RESUMO
Using selected incubation conditions we have identified intermediate steps, between the first glucose transferred to protein and the appropriate substrate for glycogen synthase. Mn2+ stimulates the addition of the first, and probably, the second glucose molecule to the acceptor protein but inhibits further elongation. In the presence of Mn2+ only one radioglucosylated protein band of M(r) 42 kDa was evident. In the absence of Mn2+, two bands of 60.7 and 64.6 kDa were obtained indicating elongation of the glucan chains. After Glc6P addition a family of glucosylated proteins with higher M(r) was obtained, as reported previously. Mn2+ inhibition of the second step, is reversed by PMSF+Glc6P addition. Under these conditions a family of radioglucosylated protein bands with M(r) far in excess of 42 kDa, similar to that obtained without Mn2+, was obtained. Therefore, two different transglucosylating activities were necessary, at least, to prepare the appropriate substrate for glycogen synthase. Based on these observations the model we proposed earlier for glycogen biogenesis is modified. The original "Glycogen Initiator" implies at present two enzymatic activities, Glycogen Initiator 1 (activated by Mn2+) and Glycogen Initiator 2 (inhibited by Mn2+).
Assuntos
Glicogênio Sintase/metabolismo , Glicogênio/biossíntese , Glicoproteínas/metabolismo , Miocárdio/metabolismo , Animais , Glucanos/metabolismo , Glucose/metabolismo , Glucosiltransferases , Manganês/metabolismo , RatosRESUMO
A rat brain extract, able to synthesize from UDP-Glc an alpha-1,4-glucan covalently bound to a protein in the absence of added primer is described. The compound formed is precipitable by dilute trichloroacetic acid (TCA). In the presence of glycogen, added as primer, this molecule is enlarged and is not precipitable by TCA. Unprimed and primed activities differ in several aspects, such as the behavior in the presence of some effectors, and the optimum pH. Umprimed and primed activities presented two pHs optima, both sharing only one. The proteoglucans synthesized under the different pHs gave different patterns after analysis under denaturing PAGE and the oligosaccharides synthesized on the protein backbone differ in the glucosyl length. It is concluded that also in rat brain, the initiation process of glycogen biosynthesis is mediated through the formation of a glycoprotein. Our present results showed that the step of the putative "Glycogen Initiator" proposed by use before, requires two enzymes UDPGlc-transglucosylating activities, Glycogen Initiator 1 and Glycogen Initiator 2, before Glycogen Synthase in the alpha-1,4-glucosidic linkages formation.
Assuntos
Encéfalo/enzimologia , Glicogênio Sintase/metabolismo , Glicogênio/biossíntese , Animais , Cromatografia Líquida de Alta Pressão , Glucose/metabolismo , Glicoproteínas/metabolismo , Concentração de Íons de Hidrogênio , Manganês/metabolismo , Proteoglicanas/metabolismo , Ratos , Ratos Endogâmicos , Uridina Trifosfato/metabolismoRESUMO
Chemical and biochemical analysis of the polysaccharide, present in rat thymus, indicate that it consists of glucose units alpha-1,4 and alpha-1,6 linked. Electron microscopy reveals the presence of a polysaccharide, similar to the beta-glycogen particles observed in liver and muscle with an average diameter of 20-30 nm. They are located in the cytoplasmic area of T-cells from the cortical region of the thymus. Enzymatic analysis indicates that the beta-particles contain a highly branched glucan with short external chains. Some of the enzymes of glycogen metabolism: synthase, phosphorylase and branching were for the first time partially purified from rat thymus and some of their properties were studied. Therefore, glycogen appeared to be synthesized in rat thymus.