Stimulative effect of potassium ion on starch synthetase of different plant origins

The stimulative effect of K⁺ on the activity of granule-boundstarch synthetase was uniformly observed in the enzyme preparationsobtained from the root, leaf and stem of sweet potato plants.The stimulation of K⁺ was most marked with the sweet potatoenzyme, while the broad bean enzyme was also appreciably stimulated.The K⁺-effect was very small in the enzyme from rice, barleyand taro plants. (Received October 25, 1968; )     

The Preparation of a Cellulose-like Polymer from 2,3,6-Tri-O (N-pheny1carbamyl)-D-glucopyranose by the Action of Phosphorus Pentoxide in Dimethyl Sulfoxide

Starch phosphorylase was purified from either freshly harvested or stored roots of sweet potato (.Ipomoea batatas (L.) Lam. cv Tain on 65). Both enzyme preparations in their native state showed on polyacrylamide gel electrophoresis a cluster of about six closely located activity bands, which had common antigenic determinants as they were simultaneously probed by monoclonal antibodies. The molecules of enzymes from stored roots were smaller than those from fresh roots. However, the two enzyme preparations had completely fused precipitin lines in double diffusion assays with an antiserum raised against the fresh root preparation. One large subunit and several small ones were found for both enzyme preparations. The small subunits appeared to be the degradation products of the large ones as revealed by peptide mapping and immunoblotting. Immunofluorescence microscopy showed that the enzyme was present in the amyloplasts and cell walls of root storage parenchyma.     

The Presence of Two Forms of Succinate Dehydrogenase in Sweet Potato Root Mitochondria

Succinate dehydrogenase was partially purified from sweet potatoroot tissue by solubilization of the enzyme from the submitochondrialparticles, ammonium sulfate fractionation, and DEAE-cellulosecolumn chromatography. Sweet potato succinate dehydrogenaseexisted in two forms; these were separated by disc polyacrylamidegel electrophoresis or by hydroxyapatite column chromatography.There was a difference in the electric charge of the molecule,but not in the molecular weights of the two forms. No differencewas detected between the two forms of succinate dehydrogenasewith respect to their K_m values for succinate, pH-optimums andsubunit compositions. The two subunits that make up the enzymehave molecular weights of about 26,000 and 65,000. ¹ This work was supported in part by Grant-in-Aid 411308 forScientific Research from the Ministry of Education, Scienceand Culture of Japan. (Received November 28, 1981; Accepted February 17, 1982)     

Biochemical studies on biogenesis of mitochondria in wounded sweet potato root tissue I. Time course analysis of increase in mitochondrial enzymes

Cytochrome oxidase and succinate dehydrogenase activities increasedduring aging of sliced tissue for sweet potato root after respectivelag phases of about 8 and 10 hr. The increase in the formerwas stepwise. On the other hand, malate dehydrogenase activityincreased slowly without a lag phase. Spectrophotometric determinationof heme contents in mitochondria indicated that the hemes increasedafter a lag phase for at least 8 hr during aging Treatment of tissue slices with cycloheximide at a concentrationof 10^–6 M prior to aging resulted in an extension of thelag phase in the increase of cytochrome oxidaseactivity andin complete inhibition of the increase of malate dehydrogenaseactivity. The antibiotic, at a concentration of 10^–5 M,completely suppressed the increases. Chloramphenicol (6 ? 10^–3M) also blocked the increases, except for that in malate dehydrogenaseactivity at an early stage of aging (Received December 22, 1970; )     

Cell-free synthesis of succinate dehydrogenase and mitochondrial adenosine triphosphatase of sweet potato

Polyadenylated mRNA was isolated from aged slices of sweet potato root tissue and translated in a wheat germ cell-free system. The synthesis of apoprotein of the flavoprotein subunit of succinate dehydrogenase and two of the subunits of mitochondrial adenosine triphosphatase were detected by indirect immunoprecipitation. The molecular weights of the immunologically identified products were 3,000 and 8,000-9,000 daltons larger than the mature flavoprotein subunit of succinate dehydrogenase and the mature subunits of adenosine triphosphatase, respectively.     

Some properties of shikimate: NADP oxidoreductase produced in sweet potato root tissue after slicing

The activity of shikimate: NADP oxidoreductase [EC 1. 1. 1.25] in sweet potato root tissue increased soon after slicing.Enzyme preparations obtained from both sliced tissue and fromfresh tissue probably contained a single enzyme component, andthey showed identical chromatographical behaviour. K_m values of the enzyme for NADP and shikimate were 1.0x10^–4Mand 1.3 x 10^–3M, respectively. Enzyme activity was potentlyinhibited by SH-inhibitors such as p-chloromercuribenzoate andoxidized glutathione. Enzyme activity was affected neither by mononucleotides suchas ATP, ADP and AMP, divalent cations, Mg⁺⁺, Ca⁺⁺ and Mn⁺⁺,nor by metabolites such as tryptophan, phenylalanine, tyrosineand t-cinnamic acid which are involved in aromatic compoundsyntheses. The enzyme rapidly lost its activity. This inactivation reactionshowed a time course consisting of two steps of the first-orderreaction. The inactivated enzyme preparation was not reactivatedby thiol compounds such as cysteine, 2-mercaptoethanol and glutathione,although these reagents, to a certain extent, protected theenzyme from inactivation. The results suggest that denaturationof the enzyme protein was involved in inactivation of the enzyme. ¹Part 74 of the phytopathological chemistry of sweet potatowith black rot and injury. ²Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Setagaya-ku, Tokyo. (Received August 5, 1968; )     

The mechanism of chilling injury in sweet potato IX. The relation of chilling to changes in mitochondrial respiratory activities

To examine the mitochondrial activity of chilling-stored sweetpotatoes a method of isolating mitochondria with a good respiratorycontrol (RC) ratio from healthy sweet potato tissue was established.Mitochondria were isolated from two varieties of sweet potatoes(Norin No. 1, moderately sensitive to chilling, and OkinawaNo. 100, very sensitive) kept at about 0°C for about 15to 40 days. Respiratory activity was measured with an oxygenelectrode apparatus. Mitochondrial activities of chilling-storedNorin No. 1, i.e. the RC ratio, respiratory rate at state 3and ADP/O ratio decreased about 2 to 3 weeks after the beginningof incubation. The decline in the RC ratio was most sensitive.Diminution of the activities when malate was used was seen earlierthan when succinate was used. When activities were measuredusing succinate at low concentration (0.2 M) of mannitol, thedecrease in activities was more conspicuous than at a high concentration(0.7 M). Similar experiments with Okinawa No. 100 also showedthe decline in these activities. However, the three kinds ofactivities simultaneously decreased, and the decline appearedfaster than in the case of Norin No. 1. ¹ This paper constitutes part 99 of the phytopathological chemistryof sweet potato with black rot and injury. ² Current address: Nomura Research Institute, Kamakura, Kanagawa247, Japan. (Received June 6, 1972; )     

Studies on factors in sweet potato root which induce spore germination of Ceratocystis fimbriata

Spore germination of Ceratocystis fimbriata was studied in termsof host-parasite specificity. The sweet potato, coffee and cacaostrains of Ceratocystis fimbriata germinated well in a fractionof sweet potato root water extract which had been passed througha column of cation exchange resin. The results showed that germinationof these strains was independent of exogenous cations. On theother hand, the prune, oak, taro and almond strains requiredfor germination both the absorbed and unabsorbed fractions ofsweet potato root water extract which were separated from eachother with a cation exchange resin column. Divalent cationssuch as Ca²⁺ Mg²⁺, Mn²⁺ and Zn²⁺ were identified as the activeprinciples in the absorbed fraction and Ca²⁺ showed the highestinductive activity for spore germination in the presence ofthe unabsorbed fraction. The active principle(s) in the unabsorbedfraction has not yet been identified. There was no relationshipbetween the Ca²⁺ and Mg²⁺ contents of the spores and the requirementof exogenous Ca²⁺ for germination. Ca²⁺ appeared to functionas a trigger of spore germination, not as a normal nutrient.These results suggest that the divalent cations such as Ca²⁺and Mg²⁺ in sweet potato contribute to the establishment ofhost-parasite specificity of this system. (Received August 10, 1977; )     

Phytochrome control of some oxido-reductases in germinating Pinus roxburghii Sarg pollen

Germination of Pinus roxburghii pollen was observed to be phytochrome-controlled.The red and far-red light affected the activities of succinatedehydrogenase and glucose-6-phosphate dehydrogenase in a mannercharacteristic of enzyme induction by phytochrome. Activitiesof peroxidase and malate dehydrogenase were not affected byred and far-red lights in this system. ¹This research was supported by the Indian Council of AgriculturalResearch, New Delhi and forms a part of the Ph.D. dissertationsubmitted to the Punjab Agricultural University, Ludhiana byA.K.D. ²Present address: Botany Department, The Australian NationalUniversity, Canberra, ACT 2600, Australia. (Received December 25, 1978; )     

Mechanism of Cold Injury in Sweet Potatoes

The biochemical mechanism of cold injury occurring in sweet potatoes stored at 0°C was studied. Oxygen uptake and RC ratio of mitochondria from sweet potatoes kept at 0°C for about 15 days declined when succinate or malate was used as substrate. As sweet potatoes suffered slight cold injury, a decrease in the respiratory rate of state 3 of mitochondria was observed. This decrease could be restored approximately to the level of that of healthy sweet potato mitochondria by the addition of cytochrome c when succinate was used as substrate. When sweet potatoes suffered severe damage, only partial recovery was observed with cytochrome c. While it was found that the respiratory rate in state 3 of mitochondria from chilled sweet potatoes was less inhibited by cyanide than that of healthy sweet potato mitochondria, the inhibition could be restored to that of healthy sweet potato mitochondria by the addition of cytochrome c. When malate was used as substrate, no effect of cytochrome c and NADH₂ was observed. There was no difference between chilled and healthy sweet potato mitochondria in enzyme activities of the electron transport system except for malate dehydrogenase.     

Isolation of microbodies from, sweet potato root tissue and their development during aging of slices

Microbodies were isolated from, sweet potato root tissue bydifferential and linear sucrose density gradient centrifugation.When the tissue was homogenized in the presence of PolyclarAT, the microbodies sedimented together with the mitochondriathrough the sucrose gradients. The microbodies had a densityof 1.25 g/cm³, and contained catalase and urate oxidase, butnot malate dehydrogenase, isocitrate lyase, glycolate oxidase,hydroxypyruvate reductase and the cyanide-insensitive palmitoylCoA-oxidation system. A small amount of o-diphenol oxidase alsoseemed to be present. Catalase, but not urate oxidase, activity in the crude extractincreased during aging of the sliced tissue. A similar resultwas obtained with the microbody fraction after linear sucrosedensity gradient centrifugation. We propose that microbodiescontaining only catalase develop during aging of sliced sweetpotato root tissue. ¹ This work was supported in part by a Grant-in-Aid (No. 311908)for Scientific Research from the Ministry of Education, Scienceand Culture, Japan. (Received June 20, 1979; )     

Growth, respiration and some enzymes of three strains of Ceratocystis fimbriata

Growth, respiratory activities and electrophoretic characteristicsof phosphatase and catalase in three strains of Ceratocystisfimbriata (sweet potato strain, coffee strain and prune strain)differing in pathogenicity on sweet potato roots were investigated.There were no significant differences in either growth kineticsor respiratory activity among the strains. Potassium cyanideand antimycin A inhibited oxygen uptake in sweet potato andprune strains. The oxygen uptake of endoconidia of coffee strainwas stimulated by these inhibitors. Mitochondria were preparedfrom endoconidia and mycelia of each strain, and enzyme activitiesof the electron transport system were measured. NADH₂: cytochromec oxidoreductase activity of coffee strain was higher than thatof the other strains. The electrophoretic phosphatase patternof coffee strain was identical with that of sweet potato strain,but differed from that of prune strain. On the other hand, thecatalase zymogram from prune strain was closely related to thatof sweet potato strain, but not to that of coffee strain. ¹This paper constitutes part 79 of the phytopathological chemistryof sweet potato with black rot and injury. (Received May 22, 1969; )     

Isolation of 1-O-trans-p-Coumaroyl-r{beta}-D-glucopyranose from Sweet Potato Roots and Examination of Its Role in Chlorogenic Acid Biosynthesis

1-O-trans-p-Coumaroyl-rß-D-glucopyranose (p-coumaroyl-D-glucose)was isolated from slices of sweet potato root which had beenincubated with trans-cinnamic acid. Pre-loaded trans-cinnamicacid efficiently trapped the radioactivity from L-[U-¹⁴C]-phenylalanineand reduced its incorporation into chlorogenic acid by 75% ofcontrol values in disks of sweet potato root. In the root diskssupplied with trans-[3-¹⁴C]-cinnamic acid, the radioactivitywas transferred first to trans-cinnamoyl- D-glucose, then top-coumaroyl-D-glucose, and subsequently to chlorogenic acidand isochlorogenic acid. These results support our earlier propositionthat p-coumaroyl-D-glucose is involved in the biosynthesis ofchlorogenic acid as an intermediate adjacent in the pathwayto trans-cinnamoyl-D-glucose in sweet potato roots. (Received April 11, 1988; Accepted August 9, 1988)     

Possible role of the cytosol pathway of acetyl-CoA supply in terpene biosynthesis in sweet potato infected with Ceratocystis fimbriata

Pyruvate was converted into acetyl-CoA by the cytosol enzymefraction prepared from sweet potato root tissue infected withCeratocystis fimbriata. The conversion was dependent on thiaminepyrophosphate, NAD⁺, ATP and CoA. Activities of pyruvate decarboxylase,aldehyde dehydrogenase and acetyl CoA synthetase increased indiseased tissue. These results indicate the operation of thecytosol pathway of acetyl-CoA supply for terpene biosynthesisin C. fimbriata-infected tissue which consists of the abovethree enzymes. ¹This paper constitutes Part 135 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. This work was supportedin part by a Grant-in-Aid from the Ministry of Education, Scienceand Culture. ²Present address: Biochemistry Department, University of Queensland,St. Lucia 4067, Queensland, Australia. (Received April 21, 1980; )     

Properties, development and cellular-localization of cinnamic acid 4-hydroxylase in cut-injured sweet potato

In sweet potato root tissue, cinnamic acid 4-hydroxylase activityincreased markedly in response to cut injury, and reached amaximum after 1 day of incubation. The patterns of developmentand successive decline were similar to those for phenylalanineammonia-lyase activity. The development of both enzyme activitieswas inhibited by cycloheximide. The activity was strictly dependenton pH of the homogenizing and reaction media. The optimum pHof the reaction was 8.0. The respective Km values for trans-cinnamicacid and NADPH were 2.6?10^-5 and 1.8?10^-6M. The activity wasnot affected by ß-mercaptoethanol and the intermediatesand product of the polyphenol biosynthetic pathway. Carbon monoxideinhibited strongly the activity and its inhibition was partiallyprevented by light. Thus, the enzyme may be involved in thecytochrome P-450 mediated electron transport system. Studiesusing differential centrifugation and sucrose density gradientcentrifugation, showed that the intracellular distribution of4-hydroxylase activity differed distinctly from that of themitochondrial marker enzyme and was not in accord with thatof NADPH-cytochrome c reductase activity. ¹This paper constitutes part 114 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. ²Present address: Laboratory of Biochemistry, Faculty of Agriculture,Nagoya University, Chikusa, Nagoya 464, Japan. (Received May 20, 1974; )     

Some investigations on inactivation of phenylalanine ammonia-lyase in cut-injured sweet potato root tissue

In sweet potato roots, activity of the phenylalanine ammonia-lyase(PAL)-inactivating system in crude enzyme solution increasedmarkedly in response to cut injury after a lag period of about10 hr and reached a maximum after 24 hr of incubation. The resultscoincided with previous results from experiments using a proteinsynthetic inhibitor. The inactivating system could be precipitatedby centrifugation and was distributed in a different patternfrom mitochondrial and microsomal marker enzymes, accordingto data from cellular fractionation by differential and sucrosedensity gradient centrifugation. The optimum pH of the inactivationwas 6.0. Previous studies showed that PAL content changed inparallel with PAL activity in vivo. However, immunochemicalstudies indicated that the inactivation was not due to proteolysis.Furthermore, proteinase activity in sweet potato tissue didnot change in response to cut injury. These results suggestedthat PAL was first inactivated by the inactivating system, thenthe inactivated PAL was rapidly decomposed by the proteinase. ¹ This paper constitutes Part 130 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. This work was supportedin part by a grant from the Ministry of Education. ² Present address: Faculty of Agriculture, Yamaguchi University,Yamaguchi 753, Japan. (Received May 14, 1977; )     

Immunocharacterization of NADH-Glutamate Dehydrogenase from Vitis vinifera L

Rabbit antiserum was raised against NADH-glutamate dehydrogenase (GDH) isoenzyme 1, purified from leaves of Vitis vinifera L. cv Soultanina and its specificity was tested. This antiserum was used for immunocharacterization of the GDH from leaf, shoot, and root tissues. The antiserum recognized the seven isoenzymes of NADH-GDH and precipitated all the enzyme activity from the three tissues tested. Western blot following SDS-PAGE revealed the same protein band for the three tissues, with a molecular mass of 42.5 kilodaltons corresponding to NADH-GDH subunit. Results, based on the immunological studies, revealed that NADH-GDH from leaf, shoot, and root tissues are closely related proteins. Furthermore, addition of ammonium ions to the culture medium of in vitro grown explants resulted in a significant increase in NADH-GDH activity in root, shoot, and leaf tissues.     

Mechanism for the induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase in HgCl2-treated sweet potaso root tissue

No 3-hydroxy-3-methylglutaryl coenzyme A reductase activitywas detected in microsomal fractions prepared from healthy andwounded sweet potato root tissues. However, there was considerableenzyme activity in the tissue discs when Hgcl₂ was applied afterincubation at 30?C for 18 hr. This increase in the enzyme activitywas followed by furano-terpene accumulation. Application ofcycloheximide to discs immediately after preparation completelyinhibited the increase in the enzyme activity when HgCl₂ wasapplied after incubation. In contrast, the increase was delayedfor about 4 hr, then the activity was enhanced, when CHI wasapplied after preliminary incubation. CHI completely inhibitedprotein synthesis when applied to the discs after the preliminaryincubation, as judged by the inhibition of the incorporationof ¹⁴C-leucine into protein and the inhibition of the increasein peroxidase activity which is synthesized de novo. These resultssuggest that the inactive precursor of HMG-CoA reductase issynthesized during the preliminary incubation in response onlyto wounding then it is converted into the active form aftertreatment with HgCl₂. (Received January 11, 1979; )     

Effects of cycloheximide, actinomycin D and ethylene on the increase and subsequent decrease in acid invertase activity in wounded sweet potato

In wounded sweet potato root tissue, the mechanisms of the increaseand subsequent decrease of acid invertase activity were examinedin compariosn with phenylalanine ammonia lyase (PAL) and peroxidaseactivities. The increase in acid invertase and PAL activitiesin response to wounding was inhibited by both cycloheximideand actinomycin D. However, the increase in peroxidase activitywas inhibited by cycloheximide but was not inhibited by actinomycinD, which repressed RNA synthesis 60 to 70%. The increase inacid invertase activity was supressed by ethylene, while PALand peroxidase activities were stimulated. The results of cycloheximideco-treatment suggest that the fluctuation in enzyme activitiesby ethylene treatment involves de nove protein synthesis. Further,the effect of ethylene on enzyme activities was under competitionby carbon dioxide. The decrease in acid invertase activity atthe late stage of incubation was stimulated by cycloheximidetreatment, but that of PAL activty was prevented. The resultssuggest the existence of different inactivating systems betweenacid invertase and PAL. ¹ This paper constitutes Part 117 in the Series "The PhytopathologicalChemistry of Sweet Potato with Black Rot and Injury". (Received September 2, 1974; )     

Induction mechanism of 3-hydroxy-3-methylglutaryl-CoA reductase in potato tuber and sweet potato root tissues

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.