. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 270 S. C. EDWARDS ET AL conditions, or incubated under phosphorylating condi- tions and subsequently stripped of phosphates with alka- line phosphatase. Specifically, aliquots of LON homoge- nates were incubated for 30 min at 30°C in media con- taining nonradioactively labeled ATP (30 pAl) with, or without, 10 nM 8-bromo cAMP (Sigma) (Edwards and Battelle, 1987). At the end of the incubation period, sev- eral aliquots were treated with 2X SDS buffer and soni- cated as described above. Other aliquots were mixed with an equal
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 270 S. C. EDWARDS ET AL conditions, or incubated under phosphorylating condi- tions and subsequently stripped of phosphates with alka- line phosphatase. Specifically, aliquots of LON homoge- nates were incubated for 30 min at 30°C in media con- taining nonradioactively labeled ATP (30 pAl) with, or without, 10 nM 8-bromo cAMP (Sigma) (Edwards and Battelle, 1987). At the end of the incubation period, sev- eral aliquots were treated with 2X SDS buffer and soni- cated as described above. Other aliquots were mixed with an equal volume of M Tris HC1 (final pH ) con- taining 2 mM phenylmethylsulfonyl fluoride, 2 mM ZnCl:, mM leupeptin, and 100 units/ml aprotinin, and then incubated for 2 h at 37°C with, or without, 81 units/ml calf intestinal alkaline phosphatase (type VII, Sigma) before the addition of SDS buffer. Samples con- taining the same amount of tissue protein were separated by SDS PAGE, blotted onto nitrocellulose, and the rela- tive level of phosphorylation of the 122 kD protein for each treatment was determined using the back phos- phorylation procedure. Tissue distribution of the 122 kD protein To determine the tissue distribution of the 122 kD pro- tein in Limulits, samples from visual and nonvisual ner- vous tissues were homogenized in SDS buffer, sonicated and stored at 4°C until they were analyzed. The amount of protein in each sample was determined by a modified Lowry procedure (Peterson, 1977) using bovine serum albumin as the standard. Tissue samples containing an equal amount of protein were subjected to SDS PAGE, and proteins were visualized by silver stain (Heukesho- ven and Dernick, 1985). Results I 'alidation of the back phosphorylation using LON homogenates Figure 1 shows that the 122 kD protein from the LON is a substrate for phosphorylation by purified catalytic subunit of cAMP PK and [f-3:PO4] ATP, even after it is separated from other tissue proteins by SDS-PA
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
