Biomembranes Part U: Cellular and Subcellular Transport: by Sidney Fleischer, Becca Fleischer (Eds.)

By Sidney Fleischer, Becca Fleischer (Eds.)

The significantly acclaimed laboratory regular, Methods in Enzymology, is among the such a lot hugely revered guides within the box of biochemistry. because 1955, each one quantity has been eagerly awaited, usually consulted, and praised by means of researchers and reviewers alike. The sequence comprises a lot fabric nonetheless appropriate this day - actually a vital book for researchers in all fields of lifestyles sciences

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It is not necessary to add such an inhibitor if the amino acid used is metabolized only very slowly (as is the case with leucine) or if the incubation period is less than about 1 min (when little metabolism will have occurred). Second, since the cell pellet on centrifugation is contaminated with a considerable quantity of extracellular water, a marker of the extracellular space should be included. Inulin, polyethylene glycol, or sucrose are suitable compounds to use for this purpose. Third, the method of termination of the transport reaction by separation of cells from the meM.

Gerok, and G. Kurz, Eur. J. Biochem. 102, 1 (1979). [4] HEPATOCYTE ALANINE TRANSPORT 31 sic membrane proteins with molecular weights of 54,000 and 49,000. Similar results were obtained when labeling was performed on intact hepatocytes followed by membrane isolation and SDS-gel analysis. The specificity of the labeling reaction was assessed by carrying out the photolysis of the 7-ADTC-membrane complex in the presence of taurocholic acid. As shown in Fig. 3, the presence of the natural substrate resulted in a large decrease in the labeling of the 68,000, 54,000, and 49,000 proteins.

4 j. D. McGivan, Biochem. J. 182, 697 (1979). [4] HEPATOCYTE ALANINE TRANSPORT 33 dium must also achieve the rapid deproteinization of the cells to prevent any possible metabolism of the substrate, and this is commonly done by centrifuging the cells through a mixture of silicone oil and dinonyl phthalate into perchloric acid. 1 ml of a mixture (1 : 1, v/v) of silicone fluid MS550 and dinonyl phthalate above this. 5 m M aminooxyacetate. 25-ml aliquot of the incubation (containing not more than 3 mg of protein) above the silicone oil layer and centrifuging for a minimum of 10 sec.

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