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T Komoda

Publications and source records attributed to T Komoda.

At least 145 records · Page 8Linked to original sources
PubMed1982

L-Phenylalanine inhibition of human alkaline phosphatases with p-nitrophenyl phosphate as substrate.

With p-nitrophenyl phosphate as the substrate, there reportedly is no organ-specific inhibition of alkaline phosphatase (EC 3.1.3.1) activity by L-phenylalanine. However, we found that at pH 10.0, with p-nitrophenyl phosphate as the substrate, L-phenylalanine obviously inhibits the alkaline phosphatase isoenzyme from human placenta, whereas there is little if any inhibition of the isoenzyme from human intestine. Because of the differing effects of substrates (p-nitrophenyl phosphate and phenyl phosphate) and their enzymic products (p-nitrophenol and phenol) for L-phenylalanine action on the placental alkaline phosphatase isoenzyme, we suggest that the isoenzyme--inhibitor--substrate complex and the effect of released phosphate on L-phenylalanine inhibition of the isoenzyme activity differ from each other.

Adult↗
PubMed1981

Multiple forms of human intestinal alkaline phosphatase: chemical and enzymatic properties, and circulating clearances of the fast- and slow-moving enzymes.

Two forms of alkaline phosphatase (orthophosphoric monoester phosphohydrolase (alkaline optimum, EC 3.1.3.1) have been purified from human small intestine by column chromatography on DEAE-cellulose and tyraminyl derivative affinity gel, and by preparative disc gel electrophoresis. Intestinal phosphatases were electrophoretically separated into two components, fast- and slow-moving enzymes, with apparent molecular weights of 140000 and 168000 and with subunit weights of 68000 and 80000, respectively. Analyses of carbohydrate and amino acid revealed marked differences in the two enzymes. Enzymatic properties and affinities for an anti-blood group antibody were also found to differ. Papain digestion released a hydrophobic small peptide from the slow-moving enzyme and its enzymatic properties resembled those of the fast-moving enzyme. Circulating clearance (T1/2) of the slow- and fast-moving enzymes from adult intestine was found to be 7.5 h and 1.3 h, respectively; that of fetal intestinal enzyme was 2.8 h. Sialidase, sialidase/beta-galactosidase, or sialidase/beta-galactosidase/N-acetyl-beta-glucosaminidase treatment of the fetal enzyme reduced the value to about 40 min. Further, digestion with alpha-fucosidase, alpha-mannosidase or both restored it to nearly the original level. Organ distribution of injected 125I-labelled enzymes indicates that the desialylated hepatic enzyme was selectively distributed in liver, while the degalactosylated intestinal enzyme was incorporated into liver lymph fluid, and small intestine. These results suggest that the pathway of circulating clearance of alkaline phosphatase has several routes.

Adult↗
PubMed1981

Inhibition of alkaline phosphatase by bismuth.

The interaction of alkaline phosphatase (EC 3.1.3.1) with bismuth was studied. Among the tested alkaline phosphatases, bismuth was found to be the most effective inhibitor of the placental enzyme. Partial denaturation of the placental enzyme by papain digestion had little effect, if any, on the inhibition. Bismuth inhibition of the placental enzyme activity was more progressive with mixed glycosidase treatment than with sialidase treatment. The pH activity profile of the mixed glycosidase-treated placental enzyme was clearly shifted in the presence of bismuth. The mixed glycosidase-treated placental enzyme/bismuth mixture was more heat labile than the non-treated placental enzyme. Based on the results of kinetic studies, the inhibition mechanism of the placental enzyme by bismuth was shown to be of the competitive type, and the Ki value and Hill coefficient of the mixed glycosidase-treated placental enzyme was found to be 92 mu mol/l and 2.25, respectively. L-Phenylalanine does not interfere with the inhibitory effect of bismuth on alkaline phosphatase. Inorganic phosphate, on the other hand, appears to disturb bismuth bindings.

Alkaline Phosphatase↗
PubMed1978

The function of carbohydrate moiety and alteration of carbohydrate composition in human alkaline phosphatase isoenzymes.

The relationship between the structure and function of alkaline phosphatase (orthoposphoric monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) isoenzymes is under investigation in a number of laboratories. The present study deals with the effects of glycosidase digestion on the alkaline phosphatase isoenzymes. Changes in physicochemcial properties, activity, affinity for various lectins and blood group antisera, carbohydrate composition and biological half-life were investigated. The desialylated hepatic enzyme was shown to be more heat labile and more sensitive to protease digestion in the presence of 0.5% sodium dodecyl sulfate than native hepatic enzyme. Helix contents of the native and desialated hepatic enzyme were calculated to be 39.0 and 30.8%, respectively, and apparent molecular weights 175,000 and 167,000, respectively. Intestinal enzyme preparations treated with alpha-mannosidase, exo-N-acetyl-Dglucosaminidase and endo-N-acetyl-D-glucosaminidase-D displayed a decrease in enzyme activity. Among these, the alpha-mannosidase-treated enzyme activity was the most clearly reduction. The maximum activity of the alpha-mannosidase-treated intestinal enzyme was observed to change from 40 mM Mg2+ to 5--10 mM Mg2+.

Alkaline Phosphatase↗
PubMed1977

Inhibition of alkaline phosphatase by sialic acid.

The interaction of human organ alkaline phosphatases (orthophosphoric-monoester phosphohydrolases (alkaline optimum), EC 3.1.3.1) with sugars was studied. Hexosamines, N-acetylneuraminic acid (NANA or sialic acid), N-acetylmuramic acid and N-acetylglycolylneuraminic acid inhibited human organ alkaline phosphatase activities. Of these, sialic acid was the most effective inhibitor. The pH profiles for the enzymes in the absence and presence of sialic acid were similar. The sialic acid - enzyme complex was more heat stable than the free enzyme between 20 and 45 degrees C. Lineweaver-Burk plots of 1/v versus 1/S at various concentrations of sialic acid showed intersecting straight lines indicating that the mechanism of inhibition was a mixed type. The Ki value obtained from the plots of 1/v versus the square of sialic acid concentration was 0.07 mM for the hepatic, sialidase-treated hepatic, and intestinal alkaline phosphatases. The respective Hill coefficients varied somewhat with the alkaline phosphatase isoenzyme. Hyperbolic curves were obtained when the percentage of remaining activity was plotted against the substrate concentration at different concentrations of sialic acid. The Hill coefficient was lowered in the presence of sialic acid. The sialidase-treated hepatic enzymes used gave the most effective conversion. Partial denaturation of the enzyme with urea, or pronase digestion had a little if any effect on the sialic acid inhibition with constant time.

Alkaline Phosphatase↗
PubMed1976

Determination of isoenzyme contents of lactic dehydrogenase activity and 2-hydroxybutyric dehydrogenase activity in lactic dehydrogenase preparations.

In this inestigation, a determination of the isoenzyme contents of LDH and HBD activities in lactate dehydrogenase preparations and the differences in the interaction of these preparations with NAD analogues were examined. The results obtained were as follows. 1. The activity ratio between oxidation and reduction in LDH reaction is shown to be similar in both H4 and M4 preparations, whereas for HBD activity, the ratio seems to be lower in the M4 preparation than in H4(H4/M = 1/2). 2. NAD and its analogues (NXD, TNAD, and TNXD) are shown to be useful coenzymes for the LDH reaction, while 3-acetyl derivatives appear to be unsuitable for this purpose because of their lower activity. HBD activity with 3-acetyl NXD is shown to be higher than with TNAD or TNXD. among these NAD analogues, 3-acetyl NXD gives the highest HBD activity, especially in the M4 preparation. 3. The LDH activity of H4 relative to M4 preparations has been shown to be maximal when 450 mM lactic acid with NAD or 15 mM lactic acid with TNXD are used. Under these conditions, the contents of LDH subunits can be estimated with considerable reliability. As to HBD activity, the content of LDH subunit having HBD activity has been estimated by determing the enzyme activity under conditions in which either 300 mM 2-hydroxybutyrate with 3-acetyl NXD or 15 mM 2-hydroxybutyrate with NAD are employed.

Hepatitis↗
PubMed1976

Partial purification of human intestinal alkaline phosphatase with affinity chromotography. Some properties and interaction of concanavalin A with alkaline phosphatase.

1. Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from human intestine was purified with concanavalin A-Sepharose and tyraminyl derivative-Sepharose affinity chromatography. The enzyme obtained with these techniques had a specific activity of approx. 513.2 mumol p-nitrophenylphosphate hydrolyzed per min per mg of protein at pH 10.0. 2. The highly purified enzyme showed one major enzymatically active band and a possible minor enzymatically active band on acrylamide gel and cellogel electrophoresis, and the two fraction types showed identical antigenicity. 3. The highly purified intestinal enzyme was compared with the purified hepatic enzyme: the saccharide content of each showed a marked difference. 4. The interaction of alkaline phosphatase with concanavalin A, a carbohydrate-binding protein, was studied. Concanavalin A showed an organ-specific behavior to alkaline phosphatase isoenzyme, i.e., the effect on the enzyme activity, and the optimum pH of the activity. 5. The concanavalin A and alkaline phosphatase complex showed a protective effect against heat denaturation and inactivation of proteinase digestion. There was no difference in stability between the intestinal enzyme and the hepatic enzyme. 6. Alkaline phosphatase preparations from human intestine and human liver can bind with concanavalin A; these interactions of concanavalin A; these interactions of concanavalin A with the enzyme occurred reversibly when alpha-methyl-D-mannoside was added. 7. The double reciprocal plots of 1/v vs. 1/s at higher concentrations of concanavalin A showed that the mechanism of inhibition was "mixed type". From the results of Dixon plots, the inhibition constant (Ki) was calculated to the 0.025 muM for human intestinal enzyme. 8. The effect of concanavalin A on L-phenylalanine inhibition of the intestinal alkaline phosphatase indicates that concanavalin A does not interfere with L-phenylalanine binding, but its effect on L-homoarginine inhibition of the hepatic enzyme seems to show that concanavalin A interfered with L-homoarginine binding.

Alkaline Phosphatase↗
PubMed1976

Partial purification and some properties of human liver alkaline phosphatase.

1. Alkaline phosphatase (EC 3.1.3.1) from human liver was solubilized from the homogenate using 0.2% Triton X-100 containing 0.2 M lithium 3,5-diiodosalicylate, and the pellet obtained was resolubilized with 20% n-butanol. The procedure resulting in 3842-fold purification included acetone fractionation, ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration, hydroxyapatite gel chromatography and further concanavalin A/Sepharose 4B affinity chromatography. 2. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 8.6, and exhibited one-seventh of the alkaline p-nitrophenylphosphatase activity in the hepatic enzyme preparation contains of the alkaline pyrophosphatase activity. 3. The highly purified enzyme was a sialic-acid containing glycoprotein. 4. Sialidase-treated hepatic enzyme clearly presented the phenomenon of delayed mobility, and the delayed enzyme fraction stained more strongly than that of non-treated hepatic alkaline phosphatase. 5. In order to investigate the role of the carbohydrate region(s) of the hepatic alkaline phosphatase molecule on substrate binding, the effect of sialidase treatment on the rate of substrate inhibition of alkaline phosphatase was studied. In the case of hepatic enzyme without sialidase, substrate inhibition of alkaline phosphatase activity was clearly shown, while in the case of the hepatic enzyme with sialidase, there was hardly any substrate inhibition in the range of 1-8 mM p-nitrophenylphosphate.

Alkaline Phosphatase↗
PubMedDate not supplied

The mechanism of elevated alkaline phosphatase activity after bile duct ligation in the rat.

Alkaline phosphatase activity in the liver and intestine increases after bile duct ligation, reportedly by increased enzyme synthesis. To ascertain the mechanism of this increased synthesis in the absence of a cDNA clone encoding the enzyme, we have estimated the concentration of liver and intestinal alkaline phosphatase mRNA by translational analysis. Monospecific antiserum to rat placental alkaline phosphatase was raised. The resulting antiserum precipitated two peptides of 53 and 56 kd after translation of liver poly(A) + RNA. The precipitation of both peptides was blocked by the single 64 kd placental alkaline phosphatase. Processing of the cell-free products by microsomal membranes produced peptides of 62 and 64 kd. Antiserum to rat intestinal alkaline phosphatase also identified two peptides as products of intestinal RNA translation. After bile duct ligation, we confirmed a transient 2-fold increase in alkaline phosphatase activity in the intestine and a more constant 7-fold increase in the liver. However, the alkaline phosphatase mRNA concentration remained unchanged in both organs. We conclude that increased alkaline phosphatase synthesis after bile duct ligation results from an enhanced rate of translation of mRNA.

Alkaline Phosphatase↗