RESUMO
A curriculum is described whereby qualified Ph.D's can become M.D.'s within a period of 18 to 24 months. It is feasible and logical not only for us, but for other schools of medicine. It is our belief that adequate safeguards have been included to preserve high quality in education while responding to the need not only for more physicians, but also for improved utilization of some of our best-trained manpower.
Assuntos
Currículo , Educação Médica , Médicos , Educação de Pós-Graduação , Estados UnidosRESUMO
There have been several reports of the incorporation of glucosamine into liver glycogen by an intraperitoneal injection of galactosamine, but it has not previously been considered that glucosamine is a normal component of liver glycogen. We now report that glucosamine occurs endogenously in rabbit- and pig-liver glycogens in the amount of about 1 nmol per 10 mg glycogen. Like the glucosamine incorporated by exogenous administration of galactosamine, the endogenous glucosamine takes the place of 1,4-linked alpha-glucose residues. It is found in both the outer and inner chains of the glycogen molecule.
Assuntos
Glucosamina/análise , Glicogênio Hepático/análise , Animais , Fenômenos Químicos , Química , Glucana 1,4-alfa-Glucosidase , Glucosamina/análogos & derivados , Marcação por Isótopo , Metilação , Coelhos , Suínos , alfa-AmilasesRESUMO
The autocatalytic protein that primes muscle-glycogen synthesis, and which glucosylates itself from UDPglucose, is inhibited by maltose. Investigation of the reason for the inhibition led to the finding that the protein will glucosylate substrates other than itself. p-Nitrophenyl alpha-glucoside, alpha-maltoside, alpha-maltotrioside and alpha-maltotetraoside each inhibit self-glucosylation of the protein by acting as alternative acceptor substrates. The alpha-maltoside is the best acceptor. The alpha-maltohexaoside did not act as an acceptor but was an effective inhibitor. These findings help to explain the self-limiting nature of the autocatalytic extension of the maltosaccharide chain of the protein and suggest that protein self-glucosylation may be an intermolecular event. They may also point to the mechanism by which the autocatalytic protein is initially glycosylated.
Assuntos
Glucose/metabolismo , Glicogênio/biossíntese , Proteínas Musculares/metabolismo , Músculos/metabolismo , Animais , Glicosilação , Cinética , Maltose/farmacologia , Coelhos , Especificidade por Substrato , Uridina Difosfato Glucose/metabolismoRESUMO
We and others have reported that glycogenin, the covalently bound protein found in muscle glycogen, also exists in muscle in a glycogen-free form (Mr 38,000-39.000) that is autocatalytic, undergoes self-glucosylation and acts as a primer for glycogen synthesis. We now report that this entity is not present in a fresh muscle extract. Instead it exists within a pro form of much higher molecular mass which breaks down spontaneously to the Mr 38,000-39,000 form. Such breakdown is accelerated by the addition of alpha-amylase and is prevented by protease inhibitors. Multiple intermediates of the breakdown process have been detected, each capable of undergoing glucosylation.
Assuntos
Glicogênio/biossíntese , Glicoproteínas/metabolismo , Músculos/metabolismo , Animais , Glucosiltransferases , Glicogênio/metabolismo , Peso Molecular , Precursores de Proteínas/metabolismo , Coelhos , alfa-Amilases/farmacologiaRESUMO
We recently reported that muscle contains a trichloroacetic acid-precipitable component having Mr approx. 400 kDa that can be glucosylated by an endogenous enzyme acting on UDPglucose. This component contains within itself the autocatalytic, self-glucosylating protein glycogenin, the primer for glycogen synthesis. We now report that this substance, to which we give the name proglycogen, is a glycogen-like molecule constituting about 15% of total glycogen. It acts as a very efficient acceptor of glucose residues added from UDPglucose. Further, that the endogenous enzyme that adds the glucose to proglycogen is not the autocatalytic protein but a glycogen synthase-like enzyme. Proglycogen may be an intermediate in the synthesis and degradation of macromolecular glycogen and may exist and be metabolized as a separate entity. Consideration should now be given to the revival of the concept that tissue contains two forms of glycogen. One is proglycogen. The other is the 'classical', macromolecular glycogen. Additionally, proglycogen and glycogen may be glucosylated by different forms of synthase.
Assuntos
Glicogênio/metabolismo , Músculos/metabolismo , Amilases/metabolismo , Animais , Precipitação Química , Glucose/metabolismo , Glucosiltransferases , Glicogênio Sintase/metabolismo , Glicoproteínas/metabolismo , Peso Molecular , Coelhos , Ácido TricloroacéticoRESUMO
Glycogenin is the protein primer for glycogen synthesis. By autocatalytic transglucosylation from UDPglucose, it creates a malto-octaose chain attached to its Tyr-194. It has been uncertain whether the autocatalysis includes the addition of the first glucose residue to Tyr-194. We now show this to be the case. However, we also demonstrate, contrary to a claim by others, that Tyr-194 is not necessary for the catalytic function and activity of glycogenin.
Assuntos
Glicoproteínas/metabolismo , Tirosina/metabolismo , Animais , Catálise , Glucosídeos/metabolismo , Glucosiltransferases , Glicogênio/biossíntese , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Glicosilação , Mutagênese , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Rabbit-muscle glycogen contains covalently bound phosphorus, equivalent to 1 phosphate group per 208 glucose residues. This often disputed, minor component was previously thought to represent a phosphomonoester group at C-6 of a glucose residue. Here we show that more than half the phosphorus is present as a phosphodiester, the remainder being monoester. A novel enzyme activity has been found in muscle that can account for the presence of the phosphodiester in glycogen. This is a UDPglucose: glycogen glucose 1-phosphotransferase that positions glucose 1-phosphate on C-6 of glucose residues in glycogen, forming a diester. The phosphomonoester groups present may arise by removal of the glucose residue originally transferred as glucose 1-phosphate.
Assuntos
Glicogênio/metabolismo , Músculos/enzimologia , Fosfotransferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos) , Animais , Ésteres/metabolismo , Glicogênio/química , Músculos/metabolismo , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , CoelhosRESUMO
Glycogenin, the self-glucosylating primer for glycogen synthesis, is expressed in wild-type E. coli as a recombinant protein in an already partly glucosylated form, owing to the presence of its substrate, UDP-glucose. By using an E. coli mutant strain lacking in UDP-glucose pyrophosphorylase activity, we have succeeded in expressing carbohydrate-free glycogenin (apo-glycogenin) in good yield. When provided with UDPxylose, it autocatalytically adds 1 xylose residue. With UDP-glucose, an average of 8 glucose residues are added. However, release of the self-synthesized maltosaccharide chains with isoamylase reveals them to be a mixture. Chains as long as 11 glucose residues (maltoundecaose) are present. The ability of recombinant apo-glycogenin to self-glucosylate is further proof that a separate enzyme is not needed for the addition of the first glucose residue to Tyr-194 of the protein.
Assuntos
Escherichia coli/genética , Glicoproteínas/biossíntese , Proteínas Recombinantes/biossíntese , Escherichia coli/enzimologia , Expressão Gênica , Glucose/análise , Glucosiltransferases , Glicogênio/biossíntese , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Mutação/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Uridina Difosfato Glucose/metabolismo , Uridina Difosfato Xilose/metabolismoRESUMO
Glycogenin, the autocatalytic, self-glucosylating primer for glycogen synthesis by glycogen synthase, is presumed, in vivo, to use UDP-glucose as the source of the glucose residues it adds to itself. When we tested its ability to utilize other nucleoside diphosphate glucoses, it emerged that purine nucleotides are not utilized but two pyrimidine nucleotides are used, in addition to UDP-glucose. These are CDP-glucose and TDP-glucose. CDP-glucose is utilized at 70% of the rate of UDP-glucose. While there is no evidence that CDP-glucose is a natural substrate for glycogenin, it has the advantage over UDP-glucose in that it can be used specifically to detect and assay glycogenin in the presence of glycogen synthase because CDP-glucose, unlike UDP-glucose, is not a substrate for the synthase.
Assuntos
Glucose/análogos & derivados , Glicoproteínas/metabolismo , Açúcares de Nucleosídeo Difosfato/metabolismo , Glucose/metabolismo , Glucosiltransferases , Glicogênio Sintase/metabolismo , Especificidade por SubstratoRESUMO
In the search for a protein primer for starch synthesis, an autocatalytic self-glucosylating protein has been isolated from sweet corn. Several tryptic peptides were obtained from the [14C]glucosylated protein and were sequenced, corresponding to over 40% of the estimated total sequence (molecular mass 42 kDa). There is no homology with the amino acid sequence of the autocatalytic glycogen primer, glycogenin, nor in respect of the nature of the union between the autocatalytically added glucose and the protein, which, in the case of the corn protein, now named amylogenin, is a novel glucose-protein bond, a single beta-glucose residue joined to an arginine residue.
Assuntos
Arginina/análogos & derivados , Arginina/metabolismo , Glucose/metabolismo , Glucosídeos/química , Zea mays/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Arginina/química , Configuração de Carboidratos , Cromatografia em Agarose , Estabilidade Enzimática , Glucose/química , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Glicosilação , Transferases Intramoleculares , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Análise de Sequência , Homologia de Sequência de Aminoácidos , Tripsina/metabolismo , Zea mays/química , Zea mays/enzimologiaRESUMO
The use of chemically modified starch substrates for measurement of serum alpha-amylase activity is described. Action of alpha-amylase on such substrates, in the presence of excess fungal glucoamylase, results in the production of glucose in direct proportion to the amount of alpha-amylase present. The glucose produced is measured by a specific enzymic assay. Results obtained by using this new assay correlate well with activities determined by a conventional saccharogenic assay. The new method is of much higher sensitivity, and is less susceptible to interference, than are most other alpha-amylase assay methods.
Assuntos
Amilases/sangue , Amilases/metabolismo , Amilose/metabolismo , Glucosidases/metabolismo , Hidrólise , Métodos , Oxirredução , Saliva/enzimologia , Amido/metabolismo , Fatores de TempoRESUMO
A new facet of the specificity of the glycogen-debranching enzyme, isoamylase, namely, the hydrolysis of a carbohydrate-amino acid linkage, is described. This bond joins the terminal, reducing-end D-glucose unit of glycogen to the hydroxyl group of tyrosine in glycogenin, the primer protein for glycogen biogenesis. The specificity was further defined by demonstrating that 4-nitrophenyl alpha-maltotrioside and higher homologs also act as substrates. The splitting of the glycogen-glycogenin bond by isoamylase indicates the alpha-anomeric configuration of the terminal D-glucose unit. It also provides a means of preparing apo-glycogenin. Pullulanase, a somewhat similar starch- and glycogen-debranching enzyme, does not split these new isoamylase substrates, permitting the 4-nitrophenyl saccharides to be used in distinguishing between isoamylase and pullulanase.
Assuntos
Glicoproteínas/metabolismo , Glicosídeo Hidrolases/metabolismo , Isoamilase/metabolismo , Glucosiltransferases , Glicogênio/metabolismo , Especificidade por SubstratoRESUMO
Salivary alpha-amylase hydrolyses amylopectin in stages. At the end of the so-called second stage, there are present glucose, maltose, and a series of alpha-limit dextrins containing (1 leads to 4)- and (1 leads to 6)-alpha-D-glucosidic bonds. The structures of the limit dextrins containing a single (1 leads to 6)-bond were examined. Six such dextrins were found. Of these, two were capable of being further hydrolysed by alpha-amylase, whereas the remaining four were true, amylase-resistant alpha-limit dextrins. The structures of the limit dextrins afforded information about those (1 leads to 4)-alpha-D-glucosidic bonds of amylopectin that are capable of being cleaved by salivary alpha-amylase and those that are resistant. In order to define further the action of alpha-amylase, the alpha-amylolytic products of 6-alpha-maltotriosyl-D-glucose, 6(3)-alpha-maltotriosylmaltotriose, and 6(3)-alpha-maltotriosylmaltotetraose were examined.
Assuntos
Amilases/metabolismo , Glândulas Salivares/enzimologia , alfa-Amilases/metabolismo , Amilopectina , Humanos , Especificidade por SubstratoRESUMO
It has long been claimed, but frequently disputed, that part of the glycogen in rat liver is insoluble in 10% trichloroacetic acid, and a physiological significance was ascribed to the existence of the two pools of glycogen, desmo-glycogen, the insoluble form, and lyo-glycogen, the soluble component. Desmo-glycogen was thought to owe its acid insolubility to a covalent binding to protein. Recent claims that glycogen, similarly insoluble in acid, can be synthesized in vitro have renewed the interest in desmo-glycogen. We have obtained trichloroacetic acid-insoluble glycogen from rat liver and find that, despite subjecting the glycogen to proteolysis, peptide material remains in close association with the glycogen through a number of purification procedures and is freed from glycogen only by enzymic decomposition of the latter. The tenacity with which the glycogen and peptide material remain in association with each other is suggestive of the occurrence of protein-bound glycogen.
Assuntos
Glicogênio Hepático/metabolismo , Aminoácidos/análise , Animais , Centrifugação com Gradiente de Concentração , Fenômenos Químicos , Química , Cromatografia em Gel , Dinitrofluorbenzeno , Eletroforese em Gel de Poliacrilamida , Glicopeptídeos/análise , Glicogênio Hepático/análise , Glicogênio Hepático/isolamento & purificação , Masculino , Ligação Proteica , Ratos , SolubilidadeRESUMO
A macromolecular (1----4)-alpha-D-[14C]glucan-protein complex was synthesized with a rat liver preparation and uridine diphosphate D-[14C]glucose. The size of the complex is contributed by both the protein and the (1----4)-alpha-D-glucosyl-oligomer components. Iodoacetamide treatment did not change the migration properties on Bio-Gel A-50m. Therefore, disulfide bonds linking glucan-protein subunits seem not to be involved. The [14C]glucan-protein, precipitated by diluted trichloroacetic acid, was digested by alpha-amylase, phosphorylase a, and proteases. The extent of proteolysis was greater for a complex having fewer D-glucose units incorporated. After proteolytic digestion of that complex, the labeled fragments behaved on electrophoresis, and ion-exchange and gel chromatography as [14C]glucosylated peptides. These findings support previous conclusions that the primer for liver glycogen synthesis is a protein on which glycogen is built up by covalent attachment.
Assuntos
Glucanos/metabolismo , Glicoproteínas/metabolismo , Glicogênio Hepático/biossíntese , Animais , Radioisótopos de Carbono , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Papel , Fígado/enzimologia , Ratos , Uridina Difosfato Glucose/metabolismoRESUMO
Glycogenin is the covalently bound protein found in muscle glycogen that is thought to be the primer for glycogen synthesis. We now report that glycogenin contains a phosphoserine residue. From a less than stoichiometric amount of phosphate in glycogenin as isolated, the content may be increased to one molecular proportion, using the catalytic subunit of cAMP-dependent protein kinase. The phosphoserine residue is present within a hitherto-undescribed amino acid sequence. In particular, the serine is not flanked by arginine, previously thought to be an essential adjunct for a serine residue to act as substrate for this kinase. We suggest that the serine phosphate may represent a means of regulating the ability of glycogenin to prime glycogen synthesis.
Assuntos
Glicoproteínas/metabolismo , Fosfosserina/metabolismo , Serina/análogos & derivados , Sequência de Aminoácidos , Animais , Bovinos , Glucosiltransferases , Glicoproteínas/isolamento & purificação , Músculos/análise , Fosfatos/análise , Fosforilação , Proteínas Quinases/metabolismo , CoelhosRESUMO
Rabbit liver glycogen is shown to contain small amounts of covalently bound phosphate and glucosamine, and to display intermolecular heterogeneity with respect to the proportions of these two trace components. The difference in phosphate content over five fractions, separated on DEAE-cellulose, ranged from 2 to 155 molecular proportions relative to glycogen of Mr 10(7), while the glucosamine content varied from 0.6 to 2.3 molecular proportions. In parallel, the average molecular size and the turbidity of the glycogen fractions increased with increasing phosphate and glucosamine contents, the molecular weight profile taking the form of a change in the ratio of two glycogen components of distinctly different sizes. The varying presence of the phosphate and glucosamine, apart from their own significance, may help in gaining an insight into the overall properties of glycogen itself, such as the relative ages of individual molecules and the existence of different metabolic pools.
Assuntos
Glicogênio Hepático/análise , Animais , Cromatografia DEAE-Celulose , Cromatografia em Gel , Glucosamina/análise , Peso Molecular , Nefelometria e Turbidimetria , Fosfatos/análise , CoelhosRESUMO
Glycogen synthesis in rabbit muscle is initiated by an autocatalytic, self-glucosylating protein (SGP). This creates a maltosaccharide primer on itself that in turn primes glycogen synthesis. Here we describe the powerful allosteric inhibition of autocatalysis by ATP and ADP, sufficient, at the physiological concentration of ATP in muscle, to inhibit autocatalysis. We also examined inhibition of self-glucosylation by analogues of the substrate UDPglucose. One of them, UDPxylose, acts as an alternative substrate and serves to block glucosylation. An improved purification procedure for the SGP is also described.
Assuntos
Glicogênio/biossíntese , Proteínas Musculares/fisiologia , Músculos/metabolismo , Regulação Alostérica/fisiologia , Animais , Catálise , Proteínas Musculares/isolamento & purificação , Açúcares de Nucleosídeo Difosfato/farmacologia , Nucleotídeos/fisiologia , CoelhosRESUMO
The reasons for the occurrence of trace amounts of glucosamine in animal liver glycogens have been explored. Human liver glycogen is now shown to contain this amino sugar. Galactosamine, known to be the source of the incorporated glucosamine, is found to give rise to glucosamine in glycogen when administered orally, or as the N-acetyl derivative. The rabbit can also incorporate glucosamine into kidney glycogen but not into glycogen in heart or skeletal muscle. These experiments led to the discovery that glucosamine is incorporated into rabbit liver glycogen in such a way that there is intermolecular heterogeneity in the content of glucosamine, suggesting that there exists more than one pool of liver glycogen.
Assuntos
Glucosamina/metabolismo , Glicogênio/metabolismo , Animais , Gatos , Galactosamina/metabolismo , Humanos , Glicogênio Hepático/metabolismo , Especificidade de Órgãos , Coelhos , Ratos , Especificidade da Espécie , SuínosRESUMO
As previously shown for rabbit liver glycogen, rabbit muscle glycogen contains a small amount of phosphate ester and there is an intermolecular heterogeneity in phosphate content such that the glycogen may be fractionated on DEAE-cellulose into components differing 10-fold in their phosphate contents. We now know that the phosphate ester is of two types, mono and di. The availability of the phosphomonoester component to hydrolysis by alkaline phosphatase is sterically regulated and is highest in the fraction of highest total phosphate content. The ability of the glycogen fractions to act as primers for glycogen synthase also varies with phosphate content, differing overall by > 2-fold. The priming ability increases with increasing phosphate content. We suggest that the phosphate content of a glycogen molecule may be related to its age and that this may be used as a metabolic marker when studying the turnover of glycogen, also that phosphate may be a signal for transport of glycogen to the lysosome. The phosphodiester grouping may act as a point of branching in glycogen, additional to the recognized interglucosidic branch, and is a candidate for the acid- and alkali-labile bond that has been reported in glycogen.