Myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1) is a plant enzyme that hydrolyses glucosinolates, principally to isothiocyanates. Myrosinase was purified to homogeneity in good yield from 8-day-old seedlings of Raphanus sativus (daikon) using a four-step procedure involving chromatographies on anion exchange, hydrophobic Phenyl-Sepharose, gel filtration and concanavalin A-Sepharose. In order to stabilize the enzyme and to avoid excessive peak broadening during chromatography, 30% (v/v) glycerol was added to dialysis and chromatography buffers. The purified enzyme was eluted as a single peak from a gel-filtration sizing column with an apparent molecular mass of 120 kDa. The enzyme was resolved into two subunits with molecular masses of 61 and 62 kDa by SDS/PAGE. Ascorbic acid activated the purified enzyme more than 100-fold. The V(max) and K(m) values for the hydrolysis of allyl glucosinolate (sinigrin) were 2.06 micromol/min per mg of protein and 23 microM in the absence of ascorbate and 280 micromol/min per mg of protein and 250 microM in the presence of 500 microM ascorbate, respectively. As the ascorbate concentration was increased from 50 to 500 microM, the V(max) and K(m) values increased in parallel, and thus the V(max)/K(m) ratio remained constant. Similarly, raising the concentrations of sinigrin increased the concentration of ascorbic acid required for half-maximal activation (K(a)). At a sinigrin concentration of 250 microM, the K(a) for ascorbic acid was 55 microM. Sulphate, a reaction product, was a competitive inhibitor of activity, having a K(i) of 60 mM with respect to sinigrin and of 27 mM with respect to ascorbate. Thus activation of myrosinase from R. sativus by ascorbic acid exemplifies an unusual and possibly unique example of linear 'uncompetitive activation' (i.e. a proportionate increase in V(max) and K(m)) of an enzyme. The enzyme also had beta-glucosidase activity and hydrolysed p-nitrophenyl-beta-d-glucopyranoside.

Myrosinase (thioglucoside glucohydrolase ; EC 3.2.3.1) is a plant enzyme that hydrolyses glucosinolates, principally to isothiocyanates. Myrosinase was purified to homogeneity in good yield from 8-day-old seedlings of Raphanus sati us (daikon) using a four-step procedure involving chromatographies on anion exchange, hydrophobic Phenyl-Sepharose, gel filtration and concanavalin A-Sepharose. In order to stabilize the enzyme and to avoid excessive peak broadening during chromatography, 30 % (v\v) glycerol was added to dialysis and chromatography buffers. The purified enzyme was eluted as a single peak from a gelfiltration sizing column with an apparent molecular mass of 120 kDa. The enzyme was resolved into two subunits with molecular masses of 61 and 62 kDa by SDS\PAGE. Ascorbic acid activated the purified enzyme more than 100-fold. The V max and K m values for the hydrolysis of allyl glucosinolate (sinigrin) were 2.06 µmol\min per mg of protein and 23 µM in the absence of ascorbate and 280 µmol\min per mg of protein and 250 µM in

Spleen cells freshly isolated from normal mice were irradiated with 20 Gy X rays in culture. Northern blot hybridizations revealed that expression of the interleukin-1 beta (IL-1 beta) gene was induced immediately after irradiation and was increased for 2 h thereafter. Dibutyryl cyclic AMP also caused a persistent expression of the IL-1 beta gene, although it differed from X rays in that it coincidentally induced expression of the c-fos gene, which was not induced by X rays. Activation of either protein kinase C or calmodulin also induced early expression of both IL-1 beta and c-fos. Myeloid cells collected from the spleen of mice with granulocytic leukemia were X-irradiated in culture as above. The leukemia cells responded to X rays as well as to other stimuli in the same manner as the spleen cells, except that IL-1 beta mRNA was no longer detected 30 min after irradiation while c-fos was detectable for 2 h. When the leukemia cells were irradiated twice with a 3-h interval between irradiations, the second irradiation led to prolonged expression of IL-1 beta without inducing c-fos expression. These results suggest that ionizing radiation elicits early expression of the IL-1 beta gene through a mechanism that does not involve protein kinase C or A, or the transcription factor, c-fos. Whole-body irradiation of mice with 50 Gy 137Cs gamma rays also induced IL-1 beta expression in spleen but not in bone marrow or liver, although there was a delay of several hours before it was amply expressed. Furthermore, a delay as long as 24 or 72 h was observed when the radiation dose was reduced to 8.5 or 4 Gy. The results of this in vivo study suggest that the rapidity of expression of the IL-1 beta gene is dependent on the dose of radiation, and that the cells in the body cannot respond to radiation as rapidly as cells in culture.