Interconversion of two forms of muscle UDPG −α-glucan transglucosylase by a phosphorylation-dephosphorylation reaction sequence

Interconversion of two forms of muscle UDPG −α-glucan transglucosylase by a phosphorylation-dephosphorylation reaction sequence

PRELIMINARYNOTES 185 Preliminary Notes PN I O O I I Interconversion of two forms of muscle U D P G -~-glucan transglucosylase by a phosphorylation ...

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Preliminary Notes PN I O O I I

Interconversion of two forms of muscle U D P G -~-glucan transglucosylase by a phosphorylation - dephosphorylation reaction sequence We have previously reported 1-3 that insulin treatment of rat diaphragm increased the activity of U D P G : a-I.4-glucan a-4-glucosyltransferase (UDPG-a-glucan transglucosylase, EC 2 . 4 . i . I I ) when measured without added glucose 6-phosphate. When the enzyme was fully stimulated by adding this cofactor in excess, no difference was detected between extracts prepared from control and insulin-treated diaphragms. It was demonstrated that the activation by insulin was not related to the increased content of glucose 6-phosphate previously observed* or to any other soluble cofactor a. To explain the activation it was postulated that the enzyme might exist in two forms and that insulin might act to regulate their interconversion. The existence of two such forms was later confirmedS, ~ by their preparation from muscle in partially purified state and their differentiation by several criteria. One form (D form) was dependent upon glucose 6-phosphate, and the other (I form) acted independently of glucose 6-phosphate. We have now found that these two forms are interconvertible: the D form is converted to the I b y a dephosphorylation reaction, and the I to the D by ATP- and Mg~+-dependent phosphorylation of the enzyme. These conversions of the enzyme occur in a variety of crude homogenates of rat muscle (Tris, sucrose, GEY AND CjEy buffer) or in extracts prepared by centrifugation at 17 ooo × g. Most of the D form is converted to the I form in io min by incubating such extracts at 3 o°. I f ATP and Mg ~+ are then added, the I form rapidly decreases in activity as the D increases. These conversions together with preliminary evidence for the phosphorylation reaction have already been reported 7. When transglycosylase was purified by isolating particles sedimenting between 17 ooo and IOO ooo × g, the D to I conversion was lost. However, the ATP- and Mge+-dependent conversion of the I form to the D form remained highly active. After the transglucosylase was purified 15o-2oo fold over a DEAE-cellulose column, the ATP- and Mg2+-dependent conversion was still present, although there was a considerable slowing of the reaction. The enzyme purified in this way was incubated with A T P labelled with a2p in the two terminal phosphates in the presence of Mg 2+. It was then precipitated with ammonium sulfate, and isolated over either a second DEAE-cellulose column or over a phosphocellulose column. These columns gave further purification yielding an overall purification of 200-4o0 fold and 6oo-8oo fold respectively. In both cases the a2p in protein coincided with transglucosylase activity in the column fractions. The amount of incorporated phosphate calculated as Pi was in the range of 1.2-2. 4 re#moles/unit of transglucosylase converted from I to D. One unit is defined as that amount of enzyme catalyzing the addition of I #mole of glucose from U D P G to glycogen in I rain under the specified assay conditions 3. When ATP labelled in the E-phosphate was used instead of doubly labelled ATP, Biochim. Biophys. Acta, 64 (1962) 185-186



only about 1% of this incorporation was observed. The DEAE-cellulose fraction used in these incorporation experiments was free of significant phosphorylase a or b activity. This ruled out contamination by this enzyme which is known to incorporate Pi from A T P during its conversion from the b form to the a form. The 32p-labelled enzyme which was isolated on either the phosphocellulose or the second DEAE-cellulose column was then incubated with a crude muscle extract and the D to I conversion reaction established. The change of radioactivity in protein was measured after precipitation with HC104 .There was a complete correspondence between enzyme conversion and release of radioactivity. For example: in IO min 51% of the D enzyme activity was converted to I while 48% of the 32p in protein was released; in 2o min 6o% of the enzyme was converted and 54% of the radioactivity released. The radioactivity which was released was established as Pi (or a phosphate ester which is rapidly cleaved enzymically or by cold HC10,) by (I) precipitation with magnesia mixture and (2) by isobutanol extraction of the molybdate complex. It has long been recognized that the phosphorylase system is under hormonal controP. The studies presented here delineate a new enzyme system which is affected b y a hormone in which the enzyme is converted from one form to another by way of phosphorylation and dephosphorylation reactions. Details of these experiments and their implications will be published shortly. The authors wish to thank Miss H. SASKO for technical assistance in these studies. This work was supported in part b y the Cleveland Diabetic Fund and the Fund for the Study of Diabetes and Related Metabolic Disorders, and b y a grant from the U.S. Public Health Service (A-2366).

Department of Pharmacology, School of Medicine, Western Reserve University, Cleveland, Ohio (U.S.A.)


1 C. VILLAR-PALASI AND J. LARNER, Biochim. Biophys. Acta, 39 (196o) 171. 2 j . LARNER, Federation Proc., 19 (196o) 971. s C. VILLAR-PALASI AND J. LARNER, Arch. Biochem. Biophys., 94 (1961) 436. a j . LARNER, C. VILLAR-PALASI AND D. J. RICHMAN, Arch. Biochem. Biophys., 86 (196o) 56. 5 IV[. ROSELL-PEREZ AND J. LARNER, Federation Proc., 21 (1962) 2o6. 6 M. ROSELL-PEREZ, C. VILLAR-PALASI AND J. LARNER, Biochemistry, in t h e press. 7 D. FRIEDMAN AND J. LARNER, Federation Proc., 21 (1962) 206. 8 E. W. SUTHERLAND AND T. W. RALL, Pharmacol. Rev., 12 (196o) 265.

Received August 2nd, 1962 Biochim. Biophys. Acta, 64 (1962) 185-186