Preparative biochemistry最新文献

Peroxidase (E.C. 1.11.1.7., hydrogen donor oxidoreductase) is widely distributed and has been isolated from many higher plants (1). The wide distribution of the enzyme suggests that it could be of great biological importance. However the role that it plays in metabolism is not clear due to the large number of reactions it catalyzes and the considerable number of isozymic species (2). In tomato plants, Evans and Aldridge (3) separated out six isoperoxidases and in a later paper Evans reported 12 isoperoxidases from tomato shoots (4). A homogeneous tomato fruit peroxidase isozyme was obtained by Jen et al. (5) using hydrophobic chromatography. Isozymes were not detected in Euphorbia characias peroxidase (6), in Ipomoea batatas peroxidase (7) and in Hordeum vulgare peroxidase (8). The simultaneous presence of Cu (II) amine oxidase and peroxidase in cell walls suggests that the peroxide generated on oxidation of the amines could be utilized by the peroxidase (6,8,9). In the graminea Oryza sativa, widely distributed, an FAD amine oxidase is present that oxidizes diamines (10). In this plant we also found two isoperoxidases called perox I and II. Only perox I was purified to homogeneity and its enzymatic, physical and chemical properties have been studied.

Spermatozoa were initially separated from fresh boar ejaculates using a 1.0 M sucrose density gradient. Spermatozoa (1 x 10(8) cells/ml) were subjected to gas cavitation (650 psi, 10 minutes), followed by a 4-step centrifugation technique to yield the final plasma membrane preparation. Purity of the plasma membrane isolate was determined using microscopic techniques (i.e. differential interference contrast and transmission electron microscopy) and marker enzymes for biochemical characterization. Plasma membranes were found to be removed primarily from the periacrosomal region of the sperm. Acrosomes appeared to remain intact on the cavitated spermatozoa. Transmission electron microscopy yielded a homogenous population of 100-200 microns unilamellar vesicles. Enzyme markers specific for plasma, acrosome and mitochondrial membranes substantial the purity observed under visual examination.

An NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) was purified from Glycine max seedlings by means of chromatographic procedures. After 1371-fold purification, the enzyme showed a single band in IEF corresponding to an isoelectric point of 6.1. A single band was also found in native-PAGE both by activity staining and Coomassie brilliant blue staining. The molecular mass determined in SDS-PAGE was 21900 Da, while in HPLC gel-filtration it was 61000 Da. The NAD(P)H:quinone oxidoreductase was able to use NADH or NADPH as the electron donor. Among the artificial quinones which are reduced by this enzyme, 6-hydroxydopa- and 6-hydroxydopamine-quinone are of particular interest because of their neurotoxic effects.

Resolution of a wide range of polypeptides, 3.5-200kDa, on a single low acrylamide and cross linkage gel of 7.7% T, 2.6% C is described here. Laemmli's (4) original discontinuous SDS polyacrylamide gel electrophoresis (SDS-PAGE) system is modified by increasing the ionic strength of both stacking and resolving gels, and replacing the usual glycine buffer with a tricine cathode buffer as described by Schagger and von Jagow (7). This system offers the advantage of a wide range of protein fractionation, with sufficient band resolution, on a single, low acrylamide concentration and cross linkage gel. Moreover, increased gel ionic concentration allows higher protein and salt load, and renders this system suitable for preparative work.

The crude extracellular extract of Aspergillus niger (syn A. ficuum) NRRL 3135 contains glucoamylase (exo-1,4-alpha-D-glucanohydrolase, EC 3.2.1.2). The enzyme, a glycoprotein, was purified 7-fold by ion-exchange chromatography, chromatofocusing, and conconavalin A affinity chromatography. The molecular weight of the enzyme was estimated to be 90 kDa by SDS-PAGE and gel permeation chromatography. The pI of the enzyme was 3.4. The temperature optimum of the enzyme was 60 degrees C and the pH optimum was 5.0. The Vmax values for soluble starch, maltose, maltotriose, maltotretraose, maltopentaose, and isomaltose were 55.2, 11.7, 32.3, 47.8, 59.2, 12.5 nKat glucose/sec, respectively and the Km values for the same substrates were 0.09%, 0.67 mM, 0.76 mM, 0.76 mM, 0.68 mM, and 122.01 mM, respectively.

The transducin heterotrimer and its alpha- and beta gamma-subunits have been purified from frozen bovine rod outer segments by modifying existing procedures. The methods described here are relatively simple and fast. The yield (ca. 8 mgs/100 retinas) and purity of the transducin heterotrimer and subunits from frozen retinas is equal to or larger than those previously obtained from fresh or frozen retinas.

Phytohemagglutinin from red kidney bean has been purified by affinity chromatography on a human alpha 1-acid glycoprotein Sepharose 4B column. Further purification of the hemagglutinin's five isolectins was achieved on a Mono S column with an 86% protein recovery. Each sequentially eluted isolectin from the ion exchange column displayed either hemagglutinating or mitogenic activity. The main activity of each fraction was the result of the combination of varying proportions of the L and E subunits.

A new purification method for rat liver glucokinase was developed. Glucokinase was purified to homogeneity in a yield of 70% in 5 days. The procedure consists of DEAE-cellulose ion-exchange chromatography, QAE-Toyopearl ion-exchange chromatography, glucosamine-Sepharose affinity chromatography, and HiLoad Superdex 200 gel filtration. Purified glucokinase had a specific activity of 200 units/mg protein and was highly stable in the presence of 100 mM glucose, 300 mM KCl, and 20% glycerol. We found that some of the methionine residues of glucokinase were oxidized to methionine sulfoxide residues during dialysis in the presence of glucose. It would appear that this oxidation is caused by formation of hydroxyl radicals in the presence of glucose and contaminating transition metal(s).

This paper illustrates the fractionation of cytoplasmic transfer ribonucleic acid from rat liver by reversed-phase high performance liquid chromatography using a gradient of acetonitrile/ammonium acetate. The procedure is fast, highly reproducible, and gives an excellent resolution of the numerous tRNA population: about 50 peaks with area peak percentages ranging from 0.001 to 5 can be monitored. Uncharged tRNA preparations exhibited a chromatographic profile different from aminoacylated tRNA, thus suggesting a possible strategy to distinguish between aminoacylated and nonacylated tRNA species. Moreover, a first approach to map the HPLC peaks was attempted by chromatographing preparations of tRNA which had been aminoacylated with individual 3H-labeled aminoacids. Here is reported the case of tRNA(Pro), which gave three well separated radioactive peaks, most likely corresponding to tRNA(Pro) isoacceptor species.

In order to be able to use triokinase for the enzymatic assay of tissue glyceraldehyde, we purified the enzyme to homogeneity from porcine kidney and characterized its biochemical properties. The purification was performed by polyethylene glycol fractionation, anion exchange chromatography, hydroxyapatite chromatography, hydrophobic chromatography, and gel filtration. The enzyme was purified 937-fold from the crude extract with an overall yield of 28%. It had a molecular weight of 122,000 and was a dimer composed of identical subunits. The optimal pH and optimal temperature were 7.0 and 60 degrees C, respectively. This enzyme was stable when incubated at pH 7.0 at 40 degrees C for 1 h in the presence of 0.1 mg/ml bovine serum albumin. No loss of activity occurred for at least 1 month when the enzyme was stored at 4 degrees C in the presence of 1 mM dithiothreitol and 15 mM NaN3 under N2. Only three compounds, i.e., D-glyceraldehyde, dihydroxyacetone, and glycolaldehyde, acted as the substrate of the enzyme, having Km's of 11, < 5, and 260 microM, respectively. The Km for ATP-Mg2+ was 68 microM. These results indicate that porcine kidney triokinase has properties advantageous for the glyceraldehyde assay using glyceraldehyde-3-phosphate dehydrogenase as a coupling enzyme.