. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. ERYTHROCYTE SHAPE TRANSFORMATION 401. 20 30 40 time (min) 50 60 Figure 11. Effect of sodium azide on shape transformation and recov- ery. N-cells were incubated for 30 or 60 min in 3 mM sodium azide in physiological medium (=expenmentals E30 and E60) or in physiological medium alone (=controls C30 and C60) prior to assay. Azide pre-incuba- tion markedly reduced the percentage of X-cells and accelerated shape reversal, with greater effect for the 60-min period. Controls maintained >90% X-cells throughout. had 95%-99%. In t
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. ERYTHROCYTE SHAPE TRANSFORMATION 401. 20 30 40 time (min) 50 60 Figure 11. Effect of sodium azide on shape transformation and recov- ery. N-cells were incubated for 30 or 60 min in 3 mM sodium azide in physiological medium (=expenmentals E30 and E60) or in physiological medium alone (=controls C30 and C60) prior to assay. Azide pre-incuba- tion markedly reduced the percentage of X-cells and accelerated shape reversal, with greater effect for the 60-min period. Controls maintained >90% X-cells throughout. had 95%-99%. In two additional experiments, controls with 9095-9395- initial activity retained 8095—9095- after 30 min, whereas experimental heated for 10 min had only 595--6% X-cells initially, and less than 295- in 30 min. Bioassays were conducted with various potential bio- chemical inhibitors added to hemolymph prior to the addi- tion of N-cells. Shape transformation was completely inhib- ited by 10 mM (or greater) EGTA, as reported previously (Dadacay et ai. 1996); at a given concentration, EDTA was less effective. The protease inhibitors initially surveyed were antipain, aprotinin, bestatin, chymostatin, leupeptin, aminoethylbenzenesulfonyl fluoride (AEBSF), and phos- phoramidon. As assayed after a 10-min exposure, only chymostatin and AEBSF were effective, at 189f and 79f X-cells, respectively (vs. 91% for controls). AEBSF pro- duced complete inhibition at 3 mg/ml (12 mM) and above, and an excess (5 mg/ml) was typically employed in other experiments. ATP-dependence of shape transformation was tested by pre-incubation of N-cells in physiological medium contain- ing 3 mM sodium azide for either 30 min or 60 min, followed by a wash, prior to mixing cells and cell-free hemolymph. Controls were similarly incubated, except without azide. As shown in Figure 11, the percentage of cells undergoing shape transformation was markedly re- duced and shape reversal accelerated in both cases com- pare
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