L-type glycogen synthase. Tissue distribution and electrophoretic mobility.

A W Tan,D D Lesikar,H R Kaslow,D Antwi

doi:10.1016/s0021-9258(17)39192-5

Abstract

We previously reported (Kaslow, H.R., and Lesikar, D.D.FEBS Lett. (1984) 172, 294-298) the generation of antisera against rat skeletal muscle glycogen synthase. Using immunoblot analysis, the antisera recognized the enzyme in crude extracts from rat skeletal muscle, heart, fat, kidney, and brain, but not liver. These results suggested that there are at least two isozymes of glycogen synthase, and that most tissues contain a form similar or identical to the skeletal muscle type, referred to as "M-type" glycogen synthase. We have now used an antiserum specific for the enzyme from liver, termed "L-type" glycogen synthase, to study its distribution and electrophoretic mobility. Immunoblot analysis using this antiserum indicates that L-type glycogen synthase is found in liver, but not skeletal muscle, heart, fat, kidney, or brain. In sodium dodecyl sulfate-polyacrylamide gels of crude liver extracts prepared with protease inhibitors, rat L-type synthase was detected with electrophoretic mobility Mapp = 85,000. In contrast, the M-type enzyme in crude skeletal muscle extracts with protease inhibitors was detected with Mapp = 86,000 and 89,000. During purification of L-type synthase, apparent proteolysis can generate forms with increased electrophoretic mobility (Mapp = 75,000), still recognized by the antiserum. These M-type and L-type antisera did not recognize a protein with Mapp greater than phosphorylase. The anti-rat L-type antisera recognized glycogen synthase in blots of crude extracts of rabbit liver, but with Mapp = 88,000, a value 3,000 greater than that found for the rat liver enzyme. The anti-rat M-type antisera failed to recognize the enzyme in blots of crude extracts of rabbit muscle. Thus, in both muscle and liver, the corresponding rat and rabbit enzymes are structurally different. Because the differences described above persist after resolving these proteins by denaturing sodium dodecyl sulfate electrophoresis, these differences reside in the structure of the proteins themselves, not in some factor bound to the protein in crude extracts.

Highlights

These results suggested that there are at least two failed to recognize rat liver glycogen synthase by immunoblot isozymes of glycogen synthase, and that most tissues analysis [6]
This result suggested that there are at leastwo contain a form similaorr identical to the skeletaml us- isozymes of glycogen synthase
Inkeeping with thenomenclacle type, referred to as uM-type” glycogen synthase. ture used for other isozymes, we referred to the enzyme in

Summary

Mn M M M rat rat rab rat

Immunoblot analysis of muscle and liver glycogen synthase from rats and rabbits. Corresponding lanes [1,2,3,4,5,6,7,8] of panels A and B contain identical portions of the same samples, with 1 milliunit of glycogen synthase/lane. Other aliquots were stored on ice until assayed for glycogen as given by Bethesda Research Laboratories are: myosin = 200,000, synthase activity. Aftecrollection, a portion of the crude phosphorylase b = 97,400, bovine serumalbumin = 68,000, and liver extract was centrifuged at 78,000 X g for 2 h a t 4 "C to pellet ovalbumin = 43,000. The glycogen synthase assaybegan within 30 min of the endof the mobility of glycogen synthase (4, 51,we have used the term Mepp final centrifugation. 20 pl of appropriately diluted extracwt as assayed (apparent relative molecular mass, or apparent M,)to describe eleca t 30 "C in a final volume of 100 pI with a reaction mixture itself trophoretic mobility

RESULTS

DISCUSSION