Cepharanthin (CR), one of biscoclaurine alkaloids, are utilized for treatment in a wide variety of diseases, and also for the stings of wasps. In order to describe the pharmacological actions of CR, it was determined the effects of CR on leukocyte migration induced by chemotactic peptides isolated from venom sac of Vespinae. Vespid chemotactic peptides were isolated from Vespa tropica (VesCP-T) and Vespa mandarinia (Ves CP-M) . Human polymorphonuclear leukocyte (PMNL) and mononuclear leukocyte (MNL) were separated from peripheral blood, and in vitro chemotaxis assay was performed with membrane filter and multi-well chemotaxis chamber. VesCP-T and VesCP-M showed significant levels of chemotactic activities for PMNL and MNL, and these chemotactic migrations to VesCPs were decreased by addition of CR at a concentration over 10-5M. However, leukocyte migration induced by formyl peptide, synthetic chemoattractant, and buffer control were also depressed. These results suggest that the depression of leukocyte chemotaxis by CR in vitro was attributed to the direct action of CR to leukocytes.

Anomalous glycosylation shift values of the signals for C-1 (the anomeric carbon atom) and C-2 (the glycosyloxylated carbon atom) were sometimes observed for 2- O -β- d -glycopyranosyl (or -β- d -xylopyranosyl)-β- d -glucopyranosides, whereas no remarkable displacements of the other sugar-carbon signals were observed in these cases. This can be explained in terms of change of the orientation of the glycosyl linkages, owing to strong, steric interaction between the 2- O -glycosyl group and the 1- O -aglycon (or -sugar) group. Various 2- O -glycosylated α- and β- l -arabinopyranosides were synthesized. In the case of α- l -arabinopyranosides, the processes of 2- O -β- d -glucosylation, -β- d -xylosylation, and -α- l -arabinosylation resulted in unexpected, upfield shifts of the C-3, -4, and -5 signals, together with displacement of the C-1 and -2 resonances. Furthermore, significant alteration of the values of 3 J H1,H2 and 1 J C1,H1 was also observed for the 2- O -glycosylated α- l -arabinopyranoside moiety, indicating an increase in the contribution of the 1 C 4 conformation of the glycosylated α- l -arabinopyranoside in these cases. On the other hand, no remarkable variation in the signals of C-3, -4, and -5, or in the 3 J H1,H2 and 1 J C1,H1 values, was found for 2- O -α- l -rhamnosylation, except for 4-epihederagenin-3-yl 2- O -α- l -rhamnopyranosyl-α- l -arabinopyranoside. In the present study, such unusual 2- O -glycosylation shifts were not encountered for the 2- O -glycosylation of β- l -arabinopyranosides.

One of the active principles isolated from spider venom, JSTX-3, and its analogs of the polyamine part were synthesized.

The effect of salt on molecular aggregation and conformation has been studied of mastoparan from wasp venom, a tetradecapeptide causing the degranulation of mast cells. The 270 MHz 1 H-NMR and CD spectra of mastoparan in solution have been analyzed. On addition of salt at high concentration in aqueous solution, the monomeric form of mastoparan in largely unordered conformation is converted to the molecular aggregate (probably tetramer) with a largely α-helical conformation. The molecular aggregation of mastoparan depends on pH and ionic strength, but not on the kind of counter anion, in contrast to the case with melittin from bee venom.

The pharmacological mechanisms of platelet aggregation induced by highly toxic proteins (CrTX-I, CrTX-II, and CrTX-III) obtained from tentacles of a jellyfish, Carybdea rastonii, were investigated. When the partially purified toxin (pCrTX) and CrTXs were added to the citrated platelet-rich plasma (PRP), aggregation was produced in a concentration-dependent manner. The activity of CrTXs was approximately 100 times more potent than pCrTX. The CrTXs-induced aggregation was little affected by indomethacin and quinacrine at concentrations sufficient to inhibit arachidonic acid- and collagen-induced aggregation. The CrTXs-induced aggregation in washed platelets was significantly augmented in the presence of Ca2+. The pretreatment with verapamil failed to modify this augmentation of aggregation. The concentration of cytoplasmic-free calcium ([Ca2+]i) of platelets was increased by CrTXs at the same concentrations that produced aggregation. This effect of CrTXs was again little affected by verapamil. CrTXs at the same concentrations as those that produced aggregation and increased [Ca2+]i caused depolarization of platelets, which was unchanged after pretreatment with sodium or potassium transport inhibitors. CrTX-I significantly increased the 22Na flux into platelets and this effect of CrTX-I was unaffected by tetrodotoxin. The CrTX-I-induced aggregation, depolarization, and increase in [Ca2+]i were all significantly attenuated in the low Na+ medium. These results suggest that CrTXs cause a massive depolarization by increasing cation permeability and this generalized depolarization permits an inward movement of Ca2+ down its electrochemical gradient which, in turn, triggers platelet aggregation.

A new tetrahydro-beta-carboline, trypargine (TRG), specifically suppresses the Na current (INa) when applied to the internal surface of the squid axon membrane without affecting the K current (IK). The binding of TRG to its receptor is potential-dependent and occurs at a site about halfway through the membrane electric field from the outside; the dissociation constant is 11 microM at 0 mV.

A proton nuclear magnetic resonance (1H-NMR) study of the interaction of Zn (II) ion with histidine-containing peptide, L-histidylglycylglycine (his-gly-gly), was carried out. The 1 : 2 complex of Zn (II)-(his-gly-gly) takes different coordination structures depending on the pH. In the region of pH 4.5-6.5, the complex is a tautomer in which the Zn (II) ion is exchanged dynamically among amide carbonyl oxygen, imidazole nitrogen, and amino nitrogens above pH 6.5.

1. The effects of the venom and its fractions of Megascolia flavifrons have been studied on synaptic transmission and axonal excitability of the giant interneuron of the cockroach. 2. The venom does not affect axonal excitability, but blocks synaptic transmission, and induces postsynaptic depolarization with a delay. 3. Five different active fractions have been recognized. 4. Three fractions of them contain substances already identified as histamine, Thr6 bradykinin and Thr6 bradykinin-Lys-Ala (megascoliakinin). 5. Three fractions contain activities, which have not yet been chemically identified. 6. All of them, and also bradykinin block synaptic transmission; histamine was not active.

The effects of dopamine (DA) and 3-methoxytyramine (3MT) on arachidonic acid (AA)-, collagen-, and adenosine 5′-diphosphate (ADP)-induced rabbit platelet aggregation were studied in relation to their metabolites in vitro. DA and 3MT did not inhibit AA- and collagen-induced platelet aggregation immediately after addition of a low concentration, but inhibition did occur, in a time-dependent and concentration-dependent manner, when DA and 3MT were incubated in platelet-rich plasma (PRP). The concentration of DA and 3MT decreased time-dependently, and the main metabolites of DA and 3MT were 3,4-dihydroxyphenylacetaldehyde (DOPAL) and 3-methoxy-4-hydroxyphenylacetaldehyde (MOPAL), respectively, as determined by high performance liquid chromatography. These aldehydes were found to have potent inhibitory activities nearly equal to those of 3,4-dihydroxyphenylethanol (DOPET) and 3-methoxy-4-hydroxyphenylethanol (MOPET) on both AA- and collagen-induced platelet aggregation. DOPET and MOPET were detected only in small concentrations. Taking into account both the anti-platelet potency and the concentrations detected in PRP of DOPAL and MOPAL, it is concluded that the time-dependent inhibition of the platelet aggregation by DA and 3MT may be due to the mediation of their corresponding main metabolites, DOPAL and MOPAL. respectively, while the concentration-dependent inhibition by DA and 3MT seems to be largely due to an innate activity.

The Pictet-Spengler reaction of Na-methyl-Nb-benzyltryptophan methyl ester hydrochloride (4a) with methyl 3-formylpropionate was carried out in 50% aqueous MeOH under reflux to afford methyl trans-2-benzyl-3-methoxycarbonyl-9-methyl-1, 2, 3, 4-tetrahydro-9H-pyrido [3, 4-b] indole-1-propionate (3a) as a major product and trans-2-benzyl-3-methoxycarbonyl-9-methyl-1, 2, 3, 4-tetrahydro-9H-pyrido [3, 4-b] indole-1-propionic acid (5a) as a minor product. In earlier work, the stereochemistry of the major product 3a had been erroneously assigned as 1, 3-cis (3b), while the minor product 5a had been incorrectly assigned as the 1, 3-trans compound 3a. Compound 3b was not formed in this reaction. The stereochemistry of the major product (3a) was confirmed by X-ray crystallographic analysis in the present work.

The total synthesis of (±)-trypargine (2) was accomplished to confirm the proposed structure, 1-(3'-guanidinopropyl)-1, 2, 3, 4-tetrahydro-β-carboline. Optically active (-)-trypargine (2a) and its enantiomer (2b) were obtained by the optical resolution of the racemate. The stereochemistry at C-1 is discussed on the basis of the optical rotatory dispersion and circular dichroism curves.

Effects of synthetic compounds similar to the structure of a spider toxin were studied on the glutamate receptors in crustacean neuromuscular synapses. Two kinds of analogues, 2,4-dihydroxyphenylacetyl-asparaginyl cadaverine (C-1) and 2,4-dihydroxyphenylacetyl-asparaginyl spermine (C-2), suppressed the excitatory postsynaptic potentials in a manner similar to natural spider toxin (JSTX). The dose-response relationship showed that the relative potency of the compounds is C-1 less than C-2 less than JSTX. While the effect of JSTX was irreversible, those of C-1 and C-2 were reversible. These synthetic compounds may serve as important tools in studying the structure and function of glutamate receptors.

To evaluate the mechanism of mosquito bite reaction in man, salivary gland extracts from female Aedes albopictus were prepared. When the extract of the gland was analysed by ion-exchange high performance liquid chromatography the amount of histamine in a pair of salivary glands of one mosquito was found to be below the limit of detection, an amount which is insufficient to produce an immediate reaction. Salivary gland extracts were fractionated to higher and lower molecular weight components. Intradermal injection tests with salivary gland extracts, which contained less than 100 ng of protein showed that the higher molecular weight fraction (molecular weight greater than 10,000) elicited an immediate and a delayed reaction similar to a bite reaction.

A new route for the synthesis of (±)-trypargine (I) is described. The Pictet-Spengler reaction of Nb-benzyltryptamine with α-ketoglutaric acid was carried out in aprotic media to afford the key intermediate, 2-benzyl-1, 2, 3, 4-tetrahydro-9H-pyrido [3, 4-b] indole-1-propionic acid (IIa), which was converted into (±)-trypargine (I) in five or six steps.

A partially purified toxin (pCrTX) was obtained from the tentacles of the jellyfish, Carybdea rastonii. When pCrTX (3 X 10(-8) - 3 X 10(-7) g/ml) was added to citrated platelet-rich plasma, aggregation was produced in a concentration-dependent manner. Scanning electron microscopic examination revealed that both pCrTX and collagen produced aggregates of platelets possessing many pseudopods. The concentration which produced 50% aggregation for pCrTX was 1.8 X 10(-7) g/ml, as compared to 2.3 X 10(-6) g/ml for collagen. The pCrTX-induced aggregation was only slightly inhibited by indomethacin and quinacrine in concentrations sufficient to inhibit arachidonic acid- and collagen-induced aggregation. pCrTX was less active in washed platelets suspended in Ca2+ free medium, whereas the pCrTX-induced aggregation was significantly augmented in the presence of Ca2+. The augmentation of aggregation by Ca2+ was only slightly attenuated by pretreatment with 100 microM verapamil. pCrTX significantly increased the concentration of cytoplasmic free Ca2+ ([Ca2+]i) and depolarized the platelet membrane in concentrations that produced aggregation. The increase in [Ca2+]i caused by pCrTX was little affected by verapamil. The depolarization by pCrTX was unchanged in the presence or absence of Ca2+, or by sodium or potassium transport inhibitors. The movement of 22Na+ into platelets was significantly increased by pCrTX. This increase in the movement of 22N+ into platelets was unaffected by tetrodotoxin. On the other hand, pCrTX-induced aggregation, depolarization and the increase in [Ca2+]i were all significantly attenuated in low Na+ medium. These results suggest that pCrTX causes a massive depolarization by increasing cation permeability indiscriminately and this generalized depolarization permits an inward movement of calcium down an electrochemical gradient which, in turn triggers platelet aggregation.

Comparative biochemistry of renins and angiotensins was discussed. Renin extracted from hog kidney was different from that from mouse submaxillary glands in immunoreactivity and carbohydrate content. Rat kidney renin was also different from hog kidney renin in the amino acid composition. The presence of big and big-big renins was pointed out immunochemically. These big renins were considered to be precursors of kidney renin. Angiotensins in mammalian and nonmammalian species produced by renal or extrarenal renin have been differentiated by some biochemical and pharmacological criteria. Some of these angiotensins were analyzed sequentially. The replacements of amino acid residues at positions 1, 5, and/or 9 of angiotensin I have been demonstrated in nonmammalian species. Specific pressor activities have been determined using synthetic angiotensins by a 4 point assay in rat. Specific pressor activities of various angiotensins were obtained from the dose-blood pressure-response curves using a single angiotensin sample per assay rat.

We examined angiotensins, the active components of the renin-angiotensin system, from renal and extrarenal sources in various vertebrates. SE-Sephadex column chromatograms, ratios of oxytocic to pressor activity, and susceptibilities to proteases were used as criteria. Angiotensins from renal and extrarenal tissues in rats, rabbits, and mice did not differ from Asp 1 -Ile 5 -angiotensin I. The active substances derived from the renal tissue of birds, reptiles, amphibians and teleosts, and from the teleostean corpuscles of Stannius, were definitely different from the known angiotensin I or II, suggesting evolution of the active components in the renin-angiotensin system. The physiological role of the renin-angiotensin system is not yet known for either mammals or lower vertebrates. We have presented a hypothesis that the renin-angiotensin system regulates glomerular filtration rate (GFR) by constricting the efferent arterioles. Plasma renin activity (PRA) was determined in bull-frogs in the dehydrated or overhydrated states. PRA was also determined in Japanese eels, transferred from fresh water to 100 and 50% sea water, or removed from water. PRA was parallel with GFR in the bullfrogs of differently hydrated states, basically supportin the above hypothesis. The results in the eels were complex. On transfer to 50% sea water or exposure to air, PRA decreased more slowly than GFR. In 100% sea water, an initial increase in PRA for 1–4 hr was observed. Intrarenal and extrarenal sites of action of angiotensin in teleosts, other than the efferent arterioles, need to be considered. The results may suggest evolution of the sites of action in the renin-angiotensin system.