Preparative biochemistry最新文献

An improved and simplified purification procedure has been developed for the isolation of diamine oxidase from pea seedlings (DAO EC 1.4.3.6). It involves ammonium sulphate precipitation, hydrophobic interaction chromatography, ion-exchange chromatography and size-exclusion chromatography. The homogeneity of the final enzyme preparations and molecular weight were determined by size-exclusion chromatography and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS PAGE). The isoelectric point of 7.35 +/- 0.05 was determined by chromatofocusing and by polyacrylamide gel isoelectric focusing.

Two molecular species of nonspecific cross-reacting antigens, NCA-90 and NCA-50 with mol. wts. of 90,000 and 50,000, respectively, were effectively extracted with phosphatidylinositol-specific phospholipase C (PI-PLC) from human lung tissues, followed by extraction with perchloric acid, immunoaffinity chromatography with anti-NCA adsorbent, and gel filtration on a TSK G3000SW column. The yields of NCA were about 2 times more than those obtained by the usual method without PI-PLC. Addition of 0.05 unit of PI-PLC to 1 g of lung tissue and incubation at 37 degrees C for 1 h with continuous shaking seem to be practically sufficient for NCA extraction. The immunochemical properties of the NCAs thus obtained were found to be identical to those of NCAs obtained by the ordinary method.

A single phase extraction procedure employing 8% (v/v) n-butanol at room temperature extracted over 90% of alkaline phosphatase activity and over 60% of 5'-nucleotidase activity from bovine milk fat globule membranes (MFGM). For 5'-nucleotidase, higher n-butanol concentrations lead to loss of activity, while lower concentrations were ineffective in extracting the enzyme. When extractions were performed at 0 degrees C, similar yields were obtained for alkaline phosphatase extraction with 8% (v/v) n-butanol, but 5'-nucleotidase extraction required 10% (v/v) n-butanol for similar yields. However, 5'-nucleotidase was less susceptible to denaturation during extraction at 0 degrees C. The Km values and substrate specificities for both alkaline phosphatase and 5'-nucleotidase were unchanged by extraction with 8% (v/v) n-butanol. The 8% (v/v) n-butanol extraction procedure provides a 3-fold purification step, and an enzyme preparation suitable for further purification.

Covalent structural information on membrane proteins is not easily acquired since it is difficult to obtain pure membrane proteins in sufficient quantities. We have therefore examined the Bio-Rad 491 prep cell continuous elution electrophoresis apparatus as a method for providing the quantities of purified alpha and beta subunits from (Na,K)-ATPase required for these studies. Twenty-four milligrams of crude (Na,K)-ATPase preparation was applied to the prep cell which consisted of a 7% Laemmli separating gel 4.5 cm in length. The prep cell was run under constant power and continuous cooling conditions. Those fractions containing the beta subunit were combined and further purified by wheat germ agglutinin affinity chromatography. Fractions containing the alpha subunit were combined and did not require further purification. The identity and the degree of purity of the proteins obtained using this approach was assessed utilizing SDS-PAGE, amino acid analysis and N-terminal sequencing. This simple and fast method provides approximately 1.8 milligrams of each purified subunit from 24 milligrams of relatively crude microsomes. Recovery of the alpha and beta subunits from the crude (Na,K)-ATPase preparation was estimated to be 28% and 81%, respectively.

Zone electrophoresis (ZE) in concentration gradients of sucrose proved to be a powerful technique in the purification of viruses although it is seldomly used as a single step for the separation of the infective agent from extraneous matter but usually as a final procedure. No difficulties were experienced in the purification of plant viruses with ZE possibly because of the resistance of the agents to the chloroform butanol treatment which the impure plant viruses received prior to the ZE. Likewise no difficulties were encountered during purification of human and animal picorna viruses because they also are resistant to chloroform or chloroform-butanol treatment prior to ZE. Purification of animal and possibly human reoviridae ZE as a final step is involved as they are associated with or even attached to the host cell membranes which are lipoidal in nature. Because the lipoidal material on the virus has an affinity for organic solvents the virus particles are caught in the emulsion when an extract of the crude virus material is shaken with the organic solvent. With these viruses polyethylene glycol M(r) 6000 (PEG) is often helpful as a means of displacing most of the host proteins as a step in the partial purification prior to ZE. Neurotropic African horse sickness virus which presented many problems to overcome was rendered free of extraneous proteins to the extent that electron micrographs could be made of it. Neurotropic Rift Valley fever virus was similarly difficult to purify as it was always associated with mouse brain components. This virus was finally purified from infected mouse blood in which the virus particles were not associated with host cell debris and other extraneous proteins. Zone electrophoresis experiments were not conducted on viruses with large particle sizes e.g. myxoviruses because they could be purified without a great deal of effort using polymer displacement, and thin layer centrifugation. Zone electrophoresis as a means of final purification was vividly displayed with the purification of five viruses of the larvae of the pine emperor moth, Nudaurelia cytheria cytheria.

Potato virus Yo was purified by centrifugation of infected and minced plant tissue in the virus extraction rotor. As the initial seeding material was heavily contaminated with tobacco mosaic virus (TMV) this virus was isolated and antibody was elicited in chickens. The chicken antibody (IgY) against TMV was used for removing this extraneous virus from the original PVYo seeding material prior to propagating PVYo in tobacco plants, CV Glutinosa.