STUDIES ON PROTEIN METABOLISM IN HIGHER PLANTS. III. CHANGES IN THE SOLUBLE PROTEIN COMPONENTS WITH LEAF GROWTH

Water soluble protein was extracted from tobacco leaves (BrightYellow) and fractionated through chromatographic and immunochemicalprocedures. Six different UV-absorbing components, 7 antigeniccomponents, and 4 enzyme activities (phosphatase, protease,peroxidase and RNase) were detected on the Sephadex chromatogramsof leaf extracts. An UV-absorbing fraction, Fr. I-2, (immunochemicallydesignated as Pr. Imm-I) corresponded to the known "FractionI " of S. G. WILDMAN. The contents of the three antigenic components,Imm-a, Imm-b and Imm-c, having estimated molecular weights of1 to 2 10⁵, showed significant fluctuations during growthof the leaves. Peroxidase and another antigenic component (Imm-f)of smaller molecular weight showed increase with age of theleaves. Properties of the protein components thus detected wereinvestigated. (Received May 11, 1967; )     

STUDIES ON PROTEIN METABOLISM IN HIGHER PLANTS IV. CHANGES IN PROTEIN COMPONENTS IN DETACHED LEAVES OF TOBACCO

Soluble protein fractions from tobacco leaves before and aftercuring were compared. The results of Sephadex G-200 or G-75chromatography and immunological experiments showed that theamount of larger molecular weight proteins diminished or greatlydecreased and that smaller molecular weight proteins accumulatedduring 3 days at 40 and 90% humidity after excision of theleaves from the stem. Fraction I, which was the largest proteinin the leaf extract and occupied about one-half of the solubleprotein before curing, was not found in the proteins after curing.On the contrary, the proteins contained in II-4 fraction, whichwere supposed to have smaller molecular weights, increased three-foldduring the curing. The origin of the smaller proteins was discussed. (Received May 11, 1967; )     

SOME PROPERTIES OF PEROXIDASE PRODUCED IN SWEET POTATO INFECTED BY THE BLACK ROT FUNGUS

Chemical and physicochemical properties of peroxidases producedback rotted sweet potato roots were investigated in comparisonwith those produced in cut one. Peroxidases in either diseased or cut tissue were composed offour major (D-A, D-B, D-C and D-D in diseased tissue and C-A,C-B, C-C and C-D in cut tissue) and several minor components.These peroxidases were separated from each other by DEAE-cellulosecolumn chromatography and other procedures. Several propertiesof the peroxidases were investigated.

1. Optimum pH's of peroxidase were in the range of 5.5 to 6.0.
2. The activity of each peroxidase was inhibited by acid, alkaliand some inhibitors such as cyanide, fluoride and azide. Azideinhibited more strongly D-A and C-A than D-B and C-B. On theother hand, cyanide and fluoride inhibited more strongly D-Band C-B than D-A and C-A.
3. Substrate specificity as determimedby using pyrogallol, guaiacol,chlorogenic acid, caffeic acidand umbelliferone differed betweenthe main peroxidases. Thedegree of indoleacetic acid oxidaseactivity of these peroxidaseswas also different from each other.
4. Light absorption spectraof the peroxidases showed that theybelonged to a-type peroxidaseexcept C-D. More precise investigationsof the spectra showedthat the spectra of D-A and C-A were differentfrom those ofD-B and C-B.

Peroxidase A (D-A), the main component in diseased tissue, waspurified by methods such as DEAE-cellulose chromatography andstarchgel electrophoresis to a grade higher than previouslyshown. It was homogeneous, according to investigations withultracentrifugation, immunochemical reaction and starch-gelelectrophoresis. Pyridine hemochrome of the peroxidase showedthat the heme in it was protoheme. Amino acid composition ofthe enzyme was determined. Peroxidase A oxidized various phenolicsubstances in the presence of H₂O₂. Indoleacetic acid oxidaseactivity of peroxidase A was inhibited by both chlorogenic acidand guaiacol. ¹Part 45 of Phytopathological Chemistry of Sweet Potato withBlack Rot. ²Present address: Central Research Institute, Japan MonopolyCorporation, Yutakamachi, Tokyo.     

Comparative studies on fraction 1 protein from spinach and tobacco leaves

Fraction 1 protein of spinach and tobacco leaves was purifiedby Sephadex G-200 and DEAE-cellulose column chromatography.Immunological comparison of purified preparations of these proteinswas carried out with precipitin analysis using an antiserumof tobacco fraction 1 protein. Two different antigenic activitieswere found in tobacco fraction 1 protein, of which one was commonlyfound in the spinach protein and the other was specific to tobaccofraction 1 protein. The immunological results suggest that fraction 1 protein iscomposed of at least two different structures, one of whichis common to both spinach and tobacco proteins and the otherof which is specific to each of these proteins. This was confirmedfrom a chemical experiment. Fraction 1 protein was divided intolarge and small polypeptide components by sodium dodecyl sulfatetreatment and subsequent Sephadex G-100 column chromatography,then the amino acid compositions of each polypeptide of theseproteins were compared. The amino acid composition of the smallpolypeptide of spinach was different from that of tobacco, whileamino acid compositions of the large polypeptides of those proteinswere similar to each other. (Received July 1, 1968; )