Biomedical subjects

K Nokihara

Publications and source records attributed to K Nokihara.

At least 37 records · Page 2Linked to original sources

PubMed1996

Purification and characterization of a medium chain acyl-coenzyme A synthetase.

Glycine conjugation is an important route of detoxification of many xenobiotic and endogenous carboxylic acids. A medium chain acyl-coenzyme A synthetase that catalyzes the first reaction of glycine conjugation was purified from bovine liver mitochondria by chromatographies on anion exchange, hydroxylapatite, affinity, and finally by gel filtration. The purified enzyme not only conjugates medium chain fatty acids, but also aromatic and arylacetic acids. The highest activity was shown with hexanoic acid. High activities were observed for benzoic acid derivatives with large alkyl and alkoxyl groups in the para- or meta-positions of the benzene ring. Ortho-substituted derivatives exhibited no activity. The enzyme was inhibited by iodoacetamide and salicylic acid, and activated by albumin. Salicylic acid was a competitive inhibitor of the enzyme, with an apparent Ki value of 37 microM. Enzyme activity increased 74% when the pH was raised from 7 to 10. Molecular weight of the purified medium chain acyl-coenzyme A synthetase was 65.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Animals↗

PubMed1996

Recognition of pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide (PACAP/VIP) hybrids and related peptides by rat brain membranes.

The binding to [125I]PACAP27 and adenylate cyclase activity have been investigated using rat brain membranes with substituted analogues of PACAP and VIP, including their hybrid peptides. Binding of [125I]PACAP27 was rapid, specific and reversible. Scatchard analysis revealed a single class of binding site, with a Kd = 457 +/- 117 pM, and a Bmax = 2.63 +/- 0.24 pmol.mg protein-1. Hybrids of PACAP, in which specific residues were substituted with the corresponding residues of VIP, and vice versa, as well as related analogues, were then tested for binding and adenylate cyclase activity. The results showed that N-terminal residues were important for recognition. In particular, multiple substituted analogues of PACAP by VIP, and vice versa, demonstrated that positions 4, 5 and 9 play a dominant role in the recognition of PACAP Type I receptor in rat brain membranes and account for the differences observed between PACAP and VIP. Substitutions in the C-terminal region at positions 24, 25 and 26 are not crucial for recognition specificity. PACAP-analogues provide evidence that positions 1 and 6 are essential for receptor recognition. The flexibility at position 21 also appears to play a role as substitution with Ala or Phe is tolerated, while Pro shows a significant loss both in binding affinity and adenylate cyclase activity.

Adenylyl Cyclases↗

PubMed1996

Immunohistochemical comparison of localization of pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) in the enteric nerve plexus of the guinea pig jejunum.

The localization and co-localization of pituitary adenylate cyclase activating polypeptides (PACAPs) and vasoactive intestinal polypeptide (VIP) in the enteric nerve plexus of the guinea pig jejunum were immunohistochemically compared by the peroxidase anti-peroxidase (PAP) method using region-specific antisera against PACAP38, PACAP27 and VIP, respectively. Immunoreactive nerve elements were demonstrated in the ganglia of both myenteric and submucous plexi in the guinea pig jejunum. Numerous immunoreactive nerve cell bodies were localized in the myenteric ganglia, while nerve cell bodies in the submucous ganglia were only slightly immunopositive. Immunoreactive nerve terminal varicosities occurred in both myenteric and submucous ganglia. Additionally, PACAPs-immunoreactive nerve cell bodies were also immunopositive with VIP. Thus, both PACAP38 and PACAP27, as well as VIP, are co-localized in the cell bodies of guinea pig jejunum.

Animals↗

PubMed1995

Fine tuning of peptide binding to HLA-B*3501 molecules by nonanchor residues.

The prerequisites of peptide HLA-B*3501 interactions have been revisited by quantitative peptide binding assays with 190 chemically synthesized peptide possessing two anchor residues corresponding to the HLA-B*3501 peptide motif and a statistical residue-position analysis of binding and nonbinding peptides. According to the peptide motif of HLA-B*3501, aliphatic hydrophobic (Leu, Ile, and Met) or aromatic residues (Tyr and Phe) specify the main anchor at the C terminus, and position 2 renders an auxiliary anchor for proline. The importance of these residues was confirmed as a minimum requirement for peptide binding. Moreover, we demonstrated that high affinity peptide binding requires more than one favorable position of positions 3, 4, and 7. Aliphatic hydrophobic residues and residues that contain -OH or -SH side chains in position 3, 7, and 4 significantly enhance binding. Positions 1 and 5, or 7 may deteriorate peptide binding if these positions are held by proline and small residues (Ala and Gly) or basic residues carrying positively charged side chains (Arg and Lys), respectively. Positions 6 and 8 were statistically free of constrains. Yet, bulky aromatic residues and basic residues with a positively charged side chain at position 8 decreased the binding affinity. These findings were used to assess the predictability of binding and nonbinding peptides. Our binding predictions of 28 nonamers were verified by experimental data. Taking into account the importance of anchor and nonanchor positions in peptide binding and their practical value in peptide binding prediction, the search for peptide epitopes becomes more efficient.

Amino Acid Sequence↗

PubMed1995

Antibody specific for phosphorylated AMPA-type glutamate receptors at GluR2 Ser-696.

Possible phosphorylation sites on the Purkinje cell alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptor subunits were identified using in vitro kinase assays of 17 synthetic peptides derived from the transmembrane-3 (TM3) domain to the end of C-terminal of a rat glutamate receptor 2 (GluR2). Only two peptides containing Ser-662 and Ser-696 were found to be efficiently phosphorylated by protein kinase C (PKC). The peptide including Ser-696 was also phosphorylated by protein kinase G (PKG). Another peptide containing Thr-692 of a rat GluRA, clone almost identical to GluR1, was phosphorylated by PKC but not by PKG. Antisera recognizing phosphorylated AMPA receptor subunits at GluR2 Ser-696 or the homologous sites of GluR1/3/4 were produced, and the specificity of one of them, named 12P3, was established by enzyme-linked immunosorbent assay (ELISA), immunoblot and immunoprecipitation analyses. 12P3-immunocytochemistry on cerebellar slices demonstrated an AMPA-induced transient AMPA receptor phosphorylation, which appeared in Purkinje cell dendrites as well as somata immediately after AMPA treatment and disappeared after 20 min. This antibody may be a useful tool to study the role of AMPA receptor phosphorylation in producing synaptic plasticity.

Amino Acid Sequence↗

PubMed1995

Instrumentation and applications of an automated C-terminal fragment peptide fractionator for C-terminal sequence analysis of proteins.

A novel automated C-terminal fragment peptide fractionator has been constructed. Digests with lysyl endopeptidase were covalently immobilized on p-phenylene diisothiocyanate polymer beads. Only the C-terminal fragment, which contains no lysyl residue, was liberated by cleavage at the first peptide bond of the immobilized fragment peptides with trifluoroacetic acid, and it was automatically collected. The whole procedure was automatically and precisely performed under microprocessor control in a nitrogen atmosphere. The resulting fragment was sequenced without further purification. Sequences of both N- and C-terminal regions can be routinely obtained for ca. 100 pmol samples by the use of a conventional automated Edman-type protein sequencer.

Amino Acid Sequence↗

PubMed1995

The effect of pituitary adenylate cyclase activating polypeptide (PACAP) on amylase secretion from guinea pig pancreatic acini.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel hypothalamic peptide, structurally related to vasoactive intestinal peptide (VIP). Our previous study in conscious dogs revealed that PACAP stimulated exocrine pancreatic secretion in a manner different from VIP. The objectives of this study were to characterize the effects of PACAP on amylase secretion and intracellular cAMP production in guinea pig pancreatic acini and to compare them with those of VIP and secretin. PACAP38 and PACAP27 (10(-10)-10(-8)M) stimulated amylase secretion from pancreatic acini in a concentration-related manner. The order of potency for amylase secretion was PACAP27 = VIP > PACAP38. The maximally stimulated amylase secretion by PACAP27 was not enhanced by VIP or secretin, but was synergistically increased by cholecystokinin and A23187. PACAP38 and PACAP27 (10(-2)-10(-7)M) increased intracellular cAMP levels in a concentration-related manner. The potency for cAMP production was PACAP38 = PACAP27 = VIP. These results suggest that PACAP38 and PACAP27, like VIP, directly stimulate amylase secretion from guinea pig pancreatic acini through alterations in cellular cAMP levels.

Amylases↗

PubMed1995

Synthesis, solution structure and biological action of PACAP-related peptide.

High quality PACAP-related peptide (PRP), a 29 amino-acid region of the PACAP precursor protein, has been synthesized in quantities sufficient for biological and structural studies. PRP has a distinct biological activity on the gallbladder that is similar to PACAP, but opposite to that of VIP and its related peptide, PHM. Its solution structure has been investigated by circular dichroism spectroscopy and 2D 1H nuclear magnetic resonance spectroscopy. In contrast to the poorly defined structure in aqueous solution alone, the limiting structure, under conditions that mimic a membrane-like environment, possesses stable secondary structure with a helical region between residues 3 and 20, that is terminated by the presence of glycine at residue 21 and is followed by a region of nascent helix. The similarities and differences in the structure of PRP, PACAP27 and GHRH(1-29) are made through comparison of their H alpha chemical shift data and differences in their biological activities assessed.

Amino Acid Sequence↗

PubMed1994

Two-dimensional electrophoresis as a complementary method of isolating peptide fragments of cleaved proteins for internal sequencing.

To determine the primary structure of proteins, usually proteolytic enzyme digests are separated by reversed-phase high-performance liquid chromatography (HPLC) and each fraction is collected and sequenced. The results obtained by different cleavages are combined to reveal the entire sequence. However, there are many N-terminal-blocked proteins and/or intact proteins or their particular fragments that are not eluted from HPLC columns. Internal fragments of such proteins were successfully isolated by the use of two-dimensional electrophoresis, after digestion. Electroblotted spots were easily sequenced to identify those difficult fragments which could not be obtained using HPLC.

Amino Acid Sequence↗

PubMed1994

HLA-B3501-peptide interactions: role of anchor residues of peptides in their binding to HLA-B3501 molecules.

Two HLA-B*3501 binding self-peptides, LPFDFTPGY (37F) and LPGPKFLQY (28H), were isolated from HLA-B*3501 molecules expressed by cultured human B lymphoid cells. Both sequences were consistent with previously reported motifs of HLA-B*3501 binding peptides which carry proline at position 2 and tyrosine at position 9 as anchor residues. Direct binding of these peptides to HLA-B*3501 molecules was quantitated by flow cytometry analysis of RMA-S cells. transfected with the HLA-B*3501 gene (RMA-S-B*3501). Both 37F and 28H peptides bound effectively to HLA-B*3501 molecules. Substitution of amino acids at position 2 and/or 9 of HLA-B*3501 binding peptides markedly reduced their binding to HLA-B*3501 molecules. These results indicate that two anchor residues, proline at position 2 and tyrosine at position 9 are critical in binding of peptides to HLA-B*3501 molecules. Insertion of up to four glycine residues at position 8 of the peptide 37F did not affect its binding affinity to HLA*3501 molecules. These results indicate that long peptides can effectively bind to HLA class I molecules provided that anchor residues are conserved.

Amino Acid Sequence↗

PubMed1994

A naturally occurring single basic amino acid substitution in the V3 region of the human immunodeficiency virus type 1 env protein alters the cellular host range and antigenic structure of the virus.

Human immunodeficiency virus type 1 circulates in vivo as a mixture of heterologous populations (quasispecies). We previously analyzed the quasispecies of the third hypervariable region (V3) in the viral envelope glycoprotein gp120 in an infected individual and found that the species with a basic amino acid substitution (lysine for aspartic acid) at a particular position evolved and became a distinct population within a short period, followed by progression to the typical immunodeficiency stage (S. Oka et al., AIDS Res. Hum. Retroviruses 10:271-277, 1994). In the present study, we examined the biological significance of this amino acid substitution by constructing recombinant viruses with specific point mutations and comparing their replication capabilities in different cell types. The results demonstrated that the single basic amino acid substitution was sufficient to render a virus fully capable of replicating in human T-cell lines under certain conditions. With an acidic amino acid at the position, the virus grew much less fast or did not grow at all in the T-cell lines. Viral neutralization assay and peptide enzyme-linked immunosorbent assays further showed that this amino acid substitution resulted in different recognition by several of the serum specimens from human immunodeficiency virus type 1-infected individuals and thus could alter the antigenic structure. An additional finding worthy of note was that at the terminal stage, the proviral sequences of peripheral blood mononuclear cells and the viral isolates from them were without exception of the late type with the basic amino acid substitution, whereas the early sequence without the substitution was retained as a major subset in the spleen. These results support the notion that basic amino acid substitutions in V3 are a strong predictor of virus tropism and may be relevant to disease progression.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Gas-phase microsequencing of peptides and proteins with a fluorescent Edman-type reagent, fluorescein isothiocyanate.

A fluorescent Edman-type reagent, fluorescein isothiocyanate (FITC), was adapted to a gas-phase sequencer fitted with a miniature reaction chamber. Fluorescein thiohydantion amino acids were identified by on-line gradient HPLC with fluorescence monitoring. The optimized protocol for the coupling reaction using FITC and phenylisothiocyanate, and the degree of washing to remove the excess reagents were established. Myoglobin (5 pmol) and lysozyme (5 pmol) were analyzed by the sequencer to obtain a 35-51% initial yield (I.Y.) and 82-88% repetitive yield (R.Y.) for the former, and 51-66% I.Y. and 91-92% R.Y. for the latter. These figures permitted 20-25 amino acid residues to be identified from the N-terminus of 5 pmol samples applied to the sequencer.

Amino Acid Sequence↗

PubMed1993

Solution structure of pituitary adenylate cyclase activating polypeptide by nuclear magnetic resonance spectroscopy.

The solution structures of the recently discovered neuropeptides PACAP38 and PACAP27 have been investigated in aqueous solution containing varying amounts of trifluoroethanol (TFE) by circular dichroism (CD) spectroscopy and a combination of 2D 1H nuclear magnetic resonance (NMR) spectroscopy, distance geometry, and refined molecular dynamics and energy minimization calculations. In aqueous solution both peptides show only small transitory amounts of stable structure while in 50% TFE they adopt ordered structures. Qualitative NOE data and the use of the chemical shift index of the alpha-protons identified the positions of alpha-helical regions. A set of low-energy conformations compatible with the quantitative NOE data were obtained for both and each set were subjected to RMS analysis to determine the positions of the secondary structure elements. PACAP38 has an initial disordered N-terminal domain of eight amino acids, followed by an alpha-helical structure stretching from Ser-9 to Val-26, which contains a discontinuity between Lys-20 and Lys-21, and in the C-terminal region there is a short alpha-helix between Gly-28 and Arg-34. The structure of PACAP27 mirrors remarkably closely that of PACAP38 and shows no fraying of the C-terminal helix. The physiological significance of the three structural domains (1-8, 9-26, and 27-38) of PACAP38 is shown by a comprehensive review of recent in vitro and in vivo investigations of PACAP analogues. The correspondence of the global structural features of PACAP with other members of this family of peptides (namely, secretin, glucagon, GHRF1-29 and VIP) is demonstrated by inspection of the chemical shift indices of the alpha-protons.

Amino Acid Sequence↗

PubMed1993

Indication of possible post-translational formation of disulphide bonds in the beta-sheet domain of human lysozyme.

Lysozyme has two distinct folding domains, and in most molecules the alpha-helical domain folds more quickly than the beta-sheet domain in vitro [Radford, Dobson and Evans (1992) Nature (London) 358, 302-307]. In order to investigate the relationship between the formation of disulphide bonds and protein folding in vivo, we carried out cysteine scanning mutagenesis to shift positions of the disulphide bonds in both the alpha-helical and beta-sheet domains of human lysozyme. Of the constructed mutants (nine in the beta-sheet domain and 13 in the alpha-helical domain), the mutant L79CC81A, in which Leu-79 and Cys-81 in the beta-sheet domain were replaced by Cys and Ala respectively, was secreted by yeast. The rest of the mutants were retained in the insoluble fraction of the cell, probably because of a failure of folding. The distance between the two alpha-carbons at positions 79 and 95 in the wild-type protein is too far to form a disulphide bond, but analysis of the primary structure revealed that the major part of L79CC81A was secreted with a non-native disulphide bond Cys79-Cys95 and two free cysteine residues at positions 65 and 77 in the beta-sheet domain. These results suggest that the beta-sheet domain of human lysozyme can tolerate the shift of locations of disulphide bonds, and the non-native folding of mutated polypeptide chains in in vivo folding. The free residues Cys-65 and Cys-77 formed a disulphide bond in vitro by air oxidation, yielding two isomers. On the basis of our previous results and present study it is suggested that the formation of Cys6-Cys128 is the first step of the in vivo correct folding of human lysozyme, and disulphide bonds in the beta-sheet domain are post-translationally formed in vivo.

Amino Acid Sequence↗

PubMed1993

Primary structure determination of mono- and diacylglycerol lipase from Penicillium camembertii.

The complete amino acid sequence of mono- and diacylglycerol lipase from Penicillium camembertii was determined. This lipase has a single polypeptide chain consisting of 276 amino acid residues with two disulfide linkages. The primary structure was revealed by sequencing the digests of the intact and S-pyridylethylated proteins by trypsin, endoproteinase Lys-C and V8 protease. The two-dimensional electrophoresis was also carried out to confirm the internal sequence. The catalytic triad of this lipase was Ser, Asp and His, and one potential N-glycosylation site was also revealed.

Amino Acid Sequence↗

PubMed1993

Matrix-assisted ultraviolet laser desorption/ionization mass spectrometry applied to multiple forms of lipases.

Matrix-assisted ultraviolet laser desorption/ionization mass spectrometry was used to investigate heterogeneous patterns and molecular masses of microbial lipases from Penicillium camembertii, Geotrichum candidum, and Pseudomonas sp. Mass spectral peaks of the native, glycosylated lipases from P. camembertii and G. candidum were broader than those of the corresponding deglycosylated enzymes, indicative of heterogeneous glycosylations. The broader peaks in the mass spectra were caused by an overlapping of unresolved peaks, derived from single glycoprotein species. Molecular masses determined for the deglycosylated proteins were in excellent agreement with those deduced from amino acid composition and sequence data, whereas with conventional biochemical methods (gelfiltration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis) only very rough estimations of molecular masses were possible. By mass spectrometric analysis of the four fractions of chromatographically separated P. camembertii lipase molecular masses of 29,990, 34,030, 31,990, and 32,140 Da were found before and 29,960, 29,980, 29,990 and 30,010 Da, respectively, after deglycosylation. Thus from the four native fractions of P. camembertii lipase three were glycoproteins. G. candidum lipase showed an average molecular mass of 63,500 Da for the heterogeneously deglycosylated native form and a molecular mass of 59,650 Da for the deglycosylated enzyme. For the Pseudomonas lipase, which could only be isolated with lipids firmly attached, a molecular mass of 32,890 Da was determined, in close agreement with that derived from the cDNA sequence.

Bacterial Proteins↗

PubMed1993

Effects of environmental temperature on atrial natriuretic peptide (ANP)-granules of auricular cardiocytes and plasma ANP level in pregnant rats.

The atrial natriuretic peptide (ANP) levels of auricular cardiocytes and plasma were examined by immunohistochemistry, ultrastructural morphometry, and radioimmunoassay (RIA) in pregnant rats (4th, 12th, 20th day of gestation) under 22 degrees C and 33 degrees C environmental conditions. Immunohistochemically, ANP immunoreactivity was stronger on the 20th day of gestation in the 22 degrees C environmental groups, but weaker on the 12th and 20th days of gestation in the 33 degrees C environmental groups. According to the results of ultrastructural morphometry, the number of ANP-granules had increased significantly by the 20th day of gestation in the 22 degrees C environmental groups, but was decreased on the 12th and 20th days of gestation in the 33 degrees C environmental groups. RIA demonstrated that the maternal plasma ANP concentration decreased gradually during pregnancy in the 22 degrees C environment. The plasma ANP concentration in the 33 degrees C environmental groups tended to be lower than that at the same stage of pregnancy in the 22 degrees C environmental groups.

Animals↗

PubMed1992

Crystallization and characterization of monoacylglycerol and diacylglycerol lipase from Penicillium camembertii.

A new lipase from Penicillium camembertii U-150, which is specific for monoacylglycerols and diacylglycerols, but not triacylglycerols, was purified as four active components using concanavalin-A-Sepharose column chromatography, crystallized in the form of needles, and its properties investigated. No significant difference was observed in substrate specificity, but molecular mass and other enzymatic properties, such as pH, heat stability and optimum pH and temperature, were clearly different between the unadsorbed and the three adsorbed components on concanavalin-A-Sepharose; the three adsorbed components were similar to each other and more stable than the unadsorbed component. On the other hand, after enzymatic removal of carbohydrates from the three adsorbed components, their enzymatic properties became similar to those of the unadsorbed component. The carbohydrates of this lipase contribute to the stability of the enzyme, but not to its enzyme activity. The amino acid compositions of the four components did not differ from each other, and tryptic mapping of the deglycosylated components and amino acid composition of the tryptic fragments were identical. The carbohydrate compositions of four intact components were, however, different from each other. All four components have the same polypeptide backbone and multiple forms of this lipase are due to the differences in composition of the carbohydrates bound in this lipase.

Amino Acids↗