Tadashi Asahi

CHARACTERIZATION OF MAJOR PROTEINS IN SWEET POTATO TUBEROUS ROOTS

The tuberous roots, but not other organs, of sweet potato contained large quantities of two proteins which accounted for more than 80% of the total proteins. The two proteins, tentatively named sporamins A and B, were monomeric forms with similar M,s (25 000). They were separated from each other by electrophoresis on polyacrylamide gels in a non-denaturing buffer or a buffer containing sodium dodecyl sulphate without being reduced by dithiothreitol. They were very similar to each other with respect to amino acid composition, peptide map and immunological properties. These proteins decreased in preference to other proteins during sprouting. The amino acid sequencing of the amino terminal part of sporamin A suggested that it consists of at least two molecular species with different combinations of a few amino acids.

Sulfur metabolism in higher plants: IV. Mechanism of sulfate reduction in chloroplasts

Abstract This is the first report of a reduction in vitro of sulfate to sulfite by an enzyme system from higher plants. The reductive system has been found in chloroplasts of spinach leaves and compared with the yeast system. Adenosine 5′-phosphosulfate, but not 3′-phosphoadenosine 5′-phosphosulfate, was formed by the chloroplasts Since the reduction of sulfate to sulfite by chloroplasts in light requires the addition of sulfate-activating enzymes from yeast, it may be inferred that 3′-phosphoadenosine 5′-phosphosulfate is the substrate for the chloroplast sulfate reductase system. Fraction C-SS of yeast was also required. NADP did not participate in reduction in light and a NADPH 2 -generating system would not substitute for the photoreduction system. The chloroplasts also reduced a low-molecular-weight protein disulfide (fraction C-SS) in light but not in darkness even with NADPH 2 as hyrogen donor. The chloroplasts contained a fraction C-SS-like substance which was reduced both by the chloroplasts in light and by fraction C-SS reductase (NADPH 2 : fraction C-SS oxidoreductase) of yeast in the presence of NADPH 2 . Evidence is presented for the existence of a number of non-dialyzable disulfides in chloroplasts that are photo-reducible but not reducible by the fraction C-SS reductase of yeast. The results suggest that electrons for the reduction of 3′-phosphoadenosine 5′-phosphosulfate are supplied from the photosynthetic electron-transport system through fraction C-SS or a fraction C-SS-like substance but not from NADPH 2 .

Molecular cloning and nucleotide sequence of cDNA for sporamin, the major soluble protein of sweet potato tuberous roots

SummarySporamin accounts for more than 80% of the total soluble proteins of tuberous roots of sweet potato, but very little, if any, in other tissues of the same plant. In vitro translation of RNA fractions from the tuberous roots in wheat germ extract and subsequent immunoprecipitation with the antibody to sporamin indicated that this protein is synthesized by membrane-bound polysomes as a precursor 4 000 daltons larger than the mature protein. A cDNA expression library was constructed from the total poly(A)+ RNA from the tuberous roots by a vector-primer method, and an essentially full-length cDNA clone for the sporamin mRNA was selected by direct immunological screening of the colonies. Northern blot analysis showed that sporamin mRNA is approximately 950 nucleotides in length and is specifically present in tuberous roots and very little, if any, in leaves, petioles and non-tuberous roots. Nucleotide sequence of the cDNA predicts a 37 amino acid extension in the precursor at the amino-terminus of the mature protein.

Respiration and Related Metabolic Activity in Wounded and Infected Tissues

Publisher Summary This chapter discusses the time-course patterns of both wound respiration and infection-induced respiration, and possible biochemical mechanisms causing that increase in respiration. Wound respiration is induced when plant tissues, especially plant storage tissues, such as tuberous roots are mechanically cut into slices and incubated under conditions of moderate temperature and high humidity. The increased respiration is related to wound-healing reactions involving the formation of lignin and suberin. Wound respiration is also probably related to callus formation, which may in turn lead to differentiation such as root formation. Respiratory response to wounding or infection is variable. Some kinds of metabolic changes precede or coincide with the increase in respiration; others follow the increase. Moreover, some of the metabolic changes are similar between wounded and infected tissues whereas others differ greatly between the two.

Isolation and characterization of two tightly linked catalase genes from castor bean that are differentially regulated.

Two catalase genes,cat1 andcat2, have been isolated from the castor bean genome. They were located in the same direction on a chromosome at a distance of 2.4 kb,cat1 being on the downstream side ofcat2. The two genes contained introns at the same positions except that one of the 7 introns incat1 is missing incat2 and the corresponding introns differed in size and sequence between the two genes. The translated regions of the two genes had the same number of nucleotides and exhibited 81.3% nucleotide sequence identity. In addition to introns, the nucleotide sequences of the 5′-and 3′-flanking regions are highly divergent between the two genes. In etiolated seedlings,cat1 mRNA was present abundantly in endosperms and cotyledons and only in a small amount in roots. Thecat1 mRNA could not be detected in hypocotyls. By contrast,cat2 mRNA is most abundant in hypocotyls and roots, while endosperms and cotyledons contained only low levels ofcat2 mRNA. Although neithercat1 norcat2 mRNA could be detected in dry seeds, both mRNAs showed temporal accumulation in the endosperm in response to germination. These results suggest that expression of two tightly linked catalase genes of castor bean,cat1 andcat2, are differentially regulated during development.

Purification and characterization of the soluble form of mitochondrial adenosine triphosphatase from sweet potato

The soluble form of mitochondrial adenosine triphosphatase was purified in an electrophoretically and immunologically pure form from sweet potato root tissue. The enzyme consisted of six kinds of subunits with different molecular weights (52,500, 51,500, 35,500, 26,000, 23,000, and 12,000), and its molecular weight was about 370,000. Adenosine triphosphatase associated with the submitochondrial particles was oligomycin-sensitive and heat-labile, whereas the soluble form of the enzyme was oligomycin-insensitive and cold-labile. The enzyme in either the membrane-bound or the soluble form showed negative cooperativity. Both experiments with polyacrylamide gel electrophoresis and immunological methods suggest that some of the subunits, probably those with molecular weights of 52,500 and 51,500, are dissociated from the enzyme protein during storage of the enzyme preparations.

Characterization of the putative alpha subunit of a heterotrimeric G protein in rice.

A recombinant protein with a cDNA that encodes the putative α subunit of a rice heterotrimeric G protein was synthesized in Escherichia coli and purified. The recombinant protein (rGrice α) with an apparent molecular mass of 45 kDa was bound with guanosine 5′-(3-O-thio)triphosphate with an apparent association constant (kapp) of 0.36. The protein also hydrolyzed GTP and its Kcat was 0.44. rGrice α was ADP-ribosylated by activated cholera toxin.Monoclonal antibodies raised against rGrice α reacted with a 45 kDa polypeptide localized in the plasma membrane of rice seedlings. The peptide map of this polypeptide after digestion with V8 protease was identical to that of rGrice α. A 45 kDa polypeptide in the plasma membrane, as well as rGrice α, was ADP-ribosylated by activated cholera toxin. The GTPase activity of the plasma membrane was stimulated 2.5-fold by mastoparan 7 but not mastoparan 17. These properties were similar to those of the α subunits of heterotrimeric G proteins in animals, suggesting that the putative α subunit is truly the α subunit itself.

Purification and characterization of sweet potato cytochrome c oxidase.

Abstract Sweet potato cytochrome c oxidase (EC 1.9.3.1) was purified 45-fold with respect to its specific activity, with a high recovery by solubilization of the enzyme from the submitochondrial particles with deoxycholate, diethylaminoethyl-cellulose column chromatography, and fractionation with ammonium sulfate. Impurities, if any, could be removed by sucrose density gradient centrifugation of the purified enzyme preparation, although a considerable inactivation of the enzyme took place during centrifugation. The purified enzyme contained approximately 12 nmol of heme a per milligram of protein and about 2.5% phospholipid. The cytochrome c oxidase consisted of at least five polypeptides with molecular weights of 39,000, 33,500, 26,000, 20,000, and 5700, as determined by polyacrylamide gel electrophoresis of the purified enzyme preparation in the presence of sodium dodecyl sulfate and urea. Phosphatidylcholine and phosphatidylethanolamine stimulated the activity over 3-fold. The optimal pH of the purified enzyme was 7.0 to 7.5 in the presence of phosphatidylcholine (egg yolk or soybean) and pH 6.5 in the presence of phosphatidylethanolamine.

Changes in properties of the inner mitochondrial membrane during mitochondrial biogenesis in aging sweet potato tissue slices in relation to the development of cyanide-insensitive respiration

Abstract Protein and phospholipid of the inner mitochondrial membrane in sweet potato root tissue increased after a lag phase during aging of the sliced tissue. The protein, but not the phospholipid, from aged slices was more insoluble in a solution containing sodium deoxycholate and sodium cholate than that from fresh tissue. There were differences in polypeptide composition between deoxycholate-cholate-soluble and -insoluble fractions as determined by polyacrylamide-gel electorophoresis of membrane fragments in the presence of sodium dodecyl sulfate. However, no difference was observed between mitochondrial membranes from fresh and aged slices. When disrupted mitochondrial membrane was subjected to equilibrium density centrifugation, two bands were obtained from aged slices but only one band from fresh tissue. The lighter band from aged slices was indentical to the single band from fresh tissue. The denser band was very poor in phospholipid, and the protein was very insoluble in deoxycholate-cholate solution. The denser membrane fragments possessed a cyanide-insensitive respiratory chain whereas the lighter did not. It is proposed that the development of the cyanide-insensitive respiration in aging slices is related to the formation of phospholipid-deficient mitochondrial membrane.

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.