Immunopurification and Initial Characterization of Dicotyledonous Phytochrome

Abstract

Antiserum was prepared against proteolytically undegraded phytochrome obtained from etiolated zucchini squash (Cucurbita pepo L., cv. Black Beauty). The antiserum was prepared by injecting into a rabbit immunoprecipitates between zucchini phytochrome and specific antiserum against undegraded oat (Avena sativa L., cv. Garry) phytochrome. Specific antiphytochrome immunoglobulins were purified from this crude serum by an affinity column consisting of conventionally purified undegraded pea phytochrome covalently linked to cyanogen bromide-activated agarose. These purified immunoglobulins were also linked to cyanogen bromide-activated agarose and were used to immunopurify zucchini, pea (Pisum sativum L., cv. Alaska), and lettuce (Lactuca sativa L., cv. Grand Rapids) phytochrome. All three dicotyledonous phytochromes exhibited a monomer size near 120,000 daltons by sodium dodecyl sulfate, polyacrylamide gel electrophoresis. Absorbance spectra of immunopurified zucchini phytochrome indicated that the ratio of visible to ultraviolet absorbance for purified zucchini phytochrome is lower than that observed for oat phytochrome. The isoelectric point of zucchini phytochrome, which was observed to be heterogeneous by this criterion, was found to be in the range of 6.5 to 7.0, higher than that observed for oat phytochrome. The electrophoretic mobility of zucchini phytochrome was found to be similar to that observed for oat and pea phytochrome under conditions that were nondenaturing and did not involve any molecular sieving effect. The amino acid analysis of zucchini phytochrome is similar to that reported previously for oat and rye (Secale cereale L., cv. Balbo) phytochrome.