Hideaki Shinshi

Relationships between peroxidase, IAA oxidase and polyphenol oxidase

Abstract Peroxidase, IAA oxidase and polyphenol oxidase activities were detected in tobacco cells in suspension culture. Using gel filtration, ion-exchange chromatography and disc gel electrophoresis, it was concluded that IAA oxidase activity is entirely due to peroxidases. Peroxidase and polyphenol oxidase zymograms using anionic gel separation were also similar, but the occurrence of true polyphenol oxidases, distinct from peroxidase, was demonstrated by cationic disc gel electrophoresis and gel filtration.

Enzymatic removal of the 5′-terminal methylated blocked structure of tobacco mosaic virus RNA and its effects on infectivity and reconstitution with coat protein

The 5’-terminus of tobacco mosaic virus (TMV) RNA, long thought to be an unphosphorylated A residue [ 1,2], was recently identified as m7GS’pppS’Gp [3,4]. The assembly reaction of TMV RNA with TMV protein is thought to start at the 5’ end and proceed to the 3’ end along the RNA chain [5-71. It would be interesting to know whether the odd structure at the 5’-terminus is related to the assembly reaction. On the other hand, elimination of the 3’-terminal base of TMV RNA by a chemical procedure (periodate oxidation and subsequent cleavage by aniline) was reported to cause marked loss of infectivity [8]. However, this chemical procedure probably eliminated both the 5’ and 3’ end of TMV RNA, so it is uncertain which elimination reaction was responsible for loss of infectivity. ’ These considerations prompted us to investigate the removal of the 5’-terminal blocked structure of TMV RNA and its effects on the assembly reaction and infectivity. Recently, a novel phosphodiesterase was purified from cultured tobacco cells [9]. This enzyme released pm7G from the 5’-terminal blocked structure of cytoplasmic polyhedrosis virus mRNA, but did not attack the polynucleotide chain of the mRNA [lo]. We used this enzyme to digest TMV RNA. We found that it preferentially cleaved the 5’-blocked structure of TMV RNA and that TMV RNA lacking the 5’blocked structure showed unchanged ability to assemble with the coat protein, but no infectivity.

Purification and properties of acid phosphatase from cultured tobacco cells

Abstract One component of acid phosphatase was purified from cultured tobacco cells. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate. The enzyme possesses high activity toward nucleoside di- and triphosphate, much less activity toward nucleoside monophosphates and sugar esters. The MWs of the phosphatase determined by Sephadex G-100 gel filtration and dodecyl sulfate gel electrophoresis were 74000 and 76000, respectively. The phosphatase showed high affinity for concanavalin A-Sepharose and single superimposed bands of protein and carbohydrate on gel electrophoresis, suggesting that it is a glycoprotein.

Properties of peroxidases from tobacco cell suspension culture

Abstract An enzyme preparation from suspension cultured tobacco cells oxidized IAA only in the presence of added cofactors, Mn 2+ and 2,4-dichlorophenol, and showed two pH optima for the oxidation at pH 4·5 and 5·5. Effects of various phenolic compounds and metal ions on IAA oxidase activity were examined. The properties of seven peroxidase fractions separated by column chromatography on DEAE-cellulose and CM-Sephadex, were compared. The peroxidases were different in relative activity toward o -dianisidine and guaiacol. All the peroxidases catalysed IAA oxidation in the presence of added cofactors. The pH optima for guaiacol peroxidation were very similar among the seven isozymes, but the optima for IAA oxidation were different. The anionic and neutral fractions showed pH optima near pH 5·5, but the cationic isozymes showed optima near pH 4·5. With guaiacol as hydrogen donor, an anionic peroxidase (A-1) and a cationic peroxidase (C-4) were very different in H 2 O 2 concentration requirements for their activity. Peroxidase A-1 was active at a wide range of H 2 O 2 concentrations, while peroxidase C-4 showed a more restricted H 2 O 2 requirement. Gel filtration and polyacrylamide gel studies indicated that the three cationic peroxidases have the same molecular weight.

Phosphodiesterase from cultured tobacco cells. Physical and chemical properties.

Phosphodiesterase isolated from suspension cultures of tobacco cells showed high affinity for concanavalin A-Sepharose and gave single superimposed bands of protein and carbohydrates on disc gel electrophoresis, suggesting that it is a glycoprotein. It contains 14% carbohydrate by weight, and has relatively high contents of basic and aromatic amino acids. Its isoelectric point is at pH 8.8, and the molecular weight of its subunits was estimated as 72 000 from a plot of the retardation coefficient on sodium dodecyl sulfate gel electrophoresis versus the molecular weight. The enzyme was catalytically active in an immobilized state on a concanavalin A-Sepharose column.

Dissociated active subunits of tobacco phosphodiesterase

The tetrameric nature of the phosphodiesterase isolated from tobacco cells is confirmed by determining the number of oligomers formed upon cross-linking the enzyme with dimethyl suberimidate. The isolation of the catalytically active monomer, which is formed by incubating the enzyme with urea and 2-mercaptoethanol, has been accomplished by gel filtration on Sephadex G-200. The isolated monomer of the phosphodiesterase is stable under nondenaturing conditions and catalytically active. The enzyme activity of the phosphodiesterase monomer is more sensitive to SDS than the tetramer. The phosphodiesterase tetramer exhibits characteristics of negative cooperativity, while the isolated monomer does not.

Phosphodiesterases of tobacco cell cultures and the intact plant

Abstract Tobacco cells grown in suspension culture contained a higher specific activity of acid phosphodiesterase than tissues of the intact plant. The activity of the cells increased with age. The presence of alkaline phosphodiesterase both in the cultured cells and the intact plant was demonstrated, and the properties were partially characterized.

Comparison of isoperoxide patterns in tobacco cell culture and in the intact plant

Abstract Analysis by ion-exchange chromatography of the enzymes from cultured tobacco cells and root or leaf tissues of the tobacco plant revealed that the cultured cells contain exclusively cationic peroxidases and the leaf tissues mainly anionic and neutral peroxidases.