Proteins in Intercellular Washing Fluids from Leaves of Barley (Hordeum vulgare L.)

Primary leaves of barley were detached, infiltrated with variousbuffers, and centrifuged to yield ‘intercellular washingfluid’ (IWF). Effective pH control of the IWF was obtainedonly with Tris, among all buffers tried. In these liquids, upto 30 proteins were detected by gradient gel electrophoresis.Intracellular protein from injured cells at the cut ends ofleaves was present in IWF but did not contribute significantlyto the total protein recovered in this liquid. The yield ofprotein in the IWF depended on the buffer used for infiltrationand on the concentration of the buffer. Higher concentrationsof buffer yielded more protein. In other experiments leaves were infiltrated with Tris, centrifuged,and then infiltrated a second time with this buffer containingvarious concentrations of the zwitterionic detergent CHAPS,a sulphobetaine derivative of cholate. Gel electrophoresis ofthe IWF obtained after the second centrifugation revealed protein‘bands’ not detected when the detergent had beenomitted from the infiltration buffer. The electrophoretic patternsof protein ‘bands’ in the gels differed dependingon the CHAPS concentration used for infiltration. The effect of CHAPS on plasmalemma integrity was studied byobserving infiltrated tissue with the electron microscope andby treating isolated protoplasts with the detergent. After infiltrationwith CHAPS at 0.6 mM or 2.0 mM no plasmalemma breaks were detectedin leaves, and isolated protoplasts survived exposure to CHAPSat these concentrations for 2 h without bursting. Evidently,CHAPS at these low concentrations did not destroy the integrityof the plasmalemma; the additional protein recovered in theIWF under these conditions probably originated in the cell wall.Infiltration of leaves with 6.0 mM CHAPS resulted in breaksof the plasmalemma, in tissue collapse and leaf tip necrosis.Isolated protoplasts burst within minutes after being exposedto CHAPS at this concentration. Key words: Cell wall permeability, Intercellular space, Detergent, CHAPS, Protoplasts