Subcellular location and identification of a large molecular weight substrate for the liver plasma membrane transglutaminase.
When the particulate fraction from a rat liver homogenate was incubated with [3H]putrescine and calcium, the radioactive amine was incorporated into the membranes via a transglutaminase-mediated reaction. Fractionation of the membranes by isopycnic density gradient centrifugation revealed that the radioactive label was coincident with the 5'-nucleotidase and transglutaminase activities which serve as markers for the plasma membrane (Slife, C. W., Dorsett, M. D., Bouquett, G. T., Register, A., Taylor, E., and Conroy, S. Arch. Biochem. Biophys. 241, 329-336). If the labeled membranes were treated with digitonin and fractionated, the radioactivity and the plasma membrane enzyme activities coincidentally shifted to a greater density. Examination of the [3H]putrescine-labeled membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that the largest amount of radioactivity was associated with a large molecular weight material that did not enter the acrylamide gel. Pulse-chase experiments indicated that the large aggregate already was present in the native membrane, or that it was formed very rapidly during the putrescine incubation. The complex did not result from putrescine cross-linking between proteins since dansylcadaverine and [3H]histamine were also selectively incorporated into it. These data show that there are protein substrates in the plasma membrane which are accessible to the membrane-associated transglutaminase and that the substrates form a large molecular weight aggregate which is not dissociated by sodium dodecyl sulfate and disulfide reducing agents.
Slife
C W
CW
Dorsett
M D
MD
Tillotson
M L
ML
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D004072:Digitonin / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D011700:Putrescine / Q000378:metabolism; D051381:Rats; D013997:Time Factors; D014018:Tissue Distribution; D011503:Transglutaminases / Q000378:metabolism*
D008565:Membrane Proteins; D011503:Transglutaminases; D004072:Digitonin; D011700:Putrescine