Failure of Lactoperoxidase to Iodinate Specifically the Plasma Membrane of Cucurbita Tissue Segments

Abstract

An attempt was made to use lactoperoxidase-catalyzed iodination of excised Cucurbita C. [pepo] hypocotyl hooks to monitor the distribution of plasma membrane fragments relative to that of phytochrome in particulate fractions from this tissue. Upon fractionation, the iodinated tissue yields a 20,000 g pellet which contains 58% of the trichloroacetic acid-precipitable 125I at a specific radioactivity 12 times that of the proteins in the supernatant. On sucrose gradients, the labeled fraction has a mean isopycnic density of 1.15 g .cntdot. cm-3. The distribution profile is distinct from that of the total particulate protein and does not coincide with either mitochondrial or endoplasmic reticulum markers. These observations satisfy operational criteria commonly accepted in other systems as indices of selective labeling of the cell surface. The sucrose gradient profiles of the phytochrome and 125I in the 20,000 g pellets are noncoincident. In the absence of more direct evidence, this indicates a lack of association of the pigment with the plasma membrane. Autoradiographic analysis indicates that the 125I is almost exclusively associated with an amorphous film (possibly phloem-exudate protein) overlying the cut cells at the point of prelabeling excision and along the outer physical surface of the hypocotyl cuticle. No evidence of plasma membrane labeling is apparent. The observed membrane-like behavior of the iodinated material on cell fractionation is attributed to the preferential posthomogenization association of this material with a particular membrane fractions(s). Apparently, in addition to the well recognized potential for spurious labeling of the internal cytoplasmic proteins of leaky cells, a further source of ambiguity in surface-labeling experiments should be considered: the potential for labeling extracellular proteins of nonplasma membrane origin but with a capacity to become associated with membranes on homogenization.