Biomedical subjects

T Yoshimura

Publications and source records attributed to T Yoshimura.

At least 415 records · Page 23Linked to original sources

PubMed1994

[Effects of "low dose" aprotinin in open heart surgery and its plasma concentration].

To determine the effects of "low dose" aprotinin in open heart surgery, we administrated it in 109 cases of CABG or prosthetic valve replacement. Patients in Group A (Aprotinin) received continuous infusion of aprotinin at the rate of 6*10(5) KIU/hr from the induction of anesthesia to the start of ECC (extra corporeal circulation), and from the end of ECC to the end of blood stanching. They also received additional 5*10(5) KIU aprotinin in ECC prime volumes. 50 patients in Group C (Control) received neither aprotinin nor placebo. Bleeding after ECC (Group A: 315.7 +/- 203.1 ml, Group C: 484.3 +/- 598.5 ml p < 0.01), the first 6 hours drainage (Group A: 273.0 +/- 210.0 ml, Group C: 404.7 +/- 243.2 ml p < 0.01), the first 24 hours drainage (Group A: 510.2 +/- 248.0 ml, Group C: 721 +/- 317.7 ml p < 0.01) was significantly reduced in Group A. Amounts of homologous blood transfusion were significantly reduced in group A. (Group A: 4.7 +/- 5.3 units, Group C: 8.5 +/- 6.4 units p < 0.01). Among the patients with autologous blood transfusion, the rate of the patients without homologous transfusion was significantly higher in Group A (group A: 40/66 cases, Group C: 8/33 cases p < 0.01). The ECC time and the operation time was significantly shorter in Group A. There were 69 patients (Group A: 40, Group C: 29) whose ECC time was longer than 120 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

PubMed1994

[A family of hereditary motor and sensory neuropathy type I with a new type of myelin P0 mutation].

A 26-year-old man had complaints of insidiously progressive muscle weakness of the legs, worse in the right leg than in the left. Slight to moderate degrees of asymmetrical muscular atrophy and weakness of the distal lower limb muscles, greater in the right leg than in the left, without fasciculation, were also observed. Pes equinovarus deformity of both feet was obvious. Muscle stretch reflexes were decreased in the upper limbs and absent in the lower limbs, without pathologic reflexes. Vibratory sensation was moderately decreased in the toes. The right median and tibial motor nerve conduction velocities were 19.4 and 10.5 m/sec, respectively, with a markedly prolonged distal latency. No nerve action potentials were elicited from stimulation of the right and left sural nerves. A fascicular biopsy of the right sural nerve was performed. The myelinated fibers showing segmental de- and remyelination were frequently found in teased fiber preparations. Both demyelinated and remyelinated axons and onion-bulbs were frequently observed by light and electron microscopy in the Epon-embedded sections. Based on the neurological examinations and nerve conduction studies of the family members, an elder brother, father and grandmother of the proband were found to be affected by polyneuropathy. However, the mother, an uncle, an aunt, and a cousin of the proband were normal. Therefore, we concluded that this family had HMSN type I with autosomal dominant inheritance.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Endocytosis of lysosomal acid phosphatase; involvement of mannose receptor and effect of lectins.

Acid phosphatase and beta-glucosidase are unique among lysosomal enzymes in that they have both high mannose and complex type sugasr chains, whereas oligosaccharide chains of lysosomal enzymes in matrix are of high mannose type. We have previously shown that beta-glucosidase was endocytosed into macrophages via an unidentified receptor different from a mannose/fucose receptor (K. Imai, Cell Struct. Funct. 13, 325-332, 1988). Here, we show that uptake of acid phosphatase purified from rat liver lysosomes into rat macrophages was inhibited by ligands for a mannose/fucose receptor and was mediated via an apparently single binding site with Kuptake of 24.7 nM. These results indicate that acid phosphatase and beta-glucosidase recognize different types of receptors even if they have similar sugar chains. Polyvalent concanavalin A which binds both to the enzyme and to macrophages specifically stimulated the uptake in a dose dependent manner, whereas wheat germ agglutinin and phytohaemagglutinin did not.

Acid Phosphatase↗

PubMed1994

[Aortic valve replacement in patients older than 75 years of age].

From 1988 to 1992, 16 patients older than 75 years underwent AVR (14 cases) or AVR+MVR (two cases). All patients were followed up for an average of 2.4 years after the operation and follow-up totaled to 38 patient-years. There were no hospital death and one late death. The survival rate was 93.8% through 1 to 5 years and 15 patients are now in NYHA class I or II. The problems of AVR for elderly patients were calcification and small annulus. Decalcification using CUSA was effective technique and supraannular fixing of bioprosthetic valve avoided from aortic annular enlargement. The improved quality of life after AVR supports the aggressive surgery in elderly population.

Aged↗

PubMed1994

Tumor-derived monocyte chemoattractant protein-1 induces intratumoral infiltration of monocyte-derived macrophage subpopulation in transplanted rat tumors.

By immunohistochemistry using anti-rat macrophage monoclonal antibodies RM-1, ED1, ED2, ED3, TRPM-3, and Ki-M2R, we studied transplanted rat tumors of 9L (rat gliosarcoma), Ad-2 (rat mammary carcinoma), and MT-P (rat malignant fibrous histiocytoma) cell lines to examine the distribution pattern of macrophages within and around the tumors. Most tumor-associated macrophages expressed RM-1, ED1, and Ia antigens, indicating activated macrophages. Based on differences in their immunophenotypical expression, these macrophages were distinguished into two major subpopulations. One expressed TRPM-3 and/or ED3, and the other was positive for ED2 and Ki-M2R. The former was considered to be monocyte-derived macrophages, whereas the latter showed the immunophenotype of tissue-fixed, resident macrophages. Infiltration and distribution patterns in the two macrophage subpopulations differed in the three different tumors. Monocyte-derived, activated macrophages infiltrated into 9L- and Ad-2-transplanted tumors, which markedly produced monocyte chemoattractant protein-1 (MCP-1). Additionally, numerous ED2- and Ki-M2R-positive macrophages were observed within the Ad-2-transplanted tumors, and some of them expressed TRPM-3. However, there were few macrophages in the MT-P-transplanted tumors that showed no MCP-1 production. In transplanted tumors of four MT-P/MCP-1 cell lines established by transfecting a rat MCP-1 gene expression vector (pCEP4/MCP-1) into the MT-P cell line, different levels of MCP-1 production were detected, which correlated well with the numbers of intratumorally infiltrated TRPM-3-positive macrophages. In contrast, ED2- and Ki-M2R-positive macrophages were not detected in any MT-P/MCP-1-transplanted tumors. MT-P/MCP-1-transplanted tumors exhibited lower growth rate than parental MT-P-transplanted tumors. These results indicate that tumor-derived MCP-1 induces intratumoral infiltration of monocyte-derived macrophages, but not macrophages with the immunophenotype of tissue-fixed, resident type. The former population of macrophages seems to have a suppressive effect on the growth of tumors.

Animals↗

PubMed1994

[A case of cerebral venous sinus thrombosis occurring in ulcerative colitis].

A 33-year-old man with a 13-year history of ulcerative colitis developed generalized clonic convulsions after transient right hemiparesis. A computed tomographic scan revealed a hemorrhagic lesion in the left frontal lobe and contrast tomography demonstrated delta-sign in the occipital area. A sagittal gadolinium-contrast magnetic resonance imaging scan demonstrated a low signal intensity area in the superior sagittal sinus. The venous phase of his carotid angiogram showed a lack of filling of the superior sagittal sinus. Coagulation studies revealed abnormal findings, elevated fibrinogen and increased aggregation of platelets. Cerebral venous or sagittal thrombosis occurring in ulcerative colitis is very rare in Japan. But thromboembolism is known as one of the extraintestinal complications of ulcerative colitis, and a hypercoagulable state may occur even in the inactive state. Ulcerative colitis should be considered as one of the etiologies of thromboembolism in addition to the results of coagulation studies for the prevention of cerebrovascular diseases.

Adult↗

PubMed1994

[A case of Cabrol's procedure in which selective cerebral perfusion was necessitated].

A 69-year-old man underwent Cabrol's procedure for annuloaortic ectasia and complicated multiple cerebral infarction. Preoperative CT scan showed atherosclerotic changes from the aortic arch to descending aorta. During extracorporeal circulation, arterial cannulation was made through left subclavian artery to prevent cerebral infarction due to detouched atheroma from the descending aorta. Intraoperatively, the ascending aorta was also atheromatous and ulcerative. In this case, cerebral infarction was most likely due to detouched atheroma from the aortic arch in retrograde blood flow during extracorporeal circulation. To prevent this complication, selective cerebral perfusion during extracorporeal circulation should have been selected.

Aged↗

PubMed1994

Effects of estrogen and progesterone on platelet-activating factor acetylhydrolase activity in ovariectomised rats.

OBJECTIVE: To evaluate the effect of estrogen and progesterone on plasma platelet-activating factor acetylhydrolase (PAF-AH) activity in ovariectomized rats. METHODS: Plasma PAF-AH activity was examined in ovariectomized rats treated with 17 alpha-ethynylestradiol (0.25 mg/kg; 6 days) or medroxyprogesterone (50 mg/kg; 6 days). The intra- and inter-assay coefficients of variation for plasma PAF-AH activity were 2.5% and 4.4%, respectively. Plasma lipoprotein profiles on day 5 were determined with commercially available kits. RESULTS: Ovariectomized rats showed no significant change in plasma PAF-AH activity over a 14 day period. Estrogen decreased the PAF-AH activity up to 85%, and progesterone increased PAF-AH activity up to 44% in ovariectomized rats. There was a positive correlation between plasma PAF-AH activity and high density lipoprotein cholesterol (r = 0.96, p < 0.001). CONCLUSION: These data suggest that estrogen and progesterone exert an effect on plasma PAF-AH activity in ovariectomized rats, possibly via high density lipoprotein cholesterol.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1993

cDNA cloning and expression of guinea pig neutrophil attractant protein-1 (NAP-1). NAP-1 is highly conserved in guinea pig.

cDNA for neutrophil attractant protein-1 (NAP-1, also known as IL-8) was cloned from Con A-stimulated guinea pig spleen cells with human NAP-1 cDNA as a probe. Guinea pig NAP-1 cDNA is composed of 1433 bp with an open reading frame which encodes for a 101-amino-acid protein. Guinea pig NAP-1 had 70% amino acid sequence similarity to human NAP-1, which was much higher than a similarity between human and guinea pig monocyte chemoattractant protein-1 (MCP-1) (56%). Nucleotide sequence similarity within the coding region was 75%. To confirm its biological activity in guinea pig, recombinant guinea pig NAP-1 was expressed in COS-7 cells then purified. N-terminal sequence analysis gave two different N-termini at position 23 (Met) or 24 (Val). The two proteins showed their peak activity for guinea pig neutrophils at the concentration of 1 microgram/ml (10-7 M). Despite its high similarity to human NAP-1, the responsiveness of human neutrophils to guinea pig NAP-1 was minimum. Recombinant guinea pig NAP-1 caused strong neutrophil infiltration after intradermal injection into guinea pig skin. Since guinea pig is classified as a rodent, it was of interest to know whether human NAP-1 cDNA hybridizes to genomic DNA of other rodents such as mouse or rat, in which a NAP-1 homologue has not been found. Under low stringency conditions, human NAP-1 cDNA hybridized to human, rabbit, and guinea pig DNA, but not to mouse or rat DNA. Unlike NAP-1, human MCP-1 cDNA hybridized to genomic DNA of rabbit, guinea pig, mouse, and rat; MCP-1 cDNA have been cloned from these species. The apparent absence of a NAP-1 gene in mouse or rat makes this chemoattractant unique among the members of the protein family to which NAP-1 and MCP-1 belong.

Amino Acid Sequence↗

PubMed1993

Quantitative study of monocyte chemoattractant protein-1 (MCP-1) in cerebrospinal fluid and cyst fluid from patients with malignant glioma.

BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) is a 76-amino acid protein that attracts monocytes. In vitro studies have reported high levels of MCP-1 messenger RNA expression, as well as the presence of MCP-1, in malignant glioma cells. PURPOSE: Our purpose was to determine whether an MCP-1 assay could be used in a clinical setting 1) to differentiate malignant from benign gliomas and from nontumor disorders of the central nervous system and 2) to detect subarachnoid dissemination of glioma cells. METHODS: MCP-1 levels in cerebrospinal fluid (CSF) and cyst fluid were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) that we had previously developed. We measured MCP-1 levels in CSF samples from 19 patients with malignant glioma (glioblastoma, 10; anaplastic astrocytoma, six; anaplastic oligodendroglioma, two; and ependymoblastoma, one), nine patients with benign glioma, and seven patients with nontumor disorders of the central nervous system. Cyst fluids from four patients with malignant glioma (anaplastic astrocytoma) were also tested. The correlation between MCP-1 concentration in the CSF and subarachnoid dissemination of malignant glioma cells was also studied. RESULTS: The MCP-1 concentration (mean +/- SE) in CSF samples from patients with malignant glioma (2.3 +/- 0.4 ng/mL) was significantly higher than that from patients with benign glioma (0.6 +/- 0.1 ng/mL) (P < .01) or from patients with no tumor (0.5 +/- 0.1 ng/mL) (P < .01). Furthermore, CSF samples from patients with subarachnoid dissemination of malignant glioma contained significantly higher amounts of MCP-1 than those from patients without dissemination (P < .05). Cyst fluids from four of the patients with malignant glioma contained high concentrations of MCP-1. CONCLUSIONS: These results indicate that MCP-1 is produced by malignant glioma in vivo as well as in vitro and suggest that testing for MCP-1 in CSF may be useful in the clinic to differentiate malignant glioma from benign glioma and to detect subarachnoid dissemination of the tumor cells. IMPLICATIONS: The MCP-1 ELISA in CSF may lead to more accurate diagnosis of malignant glioma and detection of subarachnoid dissemination of tumor cells, facilitating selection of patients with these conditions for appropriate therapy.

Adolescent↗

PubMed1993

Expression of glr (murI, dga) gene encoding glutamate racemase in Escherichia coli.

The murI (dga) gene of Escherichia coli is required for the biosynthesis of D-glutamate, an essential component of bacterial peptidoglycan (Doublet, P., van Heijetnoort, J., and Mengin-Lecreulx, D. (1992) J. Bacteriol. 174, 5772-5779; Dougherty, T. J., Thanassi, J. A., and Pucci, M. J. (1993) J. Bacteriol. 175, 111-116), but its gene product has not been identified. We found that the amino acid sequence of protein deduced from the nucleotide sequence of the open reading frame of murI gene (ORF1) shows a significant homology with that of glutamate racemase of Pediococcus pentosaceus. The amino acid sequence of glutamate racemase of Lactobacillus fermenti recently reported also shows a homology with the deduced amino acid sequence of ORFI (Gallo, K. A., and Knowles, J. R. (1993) Biochemistry 32, 3981-3990). The murI (dga) gene was ligated into a plasmid, pKK223-3, with a designed ribosome binding site and expressed in E. coli JM109 cells. Glutamate racemase was produced by the transformant cells, whereas the enzyme was not found in the host cells. Accordingly, we newly termed the gene glr, which is more relevant than murI and dga. We partially purified the enzyme to characterize it. The enzyme consists of two identical subunits with a molecular weight of about 31,000 in contrast to the P. pentosaceus enzyme, a monomer protein.

Amino Acid Isomerases↗

PubMed1993

Neutrophil attractant protein-1 and monocyte chemoattractant protein-1 in human serum. Effects of intravenous lipopolysaccharide on free attractants, specific IgG autoantibodies and immune complexes.

We recently found that normal human sera contain IgG antibodies against two chemoattractants, neutrophil attractant protein-1 (NAP-1/IL-8) and monocyte chemoattractant protein-1 (MCP-1), as well as immune complexes of these proteins. Intravenously administered LPS was reported to cause a sharp rise in serum NAP-1 concentration. Our study was designed to determine if LPS also caused an increase in MCP-1 and to measure associated changes in concentrations of antibody and immune complex. LPS caused a rise to peak within 2 to 3 h in serum concentrations of free NAP-1 and MCP-1, followed by an almost equally rapid fall toward base-line levels by about 5 h postinjection. MCP-1 concentration in sera from the 11 subjects rose to a peak of 330 +/- 52 pM. The peak value for NAP-1 was 80 +/- 11 pM. In 10 of the 11 subjects, free IgG autoantibody to MCP-1 decreased from a mean pre-LPS value of 1820 +/- 660 pM to a mean low of 53% of the respective initial values. Corresponding data for IgG anti-NAP-1 were a pre-LPS concentration of 216 +/- 7 pM, which decreased to a mean low of 44% of the respective initial values. The finding in some subjects of a rapid rise in free antibody after the nadir suggests the possibility of acute regulation of autoantibody secretion rates. Although the results suggested that LPS-induced chemoattractant combined with free antibody, serum concentrations of MCP-1-IgG or NAP-1-IgG did not increase, which points to an as yet unknown mechanism for trapping and elimination of the immune complexes.

Adult↗

PubMed1993

Role reversal for substrates and inhibitors. Slow inactivation of D-amino acid transaminase by its normal substrates and protection by inhibitors.

D-Amino acid transaminase, which catalyzes the synthesis of D-alanine and D-glutamate for the bacterial cell wall, is a candidate for the design of specific inhibitors that could be novel antimicrobial agents. Under the experimental conditions usually employed for enzyme assays, kinetic parameters for its substrates were determined for short incubation periods, when intermediates and products do not accumulate and the enzyme activity is linear with time. Such kinetic analyses indicate that the enzyme accepts most D-amino acids but D-aspartate and D-glutamate are the best substrates. Under a different type of experimental conditions when the enzyme is exposed to D-alanine, intermediates, and products for periods of hours, it slowly becomes inactivated (Martinez del Pozo, A., Yoshimura, T., Bhatia, M. B., Futaki, S., and Manning, J. M. (1992) Biochemistry 31, 6018-6023). We now report that D-aspartate, D-glutamate, and L-alanine also lead to slow inactivation. Methylation or amidation of the alpha-COOH group of D-alanine prevents inactivation, indicating that decarboxylation is required for inactivation; the slow release of CO2 from substrate is demonstrated. The alpha-methyl analog of D-alanine, D-aspartate, and D-glutamate do not lead to inactivation, showing that the alpha-hydrogen of the substrate is required, i.e. that some processing is required. Lys145, which binds pyridoxal 5'-phosphate in the wild-type enzyme, is not involved in the inactivation since two active site mutant enzymes, K145Q and K145N, are also inactivated. Reactivation of the inactive enzyme at acidic pH is accompanied by the release of ammonia corresponding to 1 mol/mol of dimeric enzyme. Competitive inhibitors, amine-containing buffers, and thiols effectively impede the inactivation. This reversal in the roles of substrates and inhibitors, i.e. when a substrate can be an inactivator and an inhibitor can act as a protector, occurs during a time period not usually used to measure steady-state kinetics or initial velocities of enzyme reactions and could have physiological relevance in cells.

Amino Acids↗

PubMed1993

Estrogen-inducible pS2 protein is not the key regulatory component in the proliferation of human breast cancer cells (MCF-7).

Exposure of human breast cancer cells (MCF-7) to tumor promoters such as 12-O-tetradecanoyl phorbol 13-acetate (TPA) for 24 h at concentrations of 1-100 nM resulted in marked inhibition of DNA synthesis but a 3-5-fold increase in the amount of pS2 protein in the medium. These results support our previous suggestion that pS2 protein is not involved in the mechanism controlling proliferation of MCF-7 cells. During treatment with TPA, the intracellular content of pS2 protein was constant, suggesting that TPA did not induce secretion of pS2 protein but rather de novo synthesis of the protein. The increase in the pS2 protein content of the medium by TPA was inhibited by simultaneous addition of cycloheximide, but not by that of actinomycin D. Northern-blot hybridization analysis showed that the amount of pS2 mRNA was unchanged by treatment of the cells with TPA. These results indicate that TPA does not induce transcription of the pS2 gene, and suggest that the main effect of TPA results from the induction of translation of pS2 mRNA.

Biological Transport↗

PubMed1993

Cloning, sequencing, and expression of human macrophage stimulating protein (MSP, MST1) confirms MSP as a member of the family of kringle proteins and locates the MSP gene on chromosome 3.

A human hepatoma (HepG2) cell line library was screened with an oligonucleotide probe for macrophage stimulating protein (MSP) to clone an MSP cDNA. Deduced sequences of isolated clones were compared with peptide fragment sequences of MSP. MSP9 cDNA encoded most of the known sequence of MSP except for a small segment of the 5' end of the open reading frame. Consequently, a hybrid 2300-base pair cDNA that encoded the complete MSP amino acid sequence was constructed from 2 clones. Culture fluid from COS-7 cells transfected with this full-length MSP cDNA had MSP biological activity, and the expressed MSP was detected by immunoprecipitation with antibody against native MSP. The deduced amino acid sequence of MSP includes 4 kringle domains, which have been found in hepatocyte growth factor and several proteins of the blood coagulation system. Among them, MSP has the highest sequence similarity to hepatocyte growth factor (45% identity). The MSP cDNA hybridized strongly to mRNA from liver, and to a lesser extent to mRNA from kidney and pancreas, suggesting that a cell type in the liver is the source of MSP. Several cloned and sequenced MSP cDNAs had insertions or deletions, suggesting that alternatively spliced MSP mRNAs may occur. This was reflected in Northern blots probed with an MSP cDNA, which showed more than one mRNA species. Furthermore, although the gene coding for MSP is on chromosome 3, the sequence of one of the cDNAs was identical with a unique sequence in chromosome 1, indicating that there may be a family of MSP genes, located on chromosomes 3 and 1.

Amino Acid Sequence↗

PubMed1993

Involvement of protein kinase C in the proliferation of cultured Schwann cells.

We investigated the signal transduction system when the proliferation of cultured Schwann cells was activated by glial growth factor (GGF). When 10 micrograms/ml of GGF was added to the culture medium, Schwann cell division was activated by about 3-fold, while protein kinase C (PKC) activity was translocated from the cytosol to the particulate fraction, and diacylglycerol (DG) production was increased in the cells. PKC activity in the particulate fraction and intracellular DG content increased to 167% and 158%, respectively. However, intracellular cyclic AMP levels remained unchanged. The GGF-induced proliferation of Schwann cells was significantly inhibited by staurosporine, a PKC inhibitor, in a dose-dependent manner, while H-8, a specific inhibitor for cyclic nucleotide-dependent protein kinases, did not show an inhibitory effect even at a high concentration. These data suggest that the signal transduction system through PKC is involved in the proliferation of Schwann cells when treated with GGF.

Animals↗

PubMed1993

cDNA cloning of guinea pig monocyte chemoattractant protein-1 and expression of the recombinant protein.

Monocyte chemoattractant protein-1 (MCP-1) cDNA was cloned from guinea pig spleen cells stimulated with Con A. The cDNA comprised 647 bp with an open reading frame that encoded a 120 amino acid protein. The sequence similarity of the first 99 amino acids to human MCP-1 is 56%. Recombinant guinea pig MCP-1 was expressed in COS-7 cells, then purified by a three step procedure with orange A-agarose, carboxymethyl-HPLC, and reversed phase-HPLC. The purified protein was found around 25 kDa as a broad band on a polyacrylamide gel under reducing conditions. Guinea pig MCP-1 attracted about 34% of input human monocytes at 5 x 10(-9) M. Guinea pig peritoneal exudate macrophages migrated toward guinea pig MCP-1 dose dependently, but only 1% of input cells responded to guinea pig MCP-1 at its optimal concentration of 5 x 10(-9) M. Human MCP-1 attracted 1% of input guinea pig peritoneal exudate macrophages at its highest concentration of 2.5 x 10(-8) M. Neither human nor guinea pig MCP-1 attracted guinea pig peritoneal resident macrophages. These results suggest that monocytes lose responsiveness to MCP-1 after differentiating to macrophages. Finally, intradermal injection of the recombinant protein into guinea pigs caused marked macrophage infiltration. Cloned and expressed guinea pig MCP-1 will help in studying the role of MCP-1 in vivo.

Amino Acid Sequence↗

PubMed1993

Kinetic and stereochemical comparison of wild-type and active-site K145Q mutant enzyme of bacterial D-amino acid transaminase.

D-Amino acid transaminase (EC 2.6.1.21), from Bacillus sp. YM-1, a thermostable enzyme with pyridoxal 5'-phosphate as coenzyme and a target for the design of novel antimicrobial agents, catalyzes the reversible transfer of an amino group between D-alanine and alpha-ketoglutarate to form pyruvate and D-glutamate, respectively. To explore the catalytic role of Lys-145, which binds the coenzyme, a site-specific mutant enzyme, K145Q (in which Lys-145 had been mutated to glutamine) constructed earlier (Futaki, S., Ueno, H., Martinez del Pozo, A., Pospischil, M. A., Manning, J. M., Ringe, D., Stoddard, B., Tanizawa, K., Yoshimura, T., and Soda, K. (1990) J. Biol. Chem. 265, 22306-22312) was compared to the wild-type enzyme for its kinetic parameters. Initial velocity studies and partial reaction isotope exchange experiments showed that the low activity of the mutant enzyme (about 1.5% the activity of the wild-type enzyme with saturating substrates) is an intrinsic property, confirming that contaminating enzymes do not account for the low activity of the K145Q mutant enzyme. The rates of the forward reaction for both wild-type and mutant enzymes were 30-40 times higher than the rates of the reverse reaction. KM values for the four substrates were 10 to 100 higher for the mutant compared to the wild-type enzyme. Whereas D-alanine is preferred over L-alanine by the wild-type enzyme (10(3) higher kcat/KM for D- over L-alanine), the K145Q enzyme does not efficiently discriminate between L- and D-alanine. Both wild-type and mutant enzymes also catalyze the slow racemization of L- and D-alanine. Proton NMR studies showed that wild-type enzyme catalyzed a time-dependent exchange of the C alpha proton of D-alanine with solvent D2O and a slow exchange of the alpha proton of L-alanine; the latter slow exchange rate is the same for the C alpha proton of both L- and D-alanine with the K145Q mutant enzyme. Thus, in addition to binding pyridoxal 5'-phosphate, the active-site Lys-145 of D-amino acid transaminase is involved in several other important functions, i.e. it optimizes catalytic efficiency and it maintains stereochemical fidelity. The steady-state kinetic results on the K145Q mutant enzyme together with the findings on the relative racemization rates and the NMR protein exchange data suggest that an alternate base catalyzes abstraction of the alpha proton of substrate in this mutant D-amino acid transaminase.

Bacillus↗