Phytochrome from green plants: assay, purification and characterization. Progress report, June 1, 1984-July 15, 1986
This research has been directed toward characterizing and purifying the molecular species of phytochrome detected in green Avena tissue. The major differences that we have found between the phytochrome extracted from green and from etiolated tissue have now been published. In addition to the initially observed spectral and immunochemical differences, we have established: (1) That the predominant (approx.805 of total) phytochrome polypeptide in green tissue has a relative molecular mass (Mr) of 118,000; (b) That the proteolytic peptide map of this 118,000-Mr species differs considerably from that of 124,000-Mr phytochrome from etiolated tissue; (c) That the green-tissue, 118,000-Mr polypeptide carries only one of three spatially separate epitopes that are present on etiolated- tissue phytochrome (i.e. an epitope in the carboxy-terminal domain recognized by Type 3 monoclonal antibodies); (d) That the minor phytochrome species in green tissue (approx.20% of total) resemblies that in etiolated tissue in that it is 124,000-Mr and is immunoprecipitable with polyclonal, anti-etiolated-oat-phytochrome antibodies, thereby accounting for the previously observed limited population of immunoprecipitable activity in green extracts; and (e) That the 118,000-Mr green-tissue molecule migrates on non-denaturing size exclusion chromatography as a approx.320 kDa entity suggesting a quaternary structure similar to etiolated tissue 125,000-Mr phytochrome. A new purification protocol that enriches the green-tissue phytochrome approx.200-fold has been developed. The preparations obtained in this way are apparently free of residential endogeneous proteolytic activity. We have examined the regulation of the level of the 118,000-Mr species during seedling development and have obtained evidence that the abundance of this species is not modulated by light, in contrast to its etiolated-tissue counterpart. 11 refs., 7 figs., 1 tab.
- Research Organization:
- Wisconsin Univ., Madison (USA)
- DOE Contract Number:
- AC02-81ER10903
- OSTI ID:
- 5430257
- Report Number(s):
- DOE/ER/10903-7; ON: DE86013955
- Country of Publication:
- United States
- Language:
- English
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Phytochrome from green plants: Assay, purification, and characterization