ABSTRACT
Absorption and transport of 3H cholesterol from the midgut to hemolymph and other tissues was studied in the locusts Schistocerca gregaria and Locusta migratoria. S. gregaria are able to absorb dietary cholesterol in the midgut and release into the hemolymph in vivo and into the incubation medium in virto. Certain proteins of midgut origin are involved in the absorption and release of cholesterol. The proteins designated as cholesterol binding proteins (CBP's) were fractionated by gel filtration chromatography using Sepharose CL-6B-200 column. Presence of a protein and its binding with cholesterol is confirmed by TCA precipitation after subsequent incubation of midgut in the incubation medium. Cholesterol binding with the proteins was also confirmed in native polyacrylamide gel electrophoresis. Biosynthesis of this protein takes place in the midgut which is inhibited by a protein synthesis inhibitor, cycloheximide. It also inhibits absorption and release of cholesterol from the midgut. The cholesterol binding activity was associated with a peak containing proteins ranging from molecular weights of 17-32 kDa in SDS-PAGE gels. Treatment of midgut with cycloheximide resulted in reduced cholesterol binding activity. Dilipidation of mucin and transport in presence of bile salts yielded a higher cholesterol binding activity. Although the absorption and release of cholesterol was observed in the hemolymph of both sexes, the ovary exhibited higher cholesterol binding as compared to testis.
Subject(s)
Animals , Cholesterol/metabolism , Chromatography/methods , Cycloheximide/pharmacology , Electrophoresis, Polyacrylamide Gel , Female , Grasshoppers , Hemolymph/metabolism , Male , Ovary/metabolism , Protein Binding , Protein Synthesis Inhibitors/pharmacology , Sepharose/pharmacology , Testis/metabolism , Time Factors , UltracentrifugationABSTRACT
Transducin serves as a mediator between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase, in the visual process. Transducin is a protein composed of three polypeptides: T alpha, T beta, and T gamma, and acts as two functional units, the alpha-subunit and the beta gamma-complex. In the present study, I describe an efficient and fast method of purifying T alpha and T beta gamma using chromatography on a blue agarose column connected in tandem with an omega-amino octylagarose column. The recombination of T alpha and T beta gamma reconstitutes the functional heterotrimeric holoprotein, as demonstrated by the recovery of three native properties of transducin: 1) its capacity to exchange guanine nucleotide, 2) its GTP hydrolytic activity, and 3) the ADP-ribosylation of T alpha catalysed by pertussis toxin