Cross-linking of the enzymes in the glycosome of Trypanosoma brucei.
Glycosomes, the microbody-like organelles containing mainly glycolytic enzymes, were purified from the long slender bloodstream form of Trypanosoma brucei EATRO 110 monomorphic strain by an improved method in which the protozoa were frozen and thawed in 15% glycerol to free, from the plasma membrane, much of the variant surface glycoprotein which used to constitute the major contaminant of our purified glycosomes. The purified glycosomes have 11 major proteins, 6 of which, tentatively identified as phosphofructose kinase, hexokinase, 3-phosphoglycerate kinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alpha-glycerophosphate dehydrogenase, constitute 87% of the total glycosomal protein. The bifunctional cross-linking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate can penetrate the glycosomal membrane and cause extensive cross-linking of all the major glycosomal proteins. The cross-linked complex, insoluble in 0.1% Triton X-100 plus 0.15 M NaCl, contains all the glycosomal enzyme activities with only partial inactivations. All the enzymes are probably cross-linked into one large complex since they all sediment rapidly to the bottom of a 5-20% (v/v) sucrose density gradient. This successful cross-linking with reagents of span lengths of 11-12 A suggests close proximities among the glycosomal enzymes which may explain the extraordinarily high rate of glycolysis in T. brucei. Whether such a close association represents specific spatial arrangement required for genuine substrate channeling among the enzymes will be verified by future kinetic studies of the cross-linked enzyme complex.
D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004120:Dimethyl Suberimidate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006019:Glycolysis; D007096:Imidoesters / Q000494:pharmacology; D008854:Microscopy, Electron; D009940:Organoids / Q000201:enzymology*; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000648:ultrastructure*
D003432:Cross-Linking Reagents; D007096:Imidoesters; D004120:Dimethyl Suberimidate; C007471:dimethyl dithiobispropionimidate