Identification of 2-guanidinoethanol in human urine

Guanidino compounds in normal human urine were analyzed by high-performance liquid chromatography; an unknown peak was observed in the chromatogram that was identical to the peak of synthetic 2-guanidinoethanol. In another experiment, the substance was purified from human urine by successive use of strongly acidic ion-exchanger, thin-layer chromatography and then weakly acidic ion-exchanger. After this it was reacted with acetylacetone to form dimethylpyrimidyl derivative. After further reaction of this derivative with trifluoroacetic anhydrate, it was analyzed by gas chromatography/mass spectrometry. The mass chromatogram and mass spectrum were identical to those of the trifluoroacetylated dimethylpyrimidyl derivative of synthetic 2-guanidinoethanol. This is the first report on the identification of 2-guanidinoethanol in human urine. The concentration of 2-guanidinoethanol in the urine of healthy humans was 5.7 +/- 1.8 (mean +/- SD) mumol/g creatinine.     

Identification of delta-guanidinovaleric acid in human urine

delta-Guanidinovaleric acid (DGVA) was identified in human urine using thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). In the TLC, all Rfs of sample from urine developed by 6 solvent systems were identical to that of authentic DGVA. In the GC/MS, the mass spectrum of the sample was identical to the trifluoroacetylated dimethylpyrimidyl derivative of DGVA butylester (M+ = 375). In the HPLC analysis, the DGVA peak was observed just before 15 min in either chromatogram obtained by analysis of human urine or authentic DGVA, and the content of DGVA in pooled human urine was calculated at 2.4 nmol/ml.     

Metabolic correction of fucosidosis fibroblasts by human α-L-fucosidase

Human α-L-fucosidase, purified from placenta, was taken up from the culture medium by skin fibroblasts from patients with fucosidosis (α-L-fucosidase deficiency). The rate of uptake was low (uptake coefficient = 6 × 10^?4 ml.mg^?1.h^?1). Intracellular α-L-fucosidase activity was directly proportional to enzyme in the medium up to an activity of at least 40 nmoles/min/ml. No evidence for saturation of specific cell-surface receptors was seen. However, uptake was reduced by 75% by 1 mM mannose-6-phosphate and by 50% by 1 mM glucose-6-phosphate, suggesting that uptake may be mediated by a receptor recognising a phosphorylated sugar or an analagous compound. Enzyme taken up by the cells was most active in subcellular fractions enriched with lysosomes and had an isozyme pattern, by isoelectric focusing, identical to that of the original enzyme preparation. Fucosidosis fibroblasts were shown to accumulate low molecular-weight, fucose-containing compounds to a level several times greater than control cells. This stored material was eluted from Sephadex G-25 as an asymmetrical peak with an elution volume of approximately twice the void volume of the column. Addition of placental α-L-fucosidase to the culture medium of fucosidosis fibroblasts prevented excessive accumulation of fucose-containing material and accelerated the breakdown of material accumulated prior to enzyme uptake.     

Chemical Structure of Mating Pheromone Peptides, αsk1 and αsk3 Pheromones,Produced by Mating Type α Cells of Saccharomyces kluyveri

Three peptides, α^sk1, α^sk2 and α^sk3 pheromones, have been isolated as α-mating pheromones of Saccharomyces kluyveri, the primary structure of the main active component, α^sk2 pheromone, having already been determined. The unknown N-terminus of α^sk1 pheromone was elucidated to be 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (β-CAR) by mass and NMR spectrometric analyses. Synthetic β-CAR-His-Trp-OH was identical with N-terminal tripeptide fragment obtained from α^sk1 pheromone, and the primary structure of α^sk1 pheromone was determined as β-CAR-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met(O)-Tyr-OH. The amino acid sequence of α^sk3 pheromone was determined as H-Trp-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met-OH by comparing the enzymatic fragments with those of α^sk2 pheromone.     

A detailed study of the periodate oxidation of sialic acids in glycoproteins

Periodate oxidation of terminalN-acetyl- andN-glycoloylneuraminic acid residues in the mucins from edible bird nest substance and pig submandibular gland, respectively, can be carried out under conditions which exclusively give rise to the formation of the C-7 analogues of these sialic acids. In contrast, the C-8 compounds can be obtained in a maximum yield of about 40%. Under identical conditions,N-glycoloylneuraminic acid is oxidized about 1.5 times faster than theN-acetylated derivative. After release of the sialic acids by acid hydrolysis, the characterization of the oxidation products was carried out by TLC, by GLC and GLC-MS of the corresponding pertrimethylsilyl derivatives, and by 500-MHz¹H-NMR spectroscopy. In addition, molar response factors for GLC analysis and extinction coefficients in the orcinol/Fe³⁺/HCl assay were determined.     

Proton magnetic resonance and optical spectroscopic studies of watr-soluble polypeptides: poly-L-lysine HBr, poly(L-glutamic acid), and copoly(L-glutamic acid42, L-lysine HBr28, L-alanine)

The helix-coil transition has been studied by high-resolution NMR for three water-soluble polypeptides. Such systems are better models for protein behavior than those in TFA-CDCl₃ solvent. An upfield shift of ～7 cps is observed for the α-CH peak of poly(L -glutamic acid) and poly-L -lysine as the helix content increases over the transition. No such shift is found for copoly(L -glutamic acid⁴², L -lysine²⁸, L -alanine³⁰). The width of the α-CH peak for poly L-lysine increases rapidly as helix content rises but for poly L -glutamic acid and the copolymer, the width of this peak remains unchanged up to 60% helicity. This demonstrates a rapid rate of interconversion between helical and random conformations in partly helical polymer for the latter two polypeptides. All three polymers however, show no apparent α-CH peak at 100% helicity. Side-chain resonance lines also broaden as helix content increases and, to a greater extent, the closer the proton is to the main chain.     

Isolation of N-acetylglutamine, pyroglutamic acid and the butyl ester of pyroglutamic acid from human brain

N-acetyl-l -glutamine, pyroglutamic acid, and the butyl ester of pyroglutamic acid were isolated in pure form from an aqueous extract of human brain. These compounds were isolated by combination of paper and ion exchange chromatography. The isolated substance identified as N-acetyl-l -glutamine did not react with the ninhydrin reagent but yielded glutamic acid and ammonia upon acid hydrolysis. An acetyl hydrazide was identified by paper chromatography from hydrazinolysates of the isolated substance. The glutamic acid liberated by hydrolysis had the l -configuration. The results of elementary analysis of the isolated compound were in full accord with the analysis calculated for synthetic N-acetyl-l -glutamine. A large amount of pyroglutamic acid and a substance identical with the butyl ester of pyroglutamic acid were isolated in pure form. The results of our studies suggest that pyroglutamic and the butyl ester derivative were artifacts formed during the isolation and purification procedures.     

Isolation of 125I-concanavalin A-labeled plasma membrane from unfertilized mouse eggs

A procedure was developed for isolation of plasma membrane (PM) preparations from unfertilized mouse eggs. Zona-free mouse eggs prepared by the method of Boldt and Wolf (Gamete Res 13:213–222, 1986) were labeled with ¹²⁵I-concanavalin A (ConA) prior to sonication and fractionation on iso-osmotic self-generated Percoll density gradients. Experiments using the ConA-specific sugar α-methylmannoside (αMM) indicated that ¹²⁵I-ConA bound specifically to the egg PM. Greater than 95% of ¹²⁵I-ConA binding to zona-free eggs was blocked in the presence of 0.1 M αMM, and incubation of eggs in αMM after ¹²⁵I-ConA labeling caused release of 85–90% of bound label. Fractionation of ¹²⁵I-ConA-labeled eggs by Percoll density gradient centrifugation yielded a single radioactive peak at density = 1.025, corresponding to egg PM material. Prolonged incubation of ¹²⁵I-ConA-labeled eggs or egg sonicates prior to fractionation did not alter the location of the radioactive peak, indicating that ¹²⁵I-ConA did not label other organelles. As a control, human erythrocytes were labeled with ¹²⁵I-ConA and fractionated under identical experimental conditions and yielded a single radioactive peak at density (1.020) comparable to that observed for ¹²⁵I-ConA-labeled eggs. These results indicate that ¹²⁵I-ConA can be used as a specific marker to support PM isolation from small numbers of zona-free mouse eggs.     

Amino acid differences between the α-chains from two hemoglobins of the yellow-cheeked vole (family cricetidae)

The yellow-cheeked vole (Microtus xanthognathus) shows two electrophoretic hemoglobin components. Electrophoresis of the polypeptide chains from the separated hemoglobin components shows identical β-chains but two α-chains of different mobility, α ^f and α ^s . The composition of soluble tryptic peptides was determined for each α-chain. Amino acid differences were found in peptides αT1 and αT9; the compositions of the remainder of the homologous peptides were identical. Differences in αT1, found at α4 (α ^s -Gly-α ^f -Val) and α5 (α ^s -Thr-α ^f -Asp), were confirmed after a run to residue 20 of the fast component in an automatic sequencer. The differences in charge between αT1 peptides can account for the electrophoretic pattern of two hemoglobins. This is the first time that it has been possible to identity the residues which can account for the charge difference between the two hemoglobins observed in a Microtus species.     

Substance P plays an important role in cell adhesion molecule 1-mediated nerve–pancreatic islet α cell interaction

Autonomic neurons innervate pancreatic islets of Langerhans and maintain blood glucose homeostasis by regulating hormone levels. We previously showed that cell adhesion molecule 1 (CADM1) mediated the attachment and interaction between nerves and aggregated pancreatic islet α cells. In this study, we cocultured αTC6 cells, a murine α cell line, with mouse superior cervical ganglion (SCG) neurons. The oscillation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) was observed in 27% and 14% of αTC6 and CADM1-knockdown αTC6 cells (αTC6^siRNA-CADM1 cells) in aggregates, respectively, within 1 min after specific SCG nerve stimulation with scorpion venom. In αTC6^siRNA-CADM1 cells, the responding rate during 3 min after SCG nerve stimulation significantly increased compared with that within 1 min, whereas the increase in the responding rate was not significantly different in αTC6 cells. This indicated that the response of αTC6 cells according to nerve stimulation occurred more rapidly and effectively than that of αTC6^siRNA-CADM1 cells, suggesting CADM1 involvement in promoting the interaction between nerves and α cells and among α cells. In addition, because we found that neurokinin (NK)-1 receptors, which are neuropeptide substance P receptors, were expressed to a similar extent by both cells, we investigated the effect of substance P on nerve–α cell interaction. Pretreatment with CP99,994 (0.1 μg/ml), an NK-1 receptor antagonist, reduced the responding rate of both cells, suggesting that substance P released from stimulated neurites was a mediator to activate αTC6 cells. In addition, α cells that were attached to neurites in a CADM1-mediated manner appeared to respond effectively to neurite activation via substance P/NK-1 receptors.     

Cloning of large-conductance Ca-activated K channel α-subunits in mouse cardiomyocytes

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels are widely distributed in cellular membranes of various tissues, but have not previously been found in cardiomyocytes. In this study, we cloned a gene encoding the mouse cardiac BK_Ca channel α-subunit (mCardBKa). Sequence analysis of the cDNA revealed an open reading frame encoding 1154 amino acids. Another cDNA variant, identical in amino acid sequence, was also identified by sequence analysis. The nucleotide sequences of the two mCardBKa cDNAs, type 1 (mCardBKa1) and type 2 (mCardBKa2), differed by three nucleotide insertions and one nucleotide substitution in the N-terminal sequence. The amino acid sequence demonstrated that mCardBKa was a unique BK_Ca channel α-subunit in mouse cardiomyocytes, with amino acids 41-1153 being identical to calcium-activated potassium channel SLO1 and amino acids 1-40 corresponding to BK_Ca channel subfamily M alpha member 1. These findings suggest that a unique BK_Ca channel α-subunit is expressed in mouse cardiomyocytes.     

Purification of endogenous inhibitors of [3H]flunitrazepam binding from bovine brain

Endogenous substances which inhibited the binding of [³H]flunitrazepam ([³H]FNZ) to bovine synaptosomal membranes have been purified from the hot acetic acid extracts of the bovine brain. Three peaks of inhibitory activity were obtained by Sephadex G-10 gel chromatography. Two of the peaks (Peak 2, and Peak 3) which had lower molecular weights that that of peak 1 were identified as inosine and hypoxanthine by TLC methods. Another peak (Peak 1) was further purified to homogeneity using both cation and anion ion-exchange chromatography and the following two-step reversed-phase HPLC. The purified substance inhibited the [³H]FNZ binding dose-dependently and competitively but did not have an effect on the binding of the peripheral-type BZ ligand [³H]Ro 5-4864. It was also shown that the substance was heat-stable and resistant to proteolytic degradation (trypsin, -chymotrypsin, pronase). However, a significant loss of inhibitory activity to [³H]FNZ binding was observed after acid hydrolysis. Molecular weight estimates based on gel filtration methods were less than 500 dalton, and the maximal ultraviolet absorption peak was at 314 nm. These results suggest that this substance is a new endogenous ligand for the central BZ receptor and may play an important role in regulating the GABAergic tone in the central nervous system.     

On sexual agglutination and mating type substances in isogamous dioecious Chlamydomonads: IV. Unilateral inactivation of the sex contact capacity in compatible and incompatible taxa by α-mannosidase and snake venom protease

Sex cell contact at fertilization is analysed in the mating type reaction of isogamous Chlamydomonas species. Contact is based upon a complementarity between special mating type substances, sex and species specific glycoprotein complexes. In three related taxa, the contact capactiy of their (+) gamete type is sensitive to snake venom protease (α-protease) and depends decisively on terminal α-glycosidically bound mannose residues. Enzymatic removal of these residues by α-mannosidase incapacitates live (+) gametes and inactivates isolated (+) mating type substance. (+) gametes inactivated by α-mannosidase or α-protease do not agglutinate with (?) gametes nor respond to isolated (?) mating type substance. Isolated (?) substance is adsorbed to and inactivated by the homologous (+) gametes. (+) gametes incapacitated by α-mannosidase or α-protease do not adsorb nor inactivate the isolated (?) substance. The agglutinability of live (?) gametes and the contact capacity of isolated (?) mating type substance is not affected by α-mannosidase or α-protease. The mannose residues react only within the species-typical complementarity. Some additional feature(s) of the (+) mating type substances must effect their species specificity and account for gametic isolation and sexual incompatibility between species.     

Leukotriene C4: The major lipoxygenase metabolite of arachidonic acid in dog spleen

Slices of dog spleen converted [¹⁴C]-arachidonic acid (AA) to a polar material which conjugated with [³H]-glutatione. Nordihydroguaiaretic acid (NDGA) and 5,8,11,14, Eicosatetraynoic acid (ETYA) but not indomethacin, inhibited the conversion of [¹⁴C]-arachidonic acid by the spleen slices into the polar material indicating that it is derived through the lipoxygenase pathway. Physicochemical analysis of the polar metabolite of arachidonic acid after thin-layer chromatography and high pressure liquid chromatography revealed that it has chemical properties identical to authentic leukotriene C₄ standard (LTC₄). The biological activity of the purified material was found to be similar to the slow reacting substance of anaphylaxis (SRS-A), viz, it caused contraction of the guinea-pig ileum which was abolished by FPL-55172, a specific SRS-A receptor antagonist. These data suggest that dog spleen slices convert arachidonic acid through lipoxygenase pathway into a polar material that appears to be identical to LTC₄.     

Persistence and effects of α-methyl-substituted amino acids on 5-hydroxytryptamine and dopamine concentrations in cockroach (Periplaneta americana) nervous tissue

The α-methylated derivatives of tryptophan, tyrosine, and dihydroxyphenylalanine were injected into cockroaches (Periplaneta americana). The levels of these compounds and those of dopamine, 5-hydroxytryptamine, tyrosine, and tryptophan in the nervous tissue, hemolymph, and fat body were measured at various times after drug administration. Levels of 5-hydroxytryptamine and tryptophan in the nervous tissue are significantly reduced by α-methyltryptophan administration. Concentrations of dopamine in nervous tissue are reduced by α-methyltyrosine administration. This effect also persists for several weeks, and α-methyltyrosine is observed in the nervous tissue 3 weeks after injection. Levels of dopamine and 5-hydroxytryptamine in the nervous tissue are unaffected by α-methyldihydroxyphenylalanine, and this compound is less persistent in nervous tissue than α-methyltyrosine or α-methyltryptophan demonstrates that these compounds can be absorbed and affect amine levels in the nervous tissue when included in the diet. Inhibition of tryptophan hydroxylation by crude enzyme preparations of cockroach nervous tissue was demonstrated with both α-methyltryptophan and α-methyltyrosine, with α-methyltryptophan being the more effective inhibitor. Aromatic amino acid decarboxylase activity toward dihydroxyphenylalanine in crude enzyme preparations of cockroach nervous tissue was strongly inhibited by α-methyldihydroxyphenylalanine and monofluoromethyldihydroxyphenylalanine, slightly inhibited by α-methyltyrosine and unaffected by α-methyltryptophan at concentrations up to 10^?3 M. The results indicate that α-methyltyrosine and α-methyltryptophan, but not α-methyldihydroxyphenylalanine, can selectively alter amine concentrations in insect nervous tissue and that insects are only poorly able to metabolize or excrete these compounds. The selective and long-lasting depletion of dopamine or 5-hydroxytryptamine by some of these compounds suggest that they may be useful in behavioral studies designed to elucidate the roles of these amines in insects.     

Nuclear magnetic resonance studies on pyridine dinucleotides. Carbon-13 assignments and structure determination of NADHX and the primary acid product of NADH.

The ¹³C spectra of β-NADH, NADHX, and the primary acid product of NADH were obtained and assigned. The conversion of the NADHX isomers to the two isomers of NADH acid product is demonstrated through the use of ¹³C-enriched compounds. The structure of NADHX is assigned as β-6-hydroxy-1,4,5,6-tetrahydronicotinamide adenine dinucleotide and the structures of the primary acid products of NADH are assigned as α-O^2′-6B-cyclotetrahydronicotinamide adenine dinucleotide and α-O^2′-6A-cyclotetrahydronicotinamide adenine dinucleotide.The structures of NADHX and the major isomer of the primary acid product, derived from studies of model compounds, are consistent with those proposed by Oppenheimer and Kaplan [Biochemistry (1974) 13, 4675, 4685]. However, the spectra of ¹³C-enriched primary acid product also demonstrated the existence of the A isomer which was not observed in the latter ¹H study. The A and B isomers were found to exist in the same ratio even when the primary acid product was formed directly from NADHX. This observation is discussed in terms of the previously proposed mechanism for the acid decomposition of NADH.     

Immunochemical studies on the specificity of soybean agglutinin

The specificity of the purified soybean agglutinin has been studied immunochemically by quantitative precipitin and quantitative precipitin inhibition assays. The lectin is precipitated by human A and Le^a blood-group substance, by the products of the second, third, fourth, and fifth stages of periodate oxidation of a human H blood-group substance (JS), and by precursor blood-group substances, as well as by a pig-submaxillary mucin having blood-group A activity, by partially hydrolyzed blood-group B substances (Pl fraction), and by group C streptococcal polysaccharide. The activity is attributable to terminal α-linked 2-acetamido-2-deoxy-d-galactopyranosyl or to α- or β-d-galactopyranosyl residues. The lectin did not precipitate with human blood-group H substances, with the product of the first stage of periodate oxidation (JS), with streptococcal group A polysaccharide, or with pig-submaxillary mucin devoid of blood-group A activity, and is poorly precipitated by blood-group B substances. Inhibition of precipitation with various monosaccharides indicated that the lectin is strongly specific for 2-acetamido-2-deoxy-d-galactose and for its oligosaccharides, and to a lesser extent for d-galactose and its oligosaccharides; the α-glycosides of both sugars were slightly more reactive than the β-glycosides of 2-acetamido-2-deoxy-d-galactose, and both α- and β-glycosides were more active than the free monosaccharides. Aromatic α- and β-glycosides of 2-acetamido-2-deoxy-d-galactose and d-galactose were better inhibitors than the corresponding methyl or ethyl compounds. The blood-group A trisaccharide α-d-GalNAcp-(1→3)-β-d-Galp-(1→3)-d-GlcNAc was more active than the disaccharide lectins by the use of precipitation with polysaccharides, as well as inhibition reactions, is essential to the understanding of their reactivity with cell-surface receptors.     

Occurrence of crustacean cardioactive peptide (CCAP) in the nervous system of an insect,Locusta migratoria

Summary Using a radioimmunoassay developed for the determination of crustacean cardioactive peptide (CCAP), immunoreactive material was detected in extracts of locust nervous tissue. Serial dilutions of a brain extract gave a displacement curve parallel to the CCAP standard curve. One locust nervous system was calculated to contain approximately 1.4 pmol CCAP-like material.In order to investigate whether the immunoreactive substance was similar or identical to the crustacean neuropeptide, isolation and complete characterization was carried out using 800 locust nervous systems. The isolation procedure consisted of pre-purification of the crude extract on a Sep-Pak cartridge, affinity chromatography on a column which was prepared by coupling of anti-CCAP antibody to CNBr-activated Sepharose, and reversed phase high performance liquid chromatography (HPLC). In the HPLC-profile immunoreactivity was confined to a single peak which co-chromatographed with authentic CCAP. The peptide was carboxymethylated and analyzed in an automated gas-phase sequencer. Its amino acid sequence, is identical to that of CCAP fromCarcinus maenas.Synthetic CCAP was tested on the isolated locust hindgut in vitro. The peptide proved to be a potent enhancer of gut contractions, with a significant effect being observable at concentrations of 10^–10 M. It is concluded that in the locust CCAP may function as a myotropic peptide.     

Occurrence and metabolism of sphagnum acid in the cell walls of bryophytes

Sphagnum acid was detected in all 30 Sphagnum species investigated. The content declines in older stem segments. Investigations have so far failed to detect this cinnamic acid derivative outside the Sphagnales. In all the Sphagnum species analysed, a second, conspicuous substance was detected, apparently identical with a degradation product of sphagnum acid produced by enzymatic reaction with peroxidase in vitro. A casual correlation between the sphagnum acid content and peroxidase activity in vivo is discussed. Glyphosate (0.5 mM) inhibits the synthesis of sphagnum acid and shikimate accumulates. Exogenously supplied phenylalanine is able to produce up to 65% reversal of the glyphosate-mediated inhibition of sphagnum acid synthesis. A mixed effect of glyphosate was found on amino acid levels. The content of sphagnum acid is also reduced by daily application of 0.1 mM l-α-aminooxy-β-phenylpropionic acid.     

Structure of α-keratin: Structural implication of the amino acid sequences of the type I and type II chain segments

Two amino acid sequences from potentially helical fragments of low-sulphur proteins from α-keratin have been analysed computationally and periods 9.4 and 28 residues long noted in the axial disposition of charged residues. Ionic interactions between chains have also been calculated and these indicate a preference for the helical fragments to aggregate in parallel with zero shift between chains in a manner essentially identical to that found for α-tropomyosin.