Trypsin treatment sensitivity

Trypsin-mediated changes in atrazine affinity are compared in Figure 2B by two methods. Trypsin-treated membranes were tested for electron transport in the presence and absence of 0.25 uM atrazine this concentration of atrazine resulted in 50% inhibition of electron transport activity in control samples. Within the first 5 min of trypsin treatment, a marked decrease in affinity to atrazine was observed as indicated by a decrease in sensitivity of electron transport to atrazine. Measurements of herb-bicide binding using radioactive atrazine showed a parallel decrease in affinity of the atrazine for trypsin-treated membranes. 

The distinctly different behavior of the phenol-type herbicides following trypsin treatment suggests that different determinants within the PS II protein complex establish the "domains" that regulate the binding properties of these inhibitors. In spite of the fact that phenol-type herbicides will displace bound radiolabeled herbicides such as diuron, these inhibitors show noncompetitive inhibition (29, 30). At present, there are three lines of evidence which favor TH e involvement of two domains within the PS II complex that participate in creating the binding sites for these herbicides (a) isolated PS II particles can be selectively depleted of a polypeptide with parallel loss of atrazine sensitivity, but not dinoseb inhibition activity (33) (b) in resistant weed biotypes, chloroplast membranes that exhibit extreme triazine resistance have increased sensitivity to the phenol-type herbicides (13) and (c) experiments with azido (photoaffinity) derivatives of phenol and triazine herbicides result in the covalent labeling of different PS II polypeptides (, 31). 

Trypsin, long known to act on photosystem II (20,21) cuts the D-1 protein at arg 238 (discussed in 18). This cut is prevented by inhibitors of the DCMQ-and triazine-type and by certain quinones, but not by some of the phenol-type (18 and references therein). Furthermore, the D-2 protein is even more sensitive to trypsin at arg 238 (18,22). (This is the reason for the accessibility of to ferricyanide after trypsin treatment of the thylakoid meiid3rane as long known (21).) Also the trypsin cut of the D-2 protein is delayed by certain inhibitors and by quinones in the Qg site (18), indicating that the Qg site on the D-1 protein comminicates via the new contact site to the D-2 protein close to either arginine. As trypsin attacks these specific sites in either polypeptide without prior denaturation and even in the membrane, it is possible to model this area of that sequence according to the known structure of the trypsin inhibitor as it docks into the active site of trypsin (23). 

Based on its mobility in gel electrophoresis and its sensitivity to trypsin treatment, peak 7 protein also appears to be identical to band-II protein (one of the four ghost proteins), which has been implicated in determining the cell shape. However, if band II protein is the tol G protein, it is probably not a shape-determining protein, since its removal does not affect the shape of the cell. 

Kaulenas and Fairbaim (1966) have presented evidence for the presence of RNase-insensitive, inactive, ribosomal aggregates in precleavage eggs of Ascaris lumbricoides. Trypsin treatment of the microsomal pellet sensitizes the aggregates to dissociation by RNase and, at the same time, activates them for protein synthesis. The proportion of RNase-resistant, inactive polysomes decreases with development. Simi-... 

Figure 6. Fluorogram of polyacrylamide gel showing trypsin sensitivity of the 34-kDal polypeptide of pea chloro-plast membranes following in vivo incorporation of S-methionine in whole leaves. Key A, control B, 2 fxg trypsin/ mL C, 10 fig trypsin/mL D, 40 fig tryp-sin/mL. Treatments were as described in Figure 4.

Figure 6. PARP-2" mice are sensitive to ionizing radiation. A) Kaplan-Meier survival curves after 8 Gy of whole body irradiation. Wilcoxon test p(PARP-2 vs PARP-2 ) < 10. (Taken from Minissier-de Murcia et al, with permission). B) Comparison ofy-ray survival curves of wild-type, PARP-T and PARP-2 mouse 3T3 fibroblasts. 10 fibroblasts from mid-log growing subcultures were plated in triplicate in 25 cm flasks and returned to the incubator overnight prior to irradiation. Following treatment, the flasks were supplied with 8 ml fresh medium and grown for exacdy 5 doubling times (relative to mock irradiated cells) with two changes of medium. Cells were harvested by trypsin-EDTA and scored visually under microscope. PARP-1, PARP-T and PARP-2 fibroblasts yielded a convex curve which fitted a linear-quadratic dose-dependent equation, as most usually found among various cell lines, with a pseudo-plateau relating to Cl arrest. PARP-2 fibroblasts showed a concave profile and fitted a double-exponential equation. PARP-2 fibroblasts were clearly the most sensitive ones in the low dose rai of radiation (insert) Bars,SD.

The mast cell receptor for IgE appears to be a glycoprotein of approximately 50,000 molecular weight (Kulczycki et aL, 1976 Conrad and Froese, 1976). These results are only tentative as it is not known if this represents part of the receptor, a subunit of it or the entire moiety. An antiserum to the native receptor would be helpful, and it appears that one has recently been prepared (Froese, 1976). The receptor has been shown to be sensitive to trypsin, pepsin, and pronase treatment (Gon-zalez-Molina and Spiegelberg, 1976 Kulczycki et aL, 1976). 

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