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Polyphenol oxidase was extracted from banana buds in the presences of Triton X-100, isoascorbate, and Polyclar AT, and two isozymes I and II have been separated and partially purified by chromatographies on Butyl Toyopearl 650 and DEAE-cellulose. I and II had different mobility in polyacrylamide gel electrophoresis with optimum pHs of 6.8 and 5.5, respectively. Both enzymes showed the apparent Km values of 0.5 mM for dopamine with substrate inhibitions at its higher concentrations. I and II were inhibited competitively by NaCI with the Ki values of 140 mM and 40 mM, respectively. I and II have a high heat stability, and 88 and 95% of the initial activities were retained after 1-hr incubation at 70°C, respectively.

To assist our choice of sprouts as healthy foodstuffs, we examined and compared the antioxidant activities of various sprouts. Most sprouts contained higher amounts of vitamin C, polyphenol and DPPH radical-scavenging activity than their mature vegetables. As food materials, young college students preferred sprouts to mature vegetables in case of vegetables whose mature forms are unfamiliar at market. Antioxidant activity of "Toumyo" sprout was highest at the 5th day after germination. Cotyledon and young root of "Toumyo" sprout, though they are usually discarded at kitchen, showed high antioxidant activities. A sensory evaluation by young college students suggested that the cotyledon could be served as meal if it is fried after boiled.

L-Galactono-gamma-lactone dehydrogenase (L-galactono-gamma-lactone:ferricytochrome c oxidoreductase [EC 1.3.2.3], GLDHase) which catalyzes the terminal step in the biosynthesis of L-ascorbic acid (AsA) has been purified from roots of sweet potato (Ipomoea batatas L., cv. Kintoki). Highly purified preparation of the GLDHase was obtained by three column chromatography steps with a recovery of ca. 1%, after solubilization from mitochondria in sweet potato roots. SDS-PAGE exhibited a single band at 56 kDa. In the native state, the apparent molecular mass of the enzyme was 56 kDa, based on a Sephadex G-100 gel filtration. The pI and optimum pH values were 5.8 and 7.9, respectively. The Km value for L-galactono-gamma-lactone was 0.12 mM. Substrate inhibition was obtained at concentrations greater than 4.2 mM. The enzyme was inhibited by p-chloromercuribenzoate (PCMB) and acriflavine, and the inhibition of acriflavine was diminished by the addition of FAD or FMN. The only effective substrate for the GLDHase was L-galactono-gamma-lactone.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC1.1.1.34), the key enzyme in isoprenoid biosynthesis, was purified from microsomes of potato tuber tissue, and a polyclonal antibody and two monoclonal antibodies against the purified enzyme were prepared. HMGR protein content was measured by immunotitration and radioimmunoassay using these antibodies. HMGR activity was very low in the fresh tissues of both potato tuber and sweet potato root. The activity in potato tuber was increased by cutting and further by additional fungal infection of the cut tissues. In sweet potato root tissue, the activity was scarcely increased after cutting alone, but was markedly increased by additional fungal infection or chemical treatment. The HMGR protein contents in both fresh potato tuber and sweet potato root tissues were also very low, and increased markedly in response to cutting and fungal infection. From these results, we proposed a hypothesis on the induction mechanism of HMGR after cutting and fungal infection in potato tuber and sweet potato root tissues.

L-Galactono-gamma-lactone dehydrogenase (EC 1.3.2.3, GLDHase) was partially purified from mitochondria of sweet potato tuberous roots over 600-fold on a specific activity basis, followed by purification of the enzyme protein of 56 kDa by a preparative SDS-PAGE. The absorption spectrum of the hydroxylapatite column-purified GLDH-ase showed peaks at 448 and 373 nm, suggesting the presence of flavin as a prosthetic group. The activity of GLDH-ase was inhibited by lycorine, an alkaloid which inhibits ascorbic acid biosynthesis in vivo. N-terminal partial sequences of four internal polypeptides generated by partial digestion of GLDHase with V8 protease were determined. The deduced nucleotide sequences were used to amplify a cDNA fragment of the GLDHase gene. The clone encoded a polypeptide of 581 amino acid residues with a molecular mass of 66 kDa. The deduced amino acid sequence showed 77% identity with that of cauliflower GLDHase, and significant homology to those of L-gulono-gamma-lactone oxidase (22% identity) from rat and L-galactono-gamma-lactone oxidase from yeast (17% identity), which are enzymes involved in L-ascorbic acid biosynthesis in these organisms. The absorption spectrum and cDNA sequence suggested that the flavin group bound noncovalently. We conclude that GLDHase, L-gulono-gamma-lactone oxidase and L-galactono-gamma-lactone oxidase are homologous in spite of the difference in substrates and electron acceptors. Genomic Southern analysis suggested that GLDHase gene exists as a single copy in the genome of sweet potato.

Nine new sesquiterpenes related biosynthetically to ipomeamarone, the well-known sweet potato phytoalexin, have been isolated from Ceratocystis fimbriata -infected sweet potato root tissue and the structures determined. Their biosynthetic relationship with previously identified furanoterpenes is discussed.

Furanoterpene-containing particles were isolated by centrifugation on a discontinuous Ficoll density gradient from a homogenate of the non-infected tissue adjacent to the infected region of Ceratocystis fimbriata-infected sweet potato root tissue. The particles were recovered at a relatively high ratio in the 2% Ficoll fraction, in which there was no contamination by mitochondria and only little by endoplasmic reticulum judging from the distribution of the activities of their marker enzymes and electron micrographs. Each particle was enveloped in a single membrane, 7-10 nm thick.

Furanoterpenes produced in sweet potato ( Ipomoea batatas ) root tissue in response to fungal infection or injury with mercuric chloride were metabolized to compounds negative to Ehrlich's reagent. The metabolism of ipomeamarone in sweet potato root tissue was enhanced in response to cut injury. The enhanced metabolism of ipomeamarone in the cut tissue was partly prevented by HgCl 2 treatment. The initial metabolite of ipomeamarone was identified as ipomeamaronol, 15-hydroxyipomeamarone. A time-course analysis of the metabolism of [ 14 C]ipomeamarone by cut tissue indicated that ipomeamarone was converted to ipomeamaronol, which was further metabolized to unknown compounds.

The change in the activity of cinnamic acid 4-hydroxylase in light- and dark-grown maize seedlings was studied during their early growth. The total enzyme activity in shoots of light-grown developing seedlings reached its highest activity on day five after planting, and then decreased gradually. The highest specific activity, on a fresh weight basis, of the enzyme was seen in day two. A similar pattern was observed in dark-grown seedlings. The enzyme activity was detected in roots and cotyledons where the activity also increased during early growth and then decreased. The increase in activity in the developing shoots of light-grown seedlings was completely suppressed by cycloheximide.

Pyruvate decarboxylase [2-oxo acid carboxy-lyase, EC 4.1.1.1] was isolated from sweet potato roots and was partially purified from healthy and diseased tissues. There was no appreciable difference in properties between the enzymes from healthy and diseased tissues. The molecular weight of the enzyme was found to be 240,000 by polyacrylamide gel electrophoresis. Since sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a molecular weight of 60,000 for the monomeric form of the enzyme, it is likely that sweet potato pyruvate decarboxylase contains 4 single polypeptide chains. The optimal pH of the decarboxylation reaction was 6.1--6.6. The Lineweaver-Burk double reciprocal plot curved upward, and the Hill coefficient was more than 1, with low concentrations of pyruvate. The enzyme was localized in the cytosol fraction. The activity of the enzyme increased in response to black-rot fungus infection, but decreased in response to cutting.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (NADPH) was solubilized by trypsin digestion from sliced potato tuber microsomes, and purified to apparent homogeneity in the absence of detergent with a recovery of 1.8%. The enzyme had a specific activity of 7,910 nmol of mevalonate formed per min per mg of protein. On molecular-sieving high-performance liquid chromatography, the activity was coincident with the single protein peak corresponding to a molecular weight of approximately 110 kDa. On SDS-polyacrylamide gel electrophoresis, the purified enzyme showed only one protein staining band corresponding to a molecular weight of approximately 55 kDa. The apparent Km value for S-HMG-CoA was 6.4 microM and that for NADPH was 25 microM.

Lactate dehydrogenase [L-lactate: NAD oxidoreductase, EC 1.1.1.27] was isolated from sweet potato root tissues. Two species of the enzyme (isozymes I and II) were separated by DE-52 cellulose column chromatography from healthy, cut, and black-rot diseased tissues. Isozymes I and II were purified from healthy and diseased tissues, respectively. Reduction of pyruvate by NADH with either isozyme I or II was inhibited by pyruvate at high concentrations, by NAD+ and by several mononucleotides. Isozyme I was inhibited by a lower concentration of adenine nucleotide than isozyme II, and Km for pyruvate was increased markedly at acidic pH in the case of isozyme I, but only slightly in the case of isozyme II. The molecular weights of both isozymes were determined to be 150,000 and they were found to be charge isomers by polyacrylamide gel electrophoresis. The enzyme activity increased in response to infection by black-rot fungus but decreased in response to cutting.