“Disaggregation” of Phytochrome in Vitro—A Consequence of Proteolysis

Abstract

The relationship between a large molecular weight (9S) and a small molecular weight (4.5S, 60,000 molecular weight) species of phytochrome was examined to determine if the larger species was an aggregate of the smaller. Alterations of pH, salt concentration, or phytochrome concentration did not cause any observable formation of the large form from the small form. However, in partially purified phytochrome extracts from Secale cereale L. and Avena sativa L., the large form was converted to the small form over time at 4 C in the dark. This breakdown was inhibitable by the protease inhibitor phenylmethanesulfonyl fluoride. When highly purified large molecular weight rye phytochrome was incubated with a neutral protease isolated from etiolated oat shoots, the large phytochrome was converted to the small form without qualitative visible absorbancy changes. The effect of the oat protease could be mimicked by a wide variety of commercial endopeptidases, including trypsin. Examination of the trypsin-induced breakdown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that as the size of the photoreversible unit changes from large to small, the size of its constituent polypeptide chains is reduced from 120,000 to 62,000 molecular weight. These experiments provide evidence that the endogenous breakdown observed in extracts is a result of contaminant protease and, consequently, that the small molecular weight species of phytochrome is an artifact due to proteolysis.Since the early work of Siegelman and Firer (41)

Cholinesterases from Plant Tissues

“…There are data concerning the formation of small forms of animal ChE by treatment with proteases (12,26). Recently, Gardner et al (6) have reported an analogous situation for phytochrome, providing evidence that the small molecular weight form of phytochrome is a result of protease activity. The above data and the fact that treatment of the bean ChE with bacterial protease resulted in partial breakdown of the enzyme suggest that the smaller molecular weight form of the bean enzyme obtained by procedure II is due to proteolysis.…”

Properties of a Solubilized Microsomal Auxin-binding Protein from Coleoptiles and Primary Leaves of Zea mays

“…We have attributed this discrepancy (5) to the protection against proteolysis afforded by our inclusion of the phenylmethylsulfonyl fluoride in the homogenization buffer. This compound is a potent inhibitor of proteases found in homogenates of grass seedlings (9).…”

“…The procedure does not change the recovery of auxinbinding sites compared to the previous procedure employing a mortar and pestle (unpublished results). The homogenization buffer was 50 mm Tris HC1, 1.0 mm Na2EDTA, 0.10 mm MgCl2, 14 mm 2-mercaptoethanol, 0.25 M sucrose (pH 8.0), normally saturated before use with phenylmethylsulfonylfluoride to retard proteolysis (9). The fluid was pressed through nylon cloth and microsomes were prepared by sequential sedimentation at 10,000g (10 min) and 48,000g (60 min) as previously described (5).…”

Auxin Receptors of Maize Coleoptile Membranes Do Not Have ATPase Activity

“…Both the coleoptiles and the primary leaves were used as starting material, since both contain the binding sites (24). Tissue was chopped and ground, as previously described (23), in one volume of homogenization buffer (50 mm Tris HCI, 1.0 mm disodium EDTA, 0.10 mM MgC62, 14 mM 2-mercaptoethanol, 0.25 M sucrose, (pH 8.0, normally saturated before use with PMSF3 to retard proteolytic degradation (8). After pressing the fluid through nylon cloth (2.7…”