RESUMO
Avian eggshell membranes are good candidates as a matrix for immobilization procedures. Chicken eggshell, a waste material available from the poultry industry as a byproduct, is a very safe and cheap raw material. While pieces of eggshell membrane, or even particles from whole eggshell, have been previously used for these purposes, we report here the use of eggshell membrane powder for E. coli ß-galactosidase immobilization with glutaraldehyde as cross-linker. A kinetic characterization is provided for eggshell membrane powder-bound enzyme compared to free enzyme. Results show a remarkable similarity between bound and free enzyme and also that the immobilized enzyme is stable and can be reused several times. Moreover, bound enzyme is able to produce glucose from skim milk serum.
Assuntos
Casca de Ovo/química , Enzimas Imobilizadas/metabolismo , Pós , Resíduos , beta-Galactosidase/metabolismo , Animais , Galinhas , Escherichia coli/enzimologia , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Microscopia Eletrônica de Varredura , Leite/químicaRESUMO
Glycogen synthesis following glucose microinjection in frog oocytes proceeds preferentially by an indirect pathway involving gluconeogenesis from triose compounds. Because of the known regulatory role of fructose-2,6-bisP on glucose utilization in most vertebrate tissues we coinjected [U-14C]glucose and fructose-2,6-bisP into oocytes and observed a marked inhibition of label incorporation into glycogen, with an I50 value of 2 microM, which is similar to the value measured for the in vitro inhibition of oocyte fructose-1,6-bisphosphatase. Other hexoses-bisP were tested: 2,5-anhydromannitol-1,6-bisP was as effective as inhibitor as fructose-2,6-bisP; glucose-1,6-bisP showed some effect although 50% inhibition was obtained at a concentration 10 times higher than with fructose-2,6-bisP; fructose-1,6-bisP had no effect at all. The inhibition pattern for the in vivo glycogen synthesis by these analogs closely matched the one obtained with partially purified oocyte fructose-1,6-bisphosphatase. The intracellular concentration of fructose-2,6-bisP in unperturbed oocytes was found to be between 0.1 and 0.2 microM. Fructose-6-phosphate,2-kinase levels measured in oocyte homogenates were between 0.02 and 0.06 mU per gram of ovary. After 60 min incubation, fructose-2,6-bisP microinjected into the oocytes was almost completely degraded, suggesting that fructose-2,6-bisphosphatase is active in vivo. The results presented in this paper indicate that fructose-2,6-bisP plays an important role in the in vivo regulation of glucose utilization in frog-grown oocytes.
Assuntos
Frutosedifosfatos/farmacologia , Glucose/metabolismo , Glicogênio/biossíntese , Oócitos/metabolismo , Animais , Anuros , Radioisótopos de Carbono , Feminino , Cinética , Oócitos/efeitos dos fármacos , Fosfofrutoquinase-2 , Monoéster Fosfórico Hidrolases/metabolismo , Técnica de Diluição de Radioisótopos , Fosfatos Açúcares/farmacologiaRESUMO
1. Eggshells are bioceramic-biopolymer composites made by a cell-mediated deposition of an extracellular matrix which drives the organisation of the inorganic phase. Ultrastructurally, eggshells are composed of shell membranes, mammillary knobs, palisade, and cuticle. Shell membranes are two nets of type X collagen-containing fibrils. On to these membranes, the mammillary knobs, that is, the crystal nucleation sites, are deposited. Type X collagen is highly cross-linked and insoluble. 2. In order to evaluate the role of type X collagen cross-linking on eggshell formation, hens were injected with different doses of beta-aminoproprionitrile, which specifically interferes with cross-link formation. 3. Changes in egg size and shape were observed. Scanning electron micrographs analysis of these eggs demonstrated marked changes in crystal growth and shell membrane structure and arrangement. A dot-blot analysis, using a monoclonal antibody against chicken type X collagen, shows a dose-dependent increase in shell membrane collagen extractability. 4. It is concluded that the formation of beta-aminoproprionitrile-sensitive cross-links among the type X collagen molecules of the shell membranes play an essential role in normal eggshell formation.
Assuntos
Aminopropionitrilo/farmacologia , Casca de Ovo/efeitos dos fármacos , Animais , Galinhas , Colágeno/análise , Casca de Ovo/anormalidades , Casca de Ovo/ultraestrutura , Feminino , Microscopia Eletrônica de Varredura , Oviposição , Óvulo/efeitos dos fármacos , Óvulo/patologiaRESUMO
Glycogen is the main end product of glucose metabolism in amphibian oocytes. However, in the first few minutes after [U-14C]glucose microinjection most of the label is found in lactate. The burst of lactate production and the shape of the time curves for the labelling of glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate and glycogen suggest a precursor-product relationship of lactate with respect to glycogen and its intermediates. Expansion (by microinjection) of the pool of glycolytic intermediates, such as dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 3-phosphoglycerate or phosphoenolpyruvate, results in a marked decrease in [U-14C]glucose incorporation into glycogen. After co-injection of doubly labelled glucoses, extensive detritiation (93%) of the glucosyl units of glycogen was observed with [2-3H, U-14C]glucose and partial detritiation with [3-3H,U-14C]glucose (34%) or [5-3H,U-14C]glucose (46%). After injection of [6-3H,U-14C]glucose, a small but significant and reproducible detritiation (13%) in glycogen was observed. Co-injection of [U-14C]glucose and 3-mercaptopicolinate resulted in marked inhibition of glycogen labelling. Half-maximal inhibition was observed at 0.58 mM 3-mercaptopicolinate, which agrees with the IC50 value (0.47 mM) for the inhibition in vitro of phosphoenolpyruvate carboxykinase activity. We concluded that in frog oocytes most of the glucosyl units are incorporated into glycogen by an indirect pathway involving breakdown of glucose to lactate, which is then converted into glycogen via gluconeogenesis. Both processes, glycolytic degradation of glucose to lactate and subsequent reconversion of the latter into hexose phosphates and glycogen, occur in the same cell.
Assuntos
Anuros/metabolismo , Glicogênio/biossíntese , Oócitos/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Gluconeogênese , Glucose/administração & dosagem , Glucose/metabolismo , Glicólise , Técnicas In Vitro , Cinética , Lactatos/metabolismo , Ácido Láctico , Microinjeções , Oócitos/efeitos dos fármacos , Fosfoenolpiruvato Carboxiquinase (GTP)/antagonistas & inibidores , Ácidos Picolínicos/farmacologiaRESUMO
1. From the liver of the teleost fish Genypterus maculatus, a partially purified preparation of arginase was obtained and characterized. 2. The Km value for arginine was found to be 9.1 mM at pH 7.5 and 11.5 mM at the optimum pH of 9.5. At both pH values, competitive inhibition was caused by ornithine and lysine, whereas proline, leucine, valine and isoleucine caused a non-competitive inhibitory effect. Branched chain amino acids were more inhibitory than proline. 3. The enzyme was found localized in the mitochondrial matrix of the liver of Genypterus maculatus. It is suggested that this localization would be of importance in the use of arginine as an energy source.