Biomedical subjects

K Takio

Publications and source records attributed to K Takio.

At least 73 records · Page 4Linked to original sources

PubMed1997

Phosphorylation of Srp1p, the yeast nuclear localization signal receptor, in vitro and in vivo.

Srp1p, the protein encoded by SRP1 of the yeast Saccharomyces cerevisiae, is a yeast nuclear localization signal (NLS) receptor protein. We have previously reported isolation of a protein kinase from yeast extracts that phosphorylates Srp1p complexed with NLS peptides/proteins. From partial amino acid sequences of the four subunits of the purified kinase, we have now identified this protein kinase to be identical to yeast casein kinase II (CKII). It was previously thought that autophosphorylation of the 36 kDa subunit of the yeast enzyme was stimulated by the substrate, GST-Srp1p. However, with the use of a more refined system, no stimulation of autophosphorylation of the 36 kDa subunit of yeast CKII was observed. Biochemical and mutational analyses localized the in vitro phosphorylation site of Srp1p by CKII to serine 67. It was shown that, in the absence of NLS peptides/proteins, phosphorylation of the intact Srp1p protein is very weak, but deletion of the C-terminal end causes great stimulation of phosphorylation without NLS peptides/proteins. Thus, the CKII phosphorylation site is apparently masked in the intact protein structure by the presence of a C-terminal region, probably between amino acids 403 and 516. Binding of NLS peptides/proteins most likely causes a change in protein conformation, exposing the CKII phosphorylation site. Mutational alterations of serine 67, the CKII phosphorylation site, to valine (S67V) and aspartic acid (S67D) were not found to cause any significant deleterious effects on cell growth. Analysis of in vivo phosphorylation showed that at least 30% of the wild type Srp1p molecules are phosphorylated in growing cells, and that the phosphorylation is mostly at the serine 67 CKII site. The ability of Srp1p purified from E coli and treated with calf intestinal phosphatase to bind a SV40 T-antigen NLS peptide was compared with that of Srp1p which was almost fully phosphorylated by CKII. No significant difference was observed. It appears that NLS binding does not require any phosphorylation of Srp1p, either by CKII or by some other protein kinase.

Amino Acid Sequence↗

PubMed1996

Isolation and characterization of the gene encoding an aminopeptidase involved in the selective toxicity of ascamycin toward Xanthomonas campestris pv. citri.

An aminopeptidase gene named XAP has been isolated from Xanthomonas campestris pv. citri, a plant pathogenic bacterium. The bacterium is one of the rare micro-organisms susceptible to ascamycin, an aminoacyl nucleoside antibiotic that inhibits protein synthesis. Sequence analysis reveals that the gene encodes a 311 amino acid protein with a calculated molecular mass of 35134 Da and approx. 50% identity for amino acids to the proline iminopeptidase from Neisseria gonorrhoeae. The XAP gene product, Xap, expressed in Escherichia coli has proline iminopeptidase activity as well as ascamycin dealanylating activity in vitro.

Adenosine↗

PubMed1996

Partial characterization of a 17 kDa acidic protein, EFP, induced by thiocarbamate in the early flowering phase in Asparagus seedlings.

Asparagus officinalis seedlings treated with either thiocarbamates or carbamates are induced to flower within 2-3 weeks of application. SDS-PAGE and 2-D PAGE resolutions of total soluble proteins of treated seedlings showed the accumulation of an acidic 17 kDa polypeptide. Partial amino acid sequence of the acidic protein showed homology to the wound-responsive A. officinalis PR 1, AoPR1. We named this protein EFP, early flowering protein, owing to its advanced appearance during the early transition from vegetative to floral phase. Carbamates and thiocarbamates that induced more than 80% flowering rates corresponded with increased level of EFP while compounds that are weak flower inducers showed reduced amount of EFP. The observation thus defined the correlationship of EFP to variable flowering rates.

Amino Acid Sequence↗

PubMed1995

The signal modulator protein 14-3-3 is a target of sphingosine- or N,N-dimethylsphingosine-dependent kinase in 3T3(A31) cells.

Phosphorylation of a 28 kDa protein in 3T3(A31) cells upon addition of semi-purified kinase was induced only by sphingosine (Sph) or N,N-dimethylsphingosine (DMS), but not by 17 other lipids tested. The 28 kDa protein, a major target of Sph- or DMS-dependent kinase, is now identified as 14-3-3 protein, which modulates a number of key protein kinases involved in regulation of intracellular signaling (Aitken A, Trends Biochem Sci 20:95-97, 1995; Morrison D, Science 266:56-57, 1994). Thus, Sph and DMS modulate the function of a versatile modulator controlling a number of signaling molecules.

14-3-3 Proteins↗

PubMed1995

Spermidine-preferential uptake system in Escherichia coli. ATP hydrolysis by PotA protein and its association with membrane.

PotA protein, one of the components of the spermidine-preferential uptake system in Escherichia coli, was purified to homogeneity, and some of its properties were examined. PotA protein showed Mg(2+)-and SH-dependent ATPase activity. The specific activity was approximately 400 nmol/min/mg of protein and the Km value for ATP was 385 microM. The nature of the ATP binding site was explored by identification of the amino acid residue photoaffinity-labeled with 8-azido-ATP. It was found that 8-azido-ATP was attached to cysteine 26. In the spermidine transport-deficient mutant E. coli NH1596, valine 135 of PotA protein, which is located between two consensus amino acid sequences for nucleotide binding (50-57 and 168-173), was replaced by methionine (Kashiwagi, K., Miyamoto, S., Nukui, E., Kobayashi, H., and Igarashi, K. (1993) J. Biol. Chem. 268, 19358-19363). This mutated PotA protein could be labeled with 8-azido-ATP, but showed very low ATPase activity. To identify which cysteine is involved in the function of potA protein, cysteines 26, 54, and 276 were replaced by alanine, threonine, and alanine, respectively. Among the three mutated PotA proteins, the mutated PotA protein C54T only lost both ATPase and spermidine uptake activities. The results taken together indicate that the adenine portion of ATP interacts with a domain close to the NH2-terminal end of PotA protein, and active centers of ATP hydrolysis are located both within and between the two consensus amino acid sequences for nucleotide binding. Association of PotA protein with membranes was strengthened by the existence of channel forming PotB and PotC proteins. ATPase of PotA protein was inhibited by spermidine, suggesting that uptake inhibition by spermidine may function during this process.

ATP-Binding Cassette Transporters↗

PubMed1995

Purification and cDNA cloning of a human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase.

A UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase (GalNAc-transferase) from human placenta was purified to apparent homogeneity using a synthetic acceptor peptide as affinity ligand. The purified GalNAc-transferase migrated as a single band with an approximate molecular weight of 52,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on a partial amino acid sequence, the cDNA encoding the transferase was cloned and sequenced from a cDNA library of a human cancer cell line. The cDNA sequence has a 571-amino acid coding region indicating a protein of 64.7 kDa with a type II domain structure. The deduced protein sequence showed significant similarity to a recently cloned bovine polypeptide GalNAc-transferase (Homa, F.L., Hollanders, T., Lehman, D.J., Thomsen, D.R., and Elhammer, A.P. (1993) J. Biol. Chem. 268, 12609-12616). A polymerase chain reaction construct was expressed in insect cells using a baculovirus vector. Northern analysis of eight human tissues differed clearly from that of the bovine GalNAc-transferase. Polymerase chain reaction cloning and sequencing of the human version of the bovine transferase are presented, and 98% similarity at the amino acid level was found. The data suggest that the purified human GalNAc-transferase is a novel member of a family of polypeptide GalNAc-transferases, and a nomenclature GalNAc-T1 and GalNAc-T2 is introduced to distinguish the members.

Amino Acid Sequence↗

PubMed1995

Structural characterization of thymidine phosphorylase purified from human placenta.

Human thymidine phosphorylase (dThdPase) is thought to be identical to an angiogenesis factor, platelet-derived endothelial cell growth factor (PD-ECGF). However, the whole amino acid sequence of dThdPase is still unknown. N-terminal amino acid sequencing of dThdPase isolated from human placenta gave the sequence Ac-AALMTPGTGAPPAPG. Comparison with the sequence predicted from the PD-ECGF cDNA reveals that residues 2-16 of dThdPase are identical to that of PD-ECGF. If dThdPase and PD-ECGF are derived from the same gene, the primary translational product of dThdPase would be processed one amino acid from the translation-initiating methionine residue and Ala-2 acetylated. Since placental and platelet PD-ECGF is reported to be processed at Thr-6 and Ala-11, respectively, and the N-terminal end is not blocked, further study is needed to clarify the reason for this discrepancy and whether the difference in N-terminal sequence affects the physiological function of these molecules.

Amino Acid Sequence↗

PubMed1995

Proline-directed and non-proline-directed phosphorylation of PHF-tau.

To gain insight into the abnormal phosphorylation of PHF-tau, we have determined the phosphorylation sites by identifying phosphopeptides by means of ion spray mass spectrometry followed by sequencing of ethane-thiol-modified peptides. Nineteen sites have been identified; all but Ser-262 are localized to the amino- and carboxyl-terminal flanking regions of the microtubule-binding domain. Eleven sites correspond to fetal type sites. Unexpectedly, 10 are non-proline-directed, whereas the others are proline-directed. Thus, the abnormal phosphorylation of PHF-tau can be considered to consist of fetal type phosphorylation and additional proline-directed and non-proline-directed phosphorylation. This non-fetal type phosphorylation may provide PHF-tau with the unusual characteristics.

Alzheimer Disease↗

PubMed1995

The complete amino acid sequences of two serine proteinase inhibitors from the fruiting bodies of a basidiomycete, Pleurotus ostreatus.

The complete amino acid sequences of two isomeric endogenous inhibitors, IA-1 and IA-2, both of which specifically inhibit an intracellular serine proteinase (proteinase A) purified from the fruiting bodies of a higher basidiomycete, Pleurotus ostreatus, were determined. Both inhibitors are acidic polypeptides with respective molecular masses of 8307 and 8244 Da, as determined by plasma desorption mass spectral analyses, and their N-terminal serine residue is blocked by acetylation. The fragments generated from the inhibitors by proteolytic and chemical cleavages were subjected to amino acid composition, sequence, and mass spectral analyses. The sequence and molecular mass information for the peptides established that the inhibitors both consisted of 76 amino acid residues and differed from each other in that aspartic acid and glutamic acid residues at residues 12 and 15 of IA-1 were replaced by glycine and aspartic acid in IA-2, respectively. The molecular masses of IA-1 and IA-2 were calculated to be 8309 and 8237, based on the sequence data. The action of carboxypeptidase Y on IA-1 resulted in a complete loss of the inhibitory activity along with successive release of glutamine and threonine from the C-terminus. Cyanogen bromide cleavage of Met38-Pro39 and Met41-Lys42 in IA-1 and hydroxylamine degradation of IA-2 completely abolished their inhibitory activity. These results suggest that the whole molecules of both inhibitors are essential to their inhibitory activities. Their structural resemblance to propeptides of subtilisin family proteinases revealed their mechanism of action.

Amino Acid Sequence↗

PubMed1995

Site-specific glycosylation of bovine butyrophilin.

Our previous studies showed that the N-linked sugar chains of most bovine glycoproteins from milk fat globule membranes (MFGM) contain the GalNAc beta 1-->4GlcNAc group [Sato et al. (1993) J. Biochem. 114, 890-900]. Since expression of the disaccharide structure is influenced by peptide sequences near the glycosylation sites [Smith and Baenziger (1992) Proc. Natl. Acad. Sci. USA 89, 329-333], the site-specificity of the N-acetylgalactosaminylated sugar chains was investigated using bovine butyrophilin, a major MFGM glycoprotein with known primary structure. Two glycopeptide fragments which contained the N-linked sugar chains linked to either Asn-55 or Asn-215 residue were obtained by digestion of the protein with Achromobacter protease I. The sugar chains released from each glycopeptide by hydrazinolysis were reduced with NaB3H4. Structural analyses of the oligosaccharides by sequential exoglycosidase digestion and methylation analysis revealed that only complex-type sugar chains with the GalNAc beta 1-->4GlcNAc structure are included in Asn-55-linked oligosaccharides, while only novel hybrid-type sugar chains detected previously in bovine MFGM glycoproteins are included in Asn-215-linked oligosaccharides. The results show that the glycosylation of butyrophilin occurs in a site-specific manner.

Amino Acid Sequence↗

PubMed1994

Purification and characterization of UDP-GlcNAc:IV3 beta Gal-Gb4Cer beta-1,6-GlcNAc transferase from mouse kidney.

The Gsl-5 gene mapped on mouse chromosome 19 controls the expression of IV6 beta Gal beta 1-4(Fuc alpha 1-3)GlcNAc, IV3 beta Gal-Gb4Cer in mouse kidney through regulation of the activity of UDP-GlcNAc:IV3 beta Gal-Gb4Cer beta-1,6-Glc-NAc transferase (GNT), which transfers GlcNAc to the C-6 position of GalNAc of IV3 beta Gal-Gb4Cer (GL-X). Here we report that GNT has been purified to apparent homogeneity from mouse kidney by means of preparation of a microsomal fraction, solubilization with Triton X-100, and sequential column chromatographies on CM-Sepharose, UDP-hexanolamine-Sepharose, and Gg4Ose-Aminocellulo-fine. GNT purified 11,000-fold from the microsomal fraction exhibited a specific activity of 26.15 mumol/min/mg protein and an apparent molecular mass of 50 kDa on SDS-polyacrylamide gel electrophoresis. The Km values for UDP-GlcNAc and GL-X were 0.36 and 0.11 mM, respectively. Among glycolipids tested as substrates, GL-X was the best and Gg4Cer the next best, but Lc3Cer, Gb4Cer, and GM1 did not act as a substrate. GNT was also able to transfer GlcNAc to Gal beta 1-3GalNAc beta 1-benzyl and Gal beta 1-3GalNAc alpha 1-p-nitrophenyl at C-6 of GalNAc through beta linkage but not to GlcNAc beta-3GalNAc alpha 1-p-nitrophenyl. The purified GNT was digested with lysyl endopeptidase, and four peptides generated were sequenced. The sequence of four peptides spanning 35 amino acid residues in total exhibited 80% homology with that of the reported human core 2 GlcNAc transferase.

Amino Acid Sequence↗

PubMed1994

Alternatively spliced isoform of P-selectin is present in vivo as a soluble molecule.

To demonstrate the presence of a soluble isoform of P-selectin predicted from cDNA sequencing (Johnston, G.I., Bliss, G.A., Newman, P.J., and McEver, R.P. (1990) J. Biol. Chem. 265, 21381-21385), we immunoisolated and compared structurally P-selectin from fresh frozen human plasma with that from washed intact platelets. Plasma P-selectin was reactive with rabbit antiserum to a synthesized peptide (residues 762-774 of mature P-selectin) but was significantly less reactive with antibody to a peptide (residues 747-760). In contrast, platelet P-selectin reacted with both antibodies. S-Pyridylethylated plasma P-selectin was fractionated by reversed phase-high performance liquid chromatography into two major species. From platelets, two virtually identical species were separated. Sequential digestion with Achromobacter protease I and then Staphylococcus V8 protease produced peptides assigned to the tail region of the protein including the putative spliced site. From the more hydrophilic species in both plasma and platelets, a peptide completely lacking the sequence of the putative spliced site was identified. In contrast, the more hydrophobic species yielded a peptide with an intact transmembrane sequence. Hence, these results provide direct evidence that the previously predicted soluble isoform of P-selectin is actually synthesized in vivo and is present as a circulating molecule.

Alternative Splicing↗

PubMed1994

Properties and structure of spermidine acetyltransferase in Escherichia coli.

Spermidine acetyltransferase (SAT) from Escherichia coli was purified about 40,000-fold. The molecular mass of native SAT was 95 kDa, and it consisted of four identical subunits. The products formed from the reaction of acetyl-CoA with spermidine by SAT were N1- and N8-acetylspermidine. The Km values for acetyl-CoA, spermidine, and spermine were 2 microM, 1.29 mM, and 220 microM, respectively. The enzymatic activity increased by 2.5-3.5-fold under the condition of poor nutrition but not in response to cold shock or high pH. By using synthetic oliogonucleotides deduced from amino acid sequences of the peptides in SAT, a polymerase chain reaction product with a length of 250 nucleotides was obtained. Using this polymerase chain reaction product, the gene encoding SAT (speG) was cloned and mapped at 35.6 min in the E. coli chromosome. E. coli cells transformed with the cloned speG gene increased SAT activity by 8-40-fold. The gene encoded a 186-amino acid protein, but SAT consisted of 185 amino acids because the initiator methionine was liberated from the protein. Thus, the predicted molecular mass was 21,756 Da. Significant similarity to aminoglycoside acetyltransferase and peptide N-acetyltransferase was observed in the amino acid sequence 87-141, and some similarity with spermidine-preferential binding protein (potD protein) in the spermidine-preferential uptake system was observed in the amino acid sequence 122-141. The results suggest that the active center of SAT may be located in the COOH-terminal portion.

Acetyl Coenzyme A↗

PubMed1994

Localization of activin and follistatin proteins in the Xenopus oocyte.

We found a binding protein for activin and follistatin in serum from female Xenopus laevis and identified it as vitellogenin, which is synthesized in the liver and transported into yolk platelets. Then, we investigated the localization of activin and follistatin proteins in early Xenopus oocytes (stage 6) by electron microscopic immunolabeling with gold colloidal particles. The protein molecules were found to be localized uniformly in oocyte yolk platelets, but not in other cytoplasmic organelles. These findings suggest a novel role of yolk platelets as a reservoir for inductive signals transported by vitellogenin in the differentiation and patterning of cells in Xenopus embryos.

Activins↗

PubMed1994

Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23.

Complementation group C of xeroderma pigmentosum (XP) represents one of the most common forms of this cancer-prone DNA repair syndrome. The primary defect is located in the subpathway of the nucleotide excision repair system, dealing with the removal of lesions from the non-transcribing sequences ('genome-overall' repair). Here we report the purification to homogeneity and subsequent cDNA cloning of a repair complex by in vitro complementation of the XP-C defect in a cell-free repair system containing UV-damaged SV40 minichromosomes. The complex has a high affinity for ssDNA and consists of two tightly associated proteins of 125 and 58 kDa. The 125 kDa subunit is an N-terminally extended version of previously reported XPCC gene product which is thought to represent the human homologue of the Saccharomyces cerevisiae repair gene RAD4. The 58 kDa species turned out to be a human homologue of yeast RAD23. Unexpectedly, a second human counterpart of RAD23 was identified. All RAD23 derivatives share a ubiquitin-like N-terminus. The nature of the XP-C defect implies that the complex exerts a unique function in the genome-overall repair pathway which is important for prevention of skin cancer.

Amino Acid Sequence↗

PubMed1994

Purification and amino- and carboxyl-terminal amino acid sequences of alanine-glyoxylate transaminase 1 from human liver.

In order to confirm the amino acid sequence predicted from the nucleotide sequence of cDNA and also to elucidate the intracellular localization and molecular evolution, human liver alanine-glyoxylate transaminase 1 (AGT1) was purified and subjected to partial amino acid sequence determination, with special attention to posttranslational modification. The enzyme was purified to homogeneity from the 10,000 x g supernatant of human liver homogenate. The purified enzyme showed only a single protein band at about 43 kDa on SDS-PAGE, indicating that it is a homodimer of two identical subunits, because the native enzyme has a molecular mass of about 80 kDa. Both the amino- and carboxyl-terminal peptides of the enzyme were isolated from a cyanogen bromide digest of the S-carboxyl-methylated protein and subjected to amino acid sequence determination. The alpha-amino group of the amino-terminal peptide was shown to be blocked by an acetyl group. The carboxyl-terminal sequence contained a putative N-glycosylation sequence (-Asn-Ala-Thr-), the only one present in the whole molecule, but this sequence was normally determined, indicating that the enzyme is not N-glycosylated. Purdue et al. [J. Cell Biol. 111, 2341-2351 (1990)] have reported that Pro-11, Gly-170, and Ile-340 in normal human AGT1 were replaced by Leu, Arg, and Met, respectively, in a patient with primary hyperoxaluria type 1. We confirmed that residue-11 was Pro. Both the amino- and carboxyl-terminal sequences of the enzyme showed extensive similarity with those of rat liver mitochondrial serine-pyruvate aminotransferase and the small chain of hydrogenase from a thermophilic unicellular cyanobacterium, Synechococcus PCC 6716.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine Transaminase↗

PubMed1994

Multiple copies of the proB gene enhance degS-dependent extracellular protease production in Bacillus subtilis.

Bacillus subtilis secretes extracellular proteases whose production is positively regulated by a two-component regulatory system, DegS-DegU, and other regulatory factors including DegR. To identify an additional regulatory gene(s) for exoprotease production, we performed a shotgun cloning in the cell carrying multiple copies of degR and found a transformant producing large amounts of the exoproteases. The plasmid in this transformant, pLC1, showed a synergistic effect with multiple copies of degR on the production of the extracellular proteases, and it required degS for its enhancing effect. The DNA region responsible for the enhancement contained the proB gene, as shown by restriction analyses and sequence determination. The proB gene encoding gamma-glutamyl kinase was followed by the proA gene encoding glutamyl-gamma-semialdehyde dehydrogenase at an interval of 39 nucleotides, suggesting that the genes constitute an operon. pLC1 contained the complete proB gene and a part of proA lacking the proA C-terminal region. It was also found that proB on the chromosome showed a synergistic effect with multiple copies of degR. We consider on the basis of these results that the metabolic intermediate, gamma-glutamyl phosphate, would transmit a signal to DegS, resulting in a higher level of phosphorylated DegU. Possible involvement of DegR in this process is discussed.

1-Pyrroline-5-Carboxylate Dehydrogenase↗

PubMed1994

Racemization: its biological significance on neuropathogenesis of Alzheimer's disease.

Amyloid beta protein (A beta) in neuritic plaques of Alzheimer's disease has been found to be racemized and/or isomerized at their Asp residues. To elucidate the effect of racemization on the aggregation properties of A beta, we synthesized three kinds of A beta peptides in which D-Asp was substituted for L-Asp residues, i.e, normal A beta 1-40, [D-Asp7]A beta 1-40 and [D-Asp23]A beta 1-40. The aggregation and fibril formation of each peptide was examined by means of spectrofluorometry and electron microscopy. Of the three peptides, normal A beta showed the gradual increase of aggregation while [D-Asp7]A beta 1-40 and [D-Asp23] A beta 1-40 showed more enhanced aggregation at the final stage when the fibril formations were detected in all peptides solutions by electron microscopy. A comparative immunohistochemical study by anti-racemized A beta antibody and anti-A beta 1-42/43 antibody further showed the in vivo incorporation of D-Asp in senile plaques of Alzheimer's disease brains, which may be involved in plaque formation at the later stage than the deposition of the longer form of A beta (A beta 1-42/43). Taken together with the recent accumulated evidence on the aggregation mechanisms of A beta, the data presented here suggest that racemization may occur after the amyloid fibril formation but enhance the aggregation process by shifting the equilibrium of A beta from the soluble form to the insoluble form in Alzheimer's disease.

Alzheimer Disease↗