In addition to their use in the polyurethane and pesticide industries, isocyanates have proven to be useful probes for the exploration of protein structure. This paper focuses on three aspects of isocyanates: their broad reactivity, their reversible interaction with cholinesterases, and the relative hydrolysis rates of alkyl and aryl isocyanates. The broad reactivity of isocyanates as well as the demonstrated affinity labeling of serine and sulfhydryl esterases are discussed. Extension of the affinity labeling studies to include the analysis of the inhibition of cholinesterases by methyl isocyanate shows that methyl isocyanate is not an effective inhibitor of any of the cholinesterases. The inhibition of cholinesterases by alkyl isocyanates shows a pattern of decreased specificity with decreased alkyl chain length. The inhibition of cholinesterases by isocyanates is shown to be reversible, with a maximum rate of reversal seen at physiological pH. This reversal is characteristic of the reaction of an isocyanate with a sulfhydryl group. Finally, the affinity labeling of proteins must compete successfully with the hydrolysis of isocyanates in aqueous solution. The hydrolysis of alkyl isocyanates is shown to be significantly slower than that of the aryl isocyanates.The application of alkyl and aryl isocyanates to the pesticide and polyurethane industries is of major commercial interest worldwide. In addition, the isocyanates have proven to be valuable tools to protein chemists making correlations between protein structure and function. It is the information from these latter studies that may prove valuable to our understanding of how isocyanates affect human and animal populations. As a reagent used for the study of protein structure and function, isocyanates have two major characteristics that make them attractive. First, they are highly reactive with a variety of functional groups found on biological macromolecules, and second, they have a finite lifetime that enables them to react with selected functional groups but not to exist long enough to cause significant, nonspecific modification of the macromolecule under study. It is the purpose of this paper to explore both of these advantages of the isocyanates as protein modification reagents, to summarize information already in the literature, and to present new data relevant to each point. Of particular interest to this symposium will be the comparison of the biochemical results for the aryl isocyanates and the alkyl isocyanates. In the former case, the emphasis will be on toluene diisocyanate (TDI)

Previous studies have indicated that phytochrome regulates Ca2" fluxes across the plasma membrane of plant cells. In this study we investigated whether phytochrome can also regulate such fluxes across mitochondrial membranes, using the Ca2"-sensitive dye murexide to monitor the uptake and release of Ca2" by mitochondria. The results showed that Ca2" fluxes in these organelles could be photoreversibly altered, red light diminishing the net uptake rate and far-red light restoring this rate to its dark control level. Treatment ofthe mitochondria with ruthenium red blocked their Ca2+ uptake. In the presence of this inhibitor, red light induced a net efflux of Ca2`from the mitochondria, and subsequent far-red light reduced this efflux to nearly zero, the dark control level. Light-induced rate changes in Ca2`flux, both with and without the inhibitor, persisted for several minutes in the dark and remained photoreversible through several irradiations for as long as 30 min. The purity of the mitochondrial preparation wasjudged to be about 80% by electron microscopic morphometry; most of the phytochrome present was localized on the mitochondria in the preparation by using immunocytochemical methods. Taken together with previous findings, the results suggest that red light activation of phytochrome would initiate an increase in the cytosolic Ca2`concentration. The results are integrated with the fact that calmodulin is a component ofplant cell cytoplasms to construct a model postulating that phytochrome directs photomorphogenesis in part through its regulation of Ca2+ and calmodulin-controlled enzyme activities.

Purified oat phytochrome was labeled with 125 I without altering the photoreversibility or absorbance properties of the pigment. The radiolabeled phytochrome was used in experiments in vitro to quantitate the binding of the pigment to both crude and purified membrane preparations from oat tissue. After the membranes were allowed to react with 125 I-labeled phytochrome, washed free of unbound material, and pelleted, they were found to have significant levels of radioactivity bound to them. Qualitative identification of phytochrome as the bound radioactive species was confirmed by autoradiography of sodium dodecyl sulfate gels after electrophoresis of the proteins contained in the washed membranes. Data supporting the specificity of the binding are that the binding shows saturation kinetics and that unlabeled phytochrome, but not bovine serum albumin, will competitively inhibit the binding of labeled phytochrome. This technique permits the detection of less than a nanogram of phytochrome and provides a new method for quantifying bound phytochrome that is independent of the spectral detectability of the pigment. It should be useful in elucidating the nature of phytochrome attachment to cellular membranes.

This study employs '25I-labeled phytochrome (126I-P) from oats to quantitate the binding of phytochrome to a membrane fraction from oats that is highly enriched for mitochondria, and it examines several parameters that influence this attachment. An early response to the photoactivation of phytochrome in plant cells is a change in the ion permeability and electrical potential of membranes in the affected cells (16,23,30). One inference from these findings is that phytochrome physically interacts with plant cell membranes en route to inducing changes in the growth patterns of plants. Support for this hypothesis comes from a wide range of studies, as discussed in several recent reviews (15,19,26). As an extension of these studies, we developed a method to quantitate the amount of phytochrome that binds to purified membranes when it is reacted with these membranes in vitro (8). The method, using 1251-P2, allowed us to determine whether this binding met certain minimal criteria for the specific interaction of a ligand with a receptor site, such as those discussed by Cuatrecasas (4). Our

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