Biomedical subjects

S Shimizu

Publications and source records attributed to S Shimizu.

At least 397 records · Page 22Linked to original sources

PubMed1997

Reliability of frozen section diagnosis of gallbladder tumor for detecting carcinoma and depth of its invasion.

BACKGROUND: An accurate frozen section diagnosis is important when deciding the surgical strategy against a gallbladder tumor intraoperatively. Little has been reported on the accuracy of frozen section diagnosis of the gallbladder. PATIENTS AND METHODS: In a total of 86 consecutive patients with gallbladder tumor, the accuracy of the frozen section diagnosis was examined. There were 32 patients with polypoid lesions and 54 with nonpolypoid tumors. RESULTS: The frozen tissue diagnosis and final diagnosis were identical in 82 of the 86 cases, that is, benign in 65 and malignant in 17. The other four cases had different diagnoses, that is, conversion from benign to malignant in two and from malignant to benign in two. The overall accuracy of frozen diagnosis was 95.3% (97.0% for benign and 94.7% for malignant). In 32 polypoid lesions, the accuracy of frozen section diagnosis was 91% (93% for benign; 89% for malignant). In 54 nonpolypoid lesions, the accuracy of diagnosis was 98% (100% for benign; 93% for malignant). The diagnosis of depth of invasion was identical only in 7 (70%) of the 10 carcinoma cases examined, while it was diverse in the remaining 3, that is, conversion from adenocarcinoma invading the subserosa to that limiting to the mucosa in one, from carcinoma within the mucosa to that infiltrating the muscle coat in one, and from carcinoma affecting the muscle layer to that invading the subserosa in the other. Alterations of frozen section diagnosis about being benign or malignant and about the depth of invasion were encountered in seven patients, five of whom had a polypoid tumor. CONCLUSIONS: The intraoperative frozen tissue diagnosis is fairly reliable as to whether lesions are malignant or benign; however, accuracy is low in patients with polypoid lesions of the gallbladder. Also, frozen section diagnosis does not reliably measure the depth of invasion of gallbladder carcinoma.

Adenocarcinoma↗

PubMed1997

Phosphoenolpyruvate prevents the decline in hepatic ATP and energy charge after ischemia and reperfusion injury in rats.

BACKGROUND: Phosphoenolpyruvate (PEP) is a high-energy metabolite in the final step of glycolysis. PEP is converted into pyruvate by pyruvate kinase. One molecule of adenosine triphosphate (ATP) is generated from one molecule of PEP. The aim of this study was to examine the effects of PEP on hepatic energy metabolism at an early phase after ischemia and reperfusion were examined in rats. MATERIALS AND METHODS: Male Wistar rats (250-350 g) were divided into two groups; after two 15-min periods of ischemia with 2 min reperfusion in between, either PEP or glucose solution (400 mmol/liter, pH 7.4) was infused into the portal vein (2.5 ml/300 g body wt/5 min). Before and 0, 5, 10, and 30 min after ischemia, arterial blood and liver tissue were collected for analyses. RESULTS: During the two ischemic periods, ATP and total adenine nucleotide (TAN) of the liver decreased from 9.10 +/- 0.50 and 14.06 +/- 0.29 to 0.99 +/- 0.50 and 10.86 +/- 0.42 mmole/g liver, respectively (P < 0.05), while adenosine monophosphate (AMP) increased from 1.18 +/- 0.15 to 8.47 +/- 0.66 mmole/g liver (P < 0. 05). Hepatic energy charge (EC) significantly decreased from 0.78 +/- 0.02 to 0.16 +/- 0.03 (P < 0.05). Serum concentrations of pyruvate and lactate were elevated from 1.18 +/- 0.15 and 18.4 +/- 0. 52 to 3.29 +/- 0.52 and 72.6 +/- 4.8 mg/dl, respectively (P < 0.05). After a 5-min infusion of PEP or glucose solution, the ATP concentration was significantly higher in the PEP group than in the glucose group (4.08 +/- 0.58 micromole/g liver vs 2.20 +/- 0.45 micromole/g liver, P < 0.01), whereas AMP concentration was significantly lower in the PEP group than in the glucose group (4.26 +/- 0.66 micromole/g liver vs 7.02 +/- 0.71 micromole/g liver, P < 0. 01). EC in the PEP group was significantly higher than that in the glucose group (0.493 +/- 0.051 vs 0.293 +/- 0.042, P < 0.01). Ten minutes after ischemia, the ATP, TAN, and EC levels were still higher in the PEP group than in the glucose group, but the difference did not reach statistical significance. At 30 min after ischemia, these values became similar in both groups. At 5, 10, and 30 min after ischemia, serum pyruvate concentrations were higher in the PEP group than in the glucose group. CONCLUSION: These findings suggest that PEP recovers hepatic energy from liver cell damage at an early phase after ischemia and reperfusion by prompt ATP production through the degradation of PEP into pyruvate in the liver.

Adenosine Monophosphate↗

PubMed1997

Pancreatic cyst as a sentinel of in situ carcinoma of the pancreas. Report of two cases.

CONCLUSION: We would like to recommend detailed examination of the pancreas including cytology of the pancreatic juice in patients with pancreatic cyst to find possible concomitant early pancreatic carcinoma. Further study is necessary to determine whether there is a rational relationship between mucinous cystadenoma of the pancreas and pancreatic adenocarcinoma. BACKGROUND: Two cases of in situ carcinoma of the pancreas first detected with pancreatic cyst as a diagnostic clue are reported. Cytologic examination of the pancreatic juice was positive for malignancy in both cases, and pancreatic cyst and in situ carcinoma were located independently. METHODS AND RESULTS: Case 1: Ultrasonography (US) and computed tomography (CT) in a 54-yr-old Japanese man with a known gastric cancer revealed a pancreatic cyst. Endoscopic retrograde cholangiopancreatography (ERCP) showed a cyst in the tail of the pancreas, and cytology of the pure pancreatic juice revealed adenocarcinoma. Intraoperatively, the pancreas was cut along the portal vein, and cytology of the pancreatic juice from the pancreas distal to the cutting line showed adenocarcinoma. Resection of the body and tail of the pancreas was performed together with total gastrectomy. Histopathologically, the cyst was mucinous cystadenoma, and the surrounding pancreatic ducts and ductules showed epithelial dysplasia of moderate-to-severe degree having foci of unequivocal in situ carcinoma. No stromal invasion was seen. Case 2: A 55-yr-old Japanese man with known hepatocellular carcinoma was diagnosed as having pancreatic cyst in the tail of the pancreas on US and CT. ERCP showed a pancreatic cyst, and cytology of the pancreatic juice highly suggested adenocarcinoma. Distal pancreatectomy and splenectomy were performed. Histopathologic diagnosis of pancreatic cyst was mucinous cystadenoma. The pancreatic ductule 2 cm proximal to the pancreatic cyst showed carcinoma in situ. The diagnostic clue of in situ carcinoma of the pancreas in these two cases was a cystic lesion of the pancreas detected by check-up US and CT of known carcinoma of the stomach and liver. Cytology of the pancreatic juice was also positive for malignancy. In situ carcinoma of the pancreas was found to be independent of the cysts because of the different locations and divergent histopathologic natures of the two lesions.

Biomarkers↗

PubMed1997

Screening of novel microbial enzymes for the production of biologically and chemically useful compounds.

Enzymes have been generally accepted as superior catalysts in organic synthesis. Micro-organisms in particular have been regarded as treasure sources of useful enzymes. The synthetic technology using microbial enzymes or micro-organisms themselves is called microbial transformation. In designing a microbial transformation process, one of the most important points is to find a suitable enzyme for the reaction of interest. Various kinds of novel enzymes for specific transformations have been discovered in micro-organisms and their potential characteristics revealed. This article reviews our current results on the discovery of novel enzymes for the production of biologically and chemically useful compounds, and emphasizes the importance of screening enzymes in a diverse microbial world.

Alcohol Oxidoreductases↗

PubMed1997

Enzymatic production of ethyl (R)-4-chloro-3-hydroxybutanoate: asymmetric reduction of ethyl 4-chloro-3-oxobutanoate by an Escherichia coli transformant expressing the aldehyde reductase gene from yeast.

The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate (CHBE) using Escherichia coli JM109 (pKAR) cells expressing the aldehyde reductase gene from Sporobolomyces salmonicolor AKU4429 as a catalyst was studied. The reduction required NADP+, glucose and glucose dehydrogenase for NADPH regeneration. In an aqueous system, the substrate was unstable, and inhibition of the reaction by the substrate was also observed. Efficient conversion of COBE to (R)-CHBE with a satisfactory enantiomeric excess (ee) was attained on incubation with transformant cells in an n-butyl acetate/water two-phase system containing the above NADPH-regeneration system. Under the optimized conditions, with the periodical addition of COBE, glucose and glucose dehydrogenase, the (R)-CHBE yield reached 1530 mM (255 mg/ml) in the organic phase, with a molar conversion yield of 91.1% and an optical purity of 91% ee. The calculated turnover of NADP+, based on the amounts of NADP+ added and CHBE formed, was about 5100 mol/mol.

Aldehyde Reductase↗

PubMed1997

Acceleration of oxidative stress-induced endothelial cell death by nitric oxide synthase dysfunction accompanied with decrease in tetrahydrobiopterin content.

The purpose of this study was to examine whether nitric oxide (NO) synthase dysfunction accompanied with decrease in tetrahydrobiopterin (BH4) content increases H2O2-induced endothelial cell death. Endothelial cell death was measured by the release of intracellular lactate dehydrogenase (LDH). Intracellular BH4 content was changed by pretreatment with 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I, or pretreatment with sepiapterin, a substrate for the salvage pathway of BH4 synthesis, and the intracellular content was measured by high performance liquid chromatography equipped with a fluorescence detector. Moreover, production of superoxide was detected by a chemiluminescence technique using MCLA, a Cypridina luciferin analogue, for the superoxide-sensitive probe. Pretreatment with DAHP (10 mM) for 24 h decreased intracellular BH4 content to 14% and increased H2O2-induced cell death. The toxic effect of DAHP was reduced by co-pretreatment with sepiapterin (100 microM) or treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 1 mM), an inhibitor of NO synthase, but not by N(G)-methyl-L-arginine (L-NMA, 1 mM), the other inhibitor of NO synthase. Moreover, production of superoxide in endothelial cells induced by Ca2+-ionophore ionomycin (1 microM) increased by the pretreatment with DAHP, and the increase in superoxide production was blocked by L-NAME (1 mM) but not L-NMA (1 mM). Co-pretreatment with sepiapterin decreased the production of superoxide. These findings suggested that dysfunction of NO synthase with a decrease in BH4 content in endothelial cells produced superoxide instead of NO and increased the oxidative stress-induced endothelial cell death.

Animals↗

PubMed1997

Cloning of bovine embryos by multiple nuclear transfer.

The in vitro development of multiple generation bovine nuclear transferred embryos to blastocysts and their survival ability after freezing and thawing were examined. Parent donor embryos which had 20 to 50 cells were recovered from superovulated cows. Follicular oocytes matured in vitro were used as recipient oocytes. The recipient oocytes enucleated at 22 to 24 h after the onset of maturation were preactivated at 33 h. Enucleated oocytes with a donor blastomere were fused 9 h after activation by an electric stimulus and the fused oocytes were cultured in vitro (first generation). Reconstituted oocytes that had developed to the 8- to 16-cell stage 3 to 4 d after fusion were used as donor embryos for the next generation. Recloning procedures were performed twice (second and third generations). The proportion of recipient oocytes successfully fused with a blastomere increased with the cycle of nuclear transfer. Eighty to 86% of fused oocytes developed to the 2-cell stage and there was no significant difference with the generation. The proportion of reconstituted embryos receiving blastomeres derived from first generation embryos had higher developmental ability in vitro, than those derived from other generations (43 vs 31% for 8 to 16-cell stage, 37 vs 20 and 21% for blastocyst stage). The number of cloned blastocysts increased with repeated nuclear transfer (once: 6.2 +/- 4.3, twice: 19.8 +/- 9.2 and three times: 30.0 +/- 14.7) but varied greatly with each parent donor embryo. The in vitro viability of cloned blastocysts after freezing and thawing (59%) was low but not significantly different from that obtained for in vitro fertilized blastocysts (72%). After transfer of either fresh or frozen-thawed cloned blastocysts to 21 recipients, 10 of them were pregnant on Day 60. Four and 3 offspring were produced from 20 fresh and 14 frozen-thawed blastocysts,respectively.

Journal Article↗

PubMed1997

Is cutaneous plasmacytosis a distinct clinical entity?

We describe a Japanese patient with cutaneous plasmacytosis whose clinical course we observed for 5 years. We also review 26 patients with this condition, including 24 Japanese and two non-Japanese, reported in detail. This review revealed that this condition has characteristic clinical and pathologic features and should be considered a distinct clinical entity. The reason for the predominant occurrence of cutaneous plasmacytosis in Japanese patients is unknown.

Female↗

PubMed1997

Retroperitoneal schwannoma mimicking hepatic tumor in the caudate lobe.

We report a 41-year-old woman with a retroperitoneal schwannoma mimicking hepatic tumor in the caudate lobe. Dynamic computed tomography in the early phase showed an enhanced tumor (2.7 cm in diameter) in the Spiegel lobe of the liver, which compressed the inferior vena cava (IVC). We also performed left hepatic angiographic computed tomography, and found that the tumor was less enhanced. The patient underwent laparotomy under a preoperative diagnosis of primary hepatic caudate tumor with faint neovascularity. At surgery, the tumor was found to be located between the left caudate lobe and the IVC, and was resected as being of a retroperitoneal origin. This case illustrates that tumor location must be determined with great care when the mass seems to exist at the dorsal edge of the liver, and especially when the tumor is hypovascular.

Adult↗

PubMed1997

Application of hydrogen absorbing alloys to medical and rehabilitation equipment.

As power sources for rehabilitation equipment, electric, hydraulic, and pneumatic actuators have been used. However a more human-sized and higher powered actuator that can reduce the equipment size is desired. A new metal hydride (MH) actuator that uses the reversible reaction between the heat energy and mechanical energy of a hydrogen absorbing alloy has recently attracted much attention. The MH actuator is characterized by its small size, low weight, noiseless operation and a compliance similar to that of the human elbow joint. Therefore, the MH actuator has the characteristic of being light and easy to use and so is suitable for use in medical and rehabilitation applications. Some lifting devices using this actuator have already been developed and are being used for the care of the aged and disabled. The characteristics of the MH actuator are presented and then some applications are introduced in this paper. It is our opinion that in our aging society the MH actuator will play an important role in the development of medical and rehabilitation equipment.

Absorption↗

PubMed1997

Germline mutations of the MEN1 gene in Japanese kindred with multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant familial cancer syndrome. The responsible gene MEN1 has recently been isolated, and its germline mutations have been identified in affected individuals in the United States, Canada and Europe. We screened for MEN1 mutations by direct nucleotide sequencing of all protein-coding regions, and identified five distinct germline mutations in five among six Japanese kindreds with familial MEN1 or familial hyperparathyroidism. The mutations were dispersed across the gene. These findings suggest that, because of the absence of an obvious founder effect, the entire MEN1 gene region should be examined for germline mutations in the probands of MEN1 and related syndromes in Japanese families.

DNA, Neoplasm↗

PubMed1997

Effect of blood blow and haematocrit on the relationship between muscle venous PO2 and oxygen uptake in dog maximally contracting gastrocnemius in situ.

1. To clarify the limiting factors for peak VO2 (VO2,peak), we measured muscle venous PO2, (PVO2) and VO2,peak under various O2 delivery conditions (arterial O2 concentration x flow) via the alteration of blood flow and haematocrit (Hct) in anaesthetized dog gastrocnemius muscle (n = 11) stimulated by 1 Hz isometrically and tetanically. 2. Two levels of flow (high and moderate) were maintained by using a pump (150 and 100 mL/min per 100 g, respectively). Haematocrit was adjusted to 45 and 30% by isovolaemic haemodilution with dextran (MW 40,000). 3. The decrease in Hct induced a 31% decrease in O2 delivery (P < 0.05), a 9-12% decrease in PVO2, a 23% decrease in VO2,peak (P < 0.05) and a 12-14% increase in O2 extraction (VO2/O2 delivery) at both flow levels. The decrease in flow induced a 24-25% decrease in O2 delivery (P < 0.05), a 15-17% decrease in PVO2 (P < 0.05), a 9% decrease in VO2,peak (P < 0.05) and a 19-22% increase in O2 extraction (P < 0.05) at both Hct levels. 4. The results suggest that the limiting factor of VO/,peak, unrelated to O2 diffusion limitation (i.e. the change in the heterogeneity of VO2 to O2 delivery ratio among capillaries), due to lowering Hct may be different from that due to lowering blood flow in maximally contracting muscle.

Animals↗

PubMed1997

Follicular dendritic cell sarcoma complicated by hyaline-vascular type Castleman's disease in a schizophrenic patient.

Follicular dendritic cell (FDC) sarcoma is an exceedingly rare neoplasm of unknown pathogenesis. A case of FDC sarcoma complicated by the hyaline-vascular type Castleman's disease occurring in a schizophrenic male is presented. Swelling of the left cervical lymph node appeared in a 44-year-old male schizophrenic who had been medicated with major tranquilizers for 20 years. He had had a history of cervical lymphadenopathy 14 years before, for which a diagnosis of hyaline-vascular type Castleman's disease had been made. The present specimen, obtained from the same site, was an enlarged lymph node heavily infiltrated with oval to spindle-shaped atypical cells but was uninvolved at the periphery. The infiltrating cells showed nodular or sheet-like growth, occasionally taking fascicular or storiform patterns. Follicular involvement was also common. Peculiarly, various amounts of small lymphocytes were intermingled with the neoplastic cells. The atypical cells expressed two FDC-specific antigens, DRC-1 and Ki-M4 antigen, together with a few other markers that are shared by FDC, including CD21 and HLA-DR. These findings clearly show the tumor to be FDC sarcoma. In addition, a peculiar fibro-hyalinous change in the lymph follicle, which is compatible with hyaline-vascular type Castleman's disease, was noted at the periphery of the lymph node where neoplastic cells had not infiltrated. Surprisingly, similar hyaline-vascular changes were observed in the previous biopsy taken 14 years ago. Meanwhile, Kaposi's sarcoma-associated herpesvirus, which is often identified from generalized Castleman's disease, was not identified in the present case by polymerase chain reaction study. Thus, this case is unique in two aspects: (i) the overlap of FDC sarcoma with hyaline-vascular type follicular changes; and (ii) its occurrence in a schizophrenic patient.

Adult↗

PubMed1997

Vascular intimal carcinomatosis: an autopsy case of unusual form of pulmonary metastasis of transitional cell carcinoma.

A 44-year-old woman with an unusual form of pulmonary metastasis is described. She presented with pulmonary thrombosis and clinical signs of disseminated intravascular coagulation (DIC) and died of cerebral hemorrhage. The autopsy study revealed transitional cell carcinoma of the left renal pelvis with pulmonary thrombosis in the large arteries. The intima of the vessels were intact on gross inspection except where the thrombi adhered to. The thrombi contained no tumor cells. However, microscopic examination identified that the metastatic carcinoma diffusely replaced the endothelium and proliferated on to the intimal surface without invasion of the wall and metastatic nodules in the parenchyma. Other examined organs had neither primary nor metastatic tumors, except for microscopic metastasis to the inferior vena cava. To date, this pattern of metastasis has not been noted in previous literature. This condition was designated as being vascular intimal carcinomatosis because of its characteristic manner of tumor proliferation on vascular intima.

Adult↗

PubMed1997

Possible involvement of nitric oxide synthase in oxidative stress-induced endothelial cell injury.

The purpose of this study was to characterize the protective effect of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, on oxidative stress-induced endothelial cell injury. Intracellular oxidative stress was induced by 1-chloro-2,4-dinitrobenzene, a glutathione (GSH) depleting agent, and the leakage of intracellular lactate dehydrogenase was measured as a marker of cell injury. Addition of 1-chloro-2,4-dinitrobenzene (100-500 microM) induced leakage of lactate dehydrogenase from endothelial cells, and the leakage of lactate dehydrogenase was strongly attenuated by L-NAME, but not by NG-methyl-L-arginine, also an inhibitor of nitric oxide synthase. However, cell injury induced by the Ca2+ ionophore ionomycin was not affected by L-NAME or NG-methyl-L-arginine. Moreover, neither L-NAME nor NG-methyl-L-arginine affected GSH depleting agent-induced or H2O2-induced cell injury in a rat foetal lung fibroblast cell line which lacks nitric oxide synthase. These results suggest that the protective effect of L-NAME is likely to be related to nitric oxide synthase, while the inhibition of nitric oxide production may not be involved in the protective effect of L-NAME, since NG-methyl-L-arginine did not affect endothelial cell injury.

Animals↗

PubMed1997

Nucleoside Oxidase, a Hydrogen Peroxide-Forming Oxidase, from Flavobacterium meningosepticum.

A novel enzyme which catalyzes the oxidation of nucleosides to nucleoside-5(prm1)-carboxylic acids, forming hydrogen peroxide, was purified to homogeneity from Flavobacterium meningosepticum T-2799. The enzyme has a molecular weight of about 500,000, and four nonidentical subunits (molecular weights of 81,000, 69,000, 33,000, and 16,000) were detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the basis of visible absorption spectra of the purified enzyme, the enzyme is concluded to be a hemoprotein. It also contains covalently bound flavin adenine dinucleotide. The various nucleosides, such as adenosine (K(infm) = 48 (mu)M), inosine (K(infm) = 66 (mu)M), guanosine (K(infm) = 21 (mu)M), thymidine (K(infm) = 50 (mu)M), uridine (K(infm) = 80 (mu)M), and cytidine (K(infm) = 50 (mu)M), were oxidized by the enzyme, but nucleotides, bases, and ribose were not.

Journal Article↗

PubMed1997

Production of 5,8,11-Eicosatrienoic Acid (Mead Acid) by a (Delta)6 Desaturation Activity-Enhanced Mutant Derived from a (Delta)12 Desaturase-Defective Mutant of an Arachidonic Acid-Producing Fungus, Mortierella alpina 1S-4.

Enhanced production of 5,8,11-eicosatrienoic acid (Mead acid, 20:3(omega)9) was attained by a mutant fungus, Mortierella alpina M209-7, derived from (Delta)12 desaturase-defective M. alpina Mut48. The 20:3(omega)9 production by M209-7 was 1.3 times greater than that by its parent strain, Mut48. This is thought to be due to its enhanced (Delta)6 desaturation activity, which was 1.4 times higher than that of Mut48. In both strains, 87 to 88% of the total lipids comprised triacylglycerol (TG) and 85% of 20:3(omega)9 was contained in TG. On optimization of the culture conditions for M209-7, earlier glucose feeding and shifting of the growth temperature from 28 to 19(deg)C on the second day were shown to be effective. Under the optimal conditions with a 10-liter jar fermentor, 20:3(omega)9 production reached 1.65 g/liter of culture medium (corresponding to 118 mg/g of dry mycelia and 28.9% of total fatty acids), which is about twice that reported previously (0.8 g/liter).

Journal Article↗

PubMed1997

L-allo-threonine aldolase from Aeromonas jandaei DK-39: gene cloning, nucleotide sequencing, and identification of the pyridoxal 5'-phosphate-binding lysine residue by site-directed mutagenesis.

We have isolated the gene encoding L-allo-threonine aldolase (L-allo-TA) from Aeromonas jandaei DK-39, a pyridoxal 5'-phosphate (PLP)-dependent enzyme that stereospecifically catalyzes the interconversion of L-allo-threonine and glycine. The gene contains an open reading frame consisting of 1,014 nucleotides corresponding to 338 amino acid residues. The protein molecular weight was estimated to be 36,294, which is in good agreement with the subunit molecular weight of the enzyme determined by polyacrylamide gel electrophoresis. The enzyme was overexpressed in recombinant Escherichia coli cells and purified to homogeneity by one hydrophobic column chromatography step. The predicted amino acid sequence showed no significant similarity to those of the currently known PLP-dependent enzymes but displayed 40 and 41% identity with those of the hypothetical GLY1 protein of Saccharomyces cerevisiae and the GLY1-like protein of Caenorhabditis elegans, respectively. Accordingly, L-allo-TA might represent a new type of PLP-dependent enzyme. To determine the PLP-binding site of the enzyme, all of the three conserved lysine residues of L-allo-TA were replaced by alanine by site-directed mutagenesis. The purified mutant enzymes, K51A and K224A, showed properties similar to those of the wild type, while the mutant enzyme K199A was catalytically inactive, with corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys199 of L-allo-TA probably functions as an essential catalytic residue forming an internal Schiff base with PLP of the enzyme to catalyze the reversible aldol reaction.

Aeromonas↗