Blue light induced ADP ribosylation of 38 and 56 kDa proteins in the soluble fraction of mycelia of Neurospora crassa

Abstract

Soluble fractions prepared from the mycelia of wild type (74-OR23-1A) and band ( bd) exhibited an increase in the rate of the ADP ribosylation of a 38 kDa protein from nicotinamide adenine [ 32P]dinucleotide ([ 32P]NAD) in the presence of 10 −7 M riboflavin caused by blue light irradiation in vitro. The soluble fraction was mixed with a reaction mixture containing 5 μCi [ 32P]NAD at 0 °C for 20 s and then it was irradiated with blue light (420 nm, 42 μmol m −2 s −1) for 12.5, 25, 50, 100, 200 or 400 s at 0 °C or for 100 s with photon irradiance of 0.42, 4.2, 6.4 or 42 μmol m −2 s −1. Immediately after irradiation, the reaction was stopped and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. An increase in the ADP ribosylation of the 38 kDa protein could be detected within 100 s of irradiation, and the enhancement in the rate of ADP ribosylation of the 38 kDa protein was proportional to the increase in the photon irradiance. By the irradiation with blue light for 200 or 400 s, the ADP ribosylation of a 56 kDa protein could also be detected. Analysis by two-dimensional gel electrophoresis of proteins after ADP ribosylation of them revealed that the 38 kDa proteins displayed at least four radioactive protein spots and the 56 kDa protein a single radioactive protein spot. Soluble fractions of mycelia prepared from blind mutants wc-1, wc-2, Δps15-1, lis-1, lis-2 and lis-3 exhibited also the enhancement of the ADP ribosylation of the 38 kDa protein by blue light irradiation, and at least wc-1, Δps15-1, lis-1 and lis-2 displayed a similar blue light response in the 56 kDa protein.