Kazuya Higashino

Studies on a variant alkaline phosphatase in sera of patients with hepatocellular carcinoma.

Abstract An alkaline phosphatase (orthophosphoric monoester phosphohydrolase, E.C. 3.1.3.1) was found in sera, ascitic fluid or cancer tissues of patients with hepatocellular carcinoma which had a different electrophoretic mobility from that of liver, bone, placental or intestinal isoenzyme. The enzyme was purified 300-fold from serum of one of these patients, and its enzymic properties were investigated. This variant alkaline phosphatase had almost identical properties to the placental isoenzyme with respect to inhibition by l -phenylalanine or l -homoarginine, inactivation by urea, susceptibility to the action of neuraminidase, molecular size, and antigenicity. The pH optimum and sensitivity to inhibition by phosphate were similar to those of the liver isoenzyme, but the enzyme was different from either one with respect to its susceptibility to l -leucine, Michaelis constant, electrophoretic mobility, and heat stability. The difference in enzymic properties among other variant alkaline phosphatases is also discussed.

The conversion of l-lysine to saccharopine and α-aminoadipate in mouse

Abstract Saccharopine [ϵ- N -( l -glutaryl-2)- l -lysine] has been found to occur in normal, untreated mouse liver. The pool of saccharopine as well as that of α-aminoadipate become labeled shortly after the administration of l -lysine-U- 14 C into intact mouse. In vitro experiments using the mouse liver homogenate have shown that l -lysine is converted to saccharopine in the presence of α-ketoglutarate and NADPH, and saccharopine to α-aminoadipate in the presence of NAD + . The oxidation of α-aminoadipic-δ-semialdehyde (Δ 1 -piperideine-6-carboxylate), the proposed reaction product of saccharopine cleavage, to α-aminoadipate is effected by either NAD + or NADP + .

Metabolism of lysine in rat liver

Abstract It was shown previously that lysine was degraded in the mitochondria in the presence of α-ketoglutarate and, under these conditions, saccharopine accumulated (1). The obligatory requirement of α-ketoglutarate for lysine breakdown as well as for saccharopine formation suggest that lysine is metabolized, in mammalian liver, by a reversal of reaction sequence described for its biosynthesis in yeast (2–6). The present report describes that saccharopine is further metabolized to α-aminoadipic acid in the mitochondria and that NAD rather than NADP is required in this reaction.

Enzymic determination of L-lysine in biological materials.

Abstract A simple and rapid method for the determination of l -lysine is presented. The method measures the amount of NADH oxidized on incubation of a lysine-containing sample with α-ketoglutarate and saccharopine dehydrogenase. A strict substrate specificity of the enzyme and the equilibrium of the reaction which is in favor of saccharopine formation make possible the specific determination of l -lysine to be done in crude biological materials.

Release of phenyl acetate esterase from liver microsomes by carbon tetrachloride

Abstract Decrease in phenyl acetate esterase activity of the microsomal fraction of mouse liver was observed within 6 h after intraperitoneal administration of carbon tetrachloride, in contrast to an increase both in the postmicrosomal supernatant (cytosol) fraction and in the serum. Incubation of microsomes with carbon tetrachloride was found to result in the release of the esterase activity into the medium, and the amount of the enzyme in the medium roughly paralleled the degree of lipoperoxidation. An adequate amount of ascorbic acid was also shown to be capable to induce in vitro lipoperoxidation, as well as the the release of enzyme. Inhibition of lipoperoxidation blocked the release of phenyl acetate esterase concomitantly. From these results, it may be suggested that carbon tetrachloride damages the microsomal membrane through lipoperoxidation resulting in phenyl acetate esterase release. This mechanism contributes at least in part to the decrease in activity of this enzyme in the liver which occurs 24 to 48 h after carbon tetrachloride treatment.

A hepatoma-associated alkaline phosphatase, the Kasahara isozyme, compared with one of the isozymes of FL amnion cells.

It was found that a human hepatoma-associated ALP (orthophosphoric monoester phosphohydrolase, E.C. 3.1.3.1) shared electrophoretic mobility, inactivation by urea, inhibition by inorganic phosphate, ethylenediaminetetraacetate, and amino acids (L-phenylalanine, L-leucine and L-homoarginine), heat stability, sensitivity to neuraminidase, pH optimum, Km value, and antigen site with fast moving ALP isozymes of FL cell strain derived from human amniotic membrane. However, 40-week-old fresh amniotic membrane lacked this isozyme. Instead, it had a placental type ALP consisting of minor components. The other ALP isozyme of FL cells had properties common to hepatoma ALP with regard to L-phenylalanine sensitivity, inhibition by ethylenediaminetetraacetate, inactivation by urea, and antigen site, but differed from it in electrophoretic mobility, sensitivity to L-leucine and L-homoarginine, and the presence of another antigen site. It was more heat stable and more sensitive to inhibition by inorganic phosphate than Hepatoma AP. The possible regulatory mechanism between the hepatoma-type ALP and the placental type ALP in the amnion cells is considered.

α-Aminoadipate aminotransferase of rat liver mitochondria

Abstract α-Aminoadipate aminotransferase was partially purified from rat liver mitochondria. The enzyme catalyzes a reversible transamination between α-aminoadipate and α-ketoglutarate. The aminotransferase has rather loosely bound coenzyme; a prolonged dialysis of enzyme against phosphate buffer resulted in an almost complete loss of its catalytic activity. The enzymic activity of the apoenzyme was largely restored by either pyridoxal or pyridoxamine phosphate. The equilibrium constant of the reaction was about 1.32 at pH 7.5 and at 37°, and the Michaelis constants for α-aminoadipate, glutamate, α-ketoadipate, and α-ketoglutarate were 9.0, 5.0, 0.5 and 1.3 mM, respectively. α-Aminoadipate aminotransferase has a strict substrate specificity. In trans-amination with α-ketoglutarate, α-aminopimelate and norleucine were about 14% and 15% active in place of α-aminoadipate, whereas the other amino acids tested were inert as substrates.

Production of fibronectin by HUH6 Cl5 cell line established from a human hepatoblastoma

Fibronectin was detected by indirect immunofluorescence on the cell surfaces of HUH6 C15 cells, established from a human hepatoblastoma and maintained with serum-free RPMI 1640 medium. Fibronectin synthesized by HUH6 Cl5 was purified by gelatin-Sepharose affinity chromatography and compared with human plasma fibronectin in respect to molecular weight, electrophoretic mobility and antigenicity. Fibronectin synthesized by this cell line was proved to be identical with human plasma fibronectin.

Serum, fecal and urinary bile acids in patients with mild and advanced liver cirrhosis

SummaryThe levels and compositions of bile acids in the serum, feces and urine were determined by gas chromatography in male patients with mild and advanced stages of liver cirrhosis, defined by usual clinical and laboratory criteria.Increased concentrations of serum bile acids, a decreased ratio of serum cholic acid plus deoxycholic acid/chenodeoxycholic acid plus lithocholic acid, a reduction of fecal bile acids, especially deoxycholic acid, and a reduction in total daily excretion of bile acids were found in patients with advanced cirrhosis. Furthermore, 3β-hydroxy-5-cholenoic acid, a presumed intermediate in the alternate pathway of chenodeoxycholic acid synthesis, was found in the urine of patients with very advanced cirrhosis. Such changes were absent in patients with mild cirrhosis except for increased concentration of serum bile acids and decreased excretion of total bile acids.The present study suggests that marked changes in bile acid metabolism occur only in advanced cirrhosis.

A novel alkaline phosphatase, a minor component of normal liver phosphatases

A novel alkaline phosphatase differing from the so-called liver-specific isoenzyme was found in four out of twenty-four normal adult livers. Although the mobility of this enzyme was the same as that of so-called liver-specific alkaline phosphatase on the polyacrylamide gel electrophoretogram, its mobility was not altered following neuraminidase treatment, while that of the liver-specific enzyme was affected by the same treatment. Both enzymes also differed in other enzymatic and immunologic properties. The enzyme, however, resembled the so-called intestinal alkaline phosphatase in many enzymatic and immunologic properties. Thus, the inhibition patterns by amino acids, EDTA and inorganic phosphate, the pH optima, KM values for phenyl phosphate and reactivity with anti-intestinal alkaline phosphatase antibody were quite similar for both enzymes. Differences in the properties of this enzyme and intestinal alkaline phosphatase were in sensitivity to denaturation by treatment with heat and urea and to inhibition by Levamisole. The possible origin of the enzyme in normal liver and its relationship to the Kasahara isoenzyme and fetal intestine-type in hepatoma is discussed.