ACTA AGRONOMICA VOLUME 35. (A MTA AGRÁRTUDOMÁNYI KÖZLEMÉNYEI, 1986)

1986 / 1-2. szám - R. NEHÉZ-S. FAZEKAS-I. ÓVÁRY-V. SZÉKESSY-HERMANN: Myosin preparations from the meristematic tissue of fresh sprouts of vine (Vitis vinifera L., cv. cardinal)

4 R. NEHÉZ et al. Sampling was performed at about 8 a.m. on 8th and 15th June 1982. It is very impor­tant that primary cell walls should be absent, and that the myosin should be prepared prac­tically from meristematic tissues. A larger quantity of sprouts (800-1000 g) was collected in a vessel containing crushed ice and stored at 0 °C until the preparation. The myosin was prepared by the method described by NEHÉZ et al. (1985), except that the precipitate (gained by dialysis) was sedimented out at a higher centrifugal rate (105,000xg) to increase the yield of crude myosin precipitated from the first, voluminous extract. Protein, lipid and P lipid content determinations, phosphorylation, and the separa­tion of basic amino acid phosphates were carried out as described by FAZEKAS et al. (1981a, 1981b). The phosphorylation is currently being investigated intensively at different ATP con­centrations and points of time, in order to obtain information about the kinetics of phosphate saturation. Results and discussion The dry matter content of fresh sprouts in the first half of June, the period of intensive growth, averaged 8.8 (+2.3)%. At this time the sprouts grew about 10-15 cm per day. The ultracentrifuging and DEAE-cellulose treatment of the crude myosin removed much accompanying matter. The purification was completed by gel­Fig. 1. TJV absorbance of gel-filtered myosin. Gel-filtration profile of vine myosin produced from meristematic tissues on a Sepharose 4B column (2.1x74 cm). The column was equi­librated with 0.5 M KCl solution containing 8 mM NaHC03 and 0.5 mM DTT. 5.5 ml total sample was poured on to the top of the column and eluted with the same buffer. 0.5 ml frac­tions were collected. The flow rate was about 18.6 ml per hour. The protein content was followed in every tube via the TJV absorbance, monitored at 280 nm. The solutions relating to the first peak were collected as myosin, and used for PAGE, phosphate saturation, time­dependent phosphate incorporation and basic amino acid phosphate investigations Abbreviations absorbance at 280 nm 0.3 0.2 J 0.1 -.La, 10 20 30 40 50 60 80 fraction number [ 5.7 ml/faction ] Chi = chloroform, DTT = dithiothreitol, HC = heavy chain, LC = light chain, MeOH = methanol, P = macroerg organic phosphate, Pi = inorganic phosphate, SDS = sodium dodecyl sulphate, TLC = thin layer chromatography Acta Agronomiea Hung. 35, 1986

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