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)     

Increase in mitochondrial activity in diseased sweet potato root tissue

When infected with Ceratocystis fimbriata, sweet potato roottissue showed an increase in respiration concomitant with anincrement in mitochondrial activities, which was not in parallelwith the increase in mitochondrial numbers. ¹ This paper constitutes part 100 of the phytopathological chemistryof sweet potato with black rot and injury. ² Present address: Department of Biochemistry, Faculty of NaturalSciences, Komensky University, Bratislava, Czechoslovakia. (Received June 21, 1972; )     

CHANGES IN ACTIVITIES OF 5-DEHYDKOQUINATE HYDRO-LYASE AND SHIKIMATE-NADP OXIDOREDUCTASE IN SLICED SWEET POTATO ROOTS

Sweet potato roots of variety Norin No. 1 were cut into discs(319 mm), which were incubated at 28–29 for 4 days.At 24-hour intervals, activities of 5-dehydroquinate hydro-lyaseand shikimate-NADP oxidoreductase in the buffer extracts ofdiscs were estimated. The activities of both enzymes were significantlydetected in the fresh root tissues. 5-Dehydroquinate hydro-lyaseactivity per fresh weight increased by three to four times within2 days after slicing, and decreased gradually. Shikimate-NADPoxidoreductase activity at the lst-day incubation period wasfound to be about three times as much as the activity foundin the fresh roots, and it remained for 4 days after slicing.The role of these enzymes in polyphenol biosynthesis in thesliced root tissues is discussed. An attempt to detect the enzymicconversion of dehydroquinic acid to quinic acid is also described. ¹This paper constitutes Part 55 of the Phytopathological Chemistryof Sweet Potato with Black Rot. ²Present address: Department of Biology, Tokyo MetropolitanUniversity, Tokyo. Nagoya     

Mechanism of chilling injury in sweet potato XI. Irreversibility of physiological deterioration

A mechanism affecting protein-lipid interaction in mitochondriais triggered in intact tissue of sweet potato roots by 2 daychilling-storage. Even after returning the once Chilled tissueto room temperature mitochondrial phospholipid is released,then the respiratory activity is decreased. ¹This paper constitutes Part 112 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. (Received September 3, 1973; )     

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.     

The mechanism of chilling injury in sweet potato VI. Changes of lipid components in the mitochondrial membrane during chilling storage

Phospholipids from the mitochondrial membrane of the sweet potato,a tropical plant, were composed mainly of phosphatidylethanolamine,phosphatidylcholine, lysophosphatidylcholine and an unknownphospholipid (S4) and their approximate contents were 39, 33,7 and 21 molar percent, respectively. Phosphatidylethanolaminebegan to decrease during the 4 day-chilling storage and thedecrease continued up to 40% during the 14 day-chilling storage.Decreases in phosphatidyicholine, lysophosphatidylcholine andS4 were indicated for the 8 day-chilling storage and their componentswere lost during the 14 day-chilling storage, by 20, 10 and20%, respectively. The amounts of these phospholipids did notseem to be changed further by prolonged chilling sotrage. Phospholipids of the mitochondrial membrane from the white potato,a temperate-zone plant, were composed mainly of phosphatidylethanolamine,phosphatidylcholine, lysophosphatidylethanolamine and an unknownphospholipid (W4), whose approximate contents were 39, 43, 3and 15 molar percent, respectively. Amounts of these four phospholipidswere not altered during the 14 day-chilling storage. ¹ This paper constitutes part 93 of the phytopathological chemistryof the sweet potato with black rot and injury. (Received August 21, 1971; )     

Mechanism of the Increase in Succinate Dehydrogenase Activity in Wounded Sweet Potato Root Tissue

The large subunit (mol wt: 65,000) of sweet potato succinatedehydrogenase was isolated by SDS-polyacrylamide gel electrophoresisof a succinate dehydrogenase preparation, which had been partiallypurified from root mitochondria by solubilizing the enzyme withEmulgen 810, DEAE-cellulose column chromatography, and polyacrylamidegel electrophoresis. Antibody to the purified large subunitwas produced in a rabbit, and the antiserum obtained was judgedto be specific to the large subunit based on the results ofdouble immunodiffusion tests and immunoelectrophoresis. Rocketimmunoelectrophoresis with the antiserum showed that the increasein succinate dehydrogenase activity during the ageing of sliced,sweet potato root tissue was due to an increase in the amountof enzyme protein. Both the increases in the activity of succinatedehydrogenase and in the amount of the large subunit proteinwere inhibited by cycloheximide or chloramphenicol. We proposethat synthesis of the large subunit of succinate dehydrogenaseon cytoplasmic ribosomes is controlled by a mitochondrial translationproduct(s). ¹ This work was supported in part by a research fund from TheIshida Foundation, Nagoya, Japan. (Received November 28, 1981; Accepted February 17, 1982)     

Studies on Microbody Development in Wounded Sweet Potato Root Tissue: Proposal for the Post-Translational Transport of Catalase into Preexisting Microbodies

Pure microbody fractions could be prepared in considerable yieldsfrom sweet potato root tissue slices incubated for 16 hr and3 days. The ratio of catalase activity to phospholipid contentin the fraction from slices incubated for 3 days was about 3times that from slices incubated for 16 hr. Total catalase activityin the former slices was about twice that in the latter. Thissuggests that catalase synthesized during incubation of theslices is transported into microbodies preexisting in intacttissue. ¹ Present address: Laboratory of Food Technology, Faculty ofApplied Biological Science, Hiroshima University, Fukuyama,Hiroshima 720, Japan. ² Present address: Terumo Co., Ltd., Omiya, Fujinomiya, Shizuoka418, Japan. (Received July 1, 1982; Accepted September 24, 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; )     

Mechanism of chilling injury in sweet potato VII. Changes in mitochondrial structure during chilling storage

Mitochondrial protein-N amounts per the contents of heme a,cytochrome b and cytochrome c, the tightly bound componentsin the mitochondrial inner membrane, were not changed during14 days of chilling stored sweet potatoes. However, the lipid-Pamount per amount of protein-N in mitochondria decreased byabout 20% during the 14 day chilling period. Via electron microscopy, two types of mitochondria were foundin the mitochondrial fraction prepared from healthy sweet potatoes.One, which had reticulately-developed cristae and extremely-electrondense matrix spaces, was called Form A. The other, which hadcristae which were not clearly distinguished from the matrixspaces, was called Form B. In the fraction prepared from 14day chill-stored sweet potatoes, a third type of mitochondriawas found besides Forms A and B. This one, which had an extremelyswollen form, was called Form C. Form C is thought to occur through the degradation of Form Aor B, concomitant with the release of phospholipid from boththe inner and outer membranes during chill-storage. It is quitelikely that Form C occurs during physiological deteriorationin sweet potato root tissues, which proceeds irreversibly duringchill-storage. ¹ This paper constitutes Part 95 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. (Received March 7, 1972; )     

Stimulation by Ethylene of Mitochondrial Development in Wounded Sweet Potato Root Tissue

Ethylene (about 100 µl per liter) markedly stimulatedincreases in respiratory, Cyt c oxidase and succinate dehydrogenaseactivities of the crude mitochondrial fraction as well as mitochondrialmembrane protein during aging of sliced sweet potato root tissue,indicating that it stimulated mitochondrial development in woundedtissue. It had such an effect even when slices were pre-agedin its absence for 1 day and thereafter aged in its presence.The mitochondrial inner membrane from slices aged in ethylene-containingair was denser than that from fresh slices, while the membranefrom slices aged in ethylene-free air was lighter. Chloramphenicolcompletely inhibited the increase in Cyt c oxidase activitywhether slices were aged in the presence or absence of ethylene.Cycloheximide did not inhibit the increase in slices aged inethylene-free air, but did by 50% in those aged in ethylene-containingair. ¹ This work was supported in part by a Grant-in-Aid (No. 411308)for Scientific Research from the Ministry of Education, Scienceand Culture, Japan. (Received April 4, 1981; Accepted July 7, 1981)     

Ultraviolet-B sensitivities in Japanese lowland rice cultivars: cyclobutane pyrimidine dimer photolyase activity and gene mutation

There is a cultivar difference in the response to ultraviolet-B(UVB: 280–320 nm) in rice (Oryza sativa L.). AmongJapanese lowland rice cultivars, Sasanishiki, a leading Japaneserice cultivar, is resistant to the damaging effects of UVB whileNorin 1, a close relative, is less resistant. We found previouslythat Norin 1 was deficient in cyclobutane pyrimidine dimer (CPD)photorepair ability and suggested that the UVB sensitivity inrice depends largely on CPD photorepair ability. In order toverify that suggestion, we examined the correlation betweenUVB sensitivity and CPD photolyase activity in 17 rice cultivarsof progenitors and relatives in breeding of UV-resistant Sasanishikiand UV-sensitive Norin 1. The amino acid at position 126 ofthe deduced amino acid sequence of CPD photolyase in cultivarsincluding such as Norin 1 was found to be arginine, the CPDphotolyase activities of which were lower. The amino acid atthat position in cultivars including such as Sasanishiki wasglutamine. Furthermore, cultivars more resistant to UVB werefound to exhibit higher photolyase activities than less resistantcultivars. These results emphasize that single amino acid alterationfrom glutamine to arginine leads to a deficit of CPD photolyaseactivity and that CPD photolyase activity is one of the mainfactors determining UVB sensitivity in rice. ¹ These authors contributed equally to the paper. ² Corresponding author: E-mail, kumagai{at}ige.tohoku.ac.jp; Fax,+81-22-217-5691.     

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; )     

Mechanism of chilling injury in sweet potato Part XII. Temperature dependency of succinoxidase activity and lipid-protein interaction in mitochondria from healthy or chilling-stored tissue

The Arrhenius plot of succinoxidase activity of intact mitochondriafrom healthy sweet potato (Ipomoea batatas Lam.) roots showedtwo transition temperatures of the activation energy at 8–10?Cand 16–18?C. Two transition temperatures were also observedin the case of sweet potatoes stored for 21 days at chillingtemperature, but the activation energy was lower than that ofhealthy tissue. The results on lipiddepleted mitochondria andlipid-rebound mitochondria indicated that the membrane lipidplayed an important role in the transition temperature of succinoxidaseactivity. On the other hand, in the binding of phospholipidto lipid-depleted mitochondria from healthy tissue, the numberof binding sites (n) and the dissociation constant (K) conspicuouslychanged at a temperature between 10 and 15?C. That is, n andK values above the temperature were considerably higher thanthose below the temperature, in spite of addition of phospholipidmicelles whose phase transition temperatures were differentfrom each other. In similar binding experiments with 14-daychilling-stored tissue, n values did not remarkably change,between higher and lower ranges of the above-mentioned temperatureand were lower at higher than at lower temperature. The resultssuggest that the conformation of the protein moiety in the mitochondrialmembrane undergoes reversible change in the early stage of chillingtreatment, which becomes irreversible with prolonged chillingtreatment. ¹This paper constitutes Part 113 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. ²Present address: Division of Breeding, Fruit Tree ResearchStation (Minist. Agric. Forest)., Hiratsuka, Kanagawa 254, Japan. (Received March 11, 1974; )     

Properties of the Dicyclohexylcarbodiimide-Binding Subunit of Cytochrome c Oxidase from Sweet Potato

Subunit IV of cytochrome c oxidase from sweet potato was boundwith dicyclohexylcarbodiimide and synthesized in isolated mitochondria.Thus, this subunit corresponds to subunit HI of the analogousmammalian and fungal enzymes. Subunit IV was a mixture of twopolypeptides (subunits IVa and IVb) which differed slightlyin structure. ¹Present address: Research Institute of Molecular Genetics,Shimane University, Matsue, 690 Japan. ²Present address: Institute of Low Temperature Science, HokkaidoUniversity, Sapporo, 060 Japan.     

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; )     

Elucidation of the structure of a possible intermediate in chlorogenic acid biosynthesis in sweet potato root tissue

The structure of a possible intermediate, compound V, in chlorogenicacid biosynthesis in sweet potato root tissue was determinedas ß-1-cinnamoyl-D-glucose. The role of the compoundin chlorogenic acid biosynthesis is also discussed. ¹ This paper constitutes Part 101 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. (Received June 28, 1972; )     

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; )