Characterization of hypusine-containing 21,000-dalton protein in Neurospora crassa
The deoxyhypusine/hypusine-containing 21,000-dalton protein was labeled by ({sup 3}H)spermidine both in vitro and in vivo in polyamine depleted Neurospora crassa arg-12 ota aga and aga mutants. The in vitro labeling of the 21,000-dalton protein could be dramatically stimulated by NAD{sup +} and NADP{sup +} but not by FMN or FAD. The in vitro labeled 21,000-dalton protein contained the radioactivity of deoxyhypusine and hypusine with a ratio of 2 to 1. The in vivo labeled protein resulted only in hypusine. Three isoform structures of the in vitro labeled 21,000-dalton protein were found with pl values ranging from 5.2 to 6.5. In contrast, the 21,000-dalton protein metabolically labeled in vivo with ({sup 3}H)ornithine gave only one spot with a pl value of 3.5. The deoxyhypusine-modifying enzyme is heat labile and has a half life of 40 min. The complete inhibition of deoxyhypusine-modifying enzyme by NEM and pCMBS suggests that the sulfhydryl group is required for the activity. The unmodified 21,000-dalton protein is heat stable and has a half life of more than 5 hrs. In order to determine the functional role of hypusine modification of 21,000-dalton protein, a protein synthesizing cell-free system has been established to translate endogenous mRNA to discrete polypeptides ranging up to 200,000-dalton. The inhibitory effect of spermidine on protein synthesis in cell-free system of unmodified 21,000-dalton protein is in contrast to the stimulatory effect on protein synthesis in cell-free system of modified protein, suggesting that unmodified 21,000-dalton protein might be responsible for the inhibitory effect of spermidine on protein synthesis in cell-free system containing unmodified 21,000-dalton protein.
- Research Organization:
- Rutgers-the State Univ., New Brunswick, NJ (United States)
- OSTI ID:
- 5545665
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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MESSENGER-RNA
MUTANTS
NAD
NADP
ORNITHINE
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CHEMISTRY
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ORGANIC ACIDS
ORGANIC COMPOUNDS
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550201* - Biochemistry- Tracer Techniques