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Purification of glutamate dehydrogenase from ox brain and liver. Evidence that commercially available preparations of the enzyme from ox liver have suffered proteolytic cleavage.

ABSTRACT: 1. A rapid procedure, involving ion-exchange chromatography on DEAE-cellulose and affinity chromatography on GTP-Sepharose, was used to purify glutamate dehydrogenase from ox brain and liver. 2. Preparations purified in this way differed from those of the ox liver enzyme that were obtained from commercial suppliers in their mobilities on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. This difference appears to result from the occurrence of limited proteolysis during the preparation of the latter enzyme samples. 3. N-Terminal sequence analysis showed the presence of four amino acid residues in the enzyme prepared in this study that were not present in those obtained from the commercial sources and which have not been reported in previous studies on the sequence of the ox liver enzyme. 4. A preliminary examination of the enzyme prepared in this way indicated that the Michaelis constants for the substrates are similar to those obtained from the commercial preparation, but that the response to allosteric effectors was modified.

SUBMITTER: McCarthy AD

PROVIDER: S-EPMC1162252 | biostudies-other | 1980 Nov

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Describing and evaluating miRNA inventories with Next Generation Sequencing is a goal of scientists from a wide range of fields. It requires high purity, high quality, and high yield RNA extractions that do not only contain abundant ribosomal RNAs but are also enriched in miRNAs. Here we compare 6 disparate and commercially available totalRNA extraction kits for their suitability for miRNA-preparations from Gyrodactylus salaris, an important but small (500 ?m in length) monogenean pathogen of Norwegian Atlantic salmon (Salmo salar). FINDINGS: We evaluated 1 salt precipitation method (MasterPure™ Complete RNA Purification Kit, Epicentre), 2 Phenol based extraction methods (mirVana Kit, Ambion, and Trizol Plus Kit, Invitrogen), 1 paramagnetic bead extraction method (RNA Tissue kit, GeneMole) and 2 purification methods based on spin column chromatography using a proprietary resin as separation matrix (Phenol-free Total RNA Purification Kit, Amresco, and ZR MicroPrep Kit, Zymo Research). The quality of the extractions from 1, 10 and 100 individuals, respectively, was assessed in terms of totalRNA yield, RNA integrity, and smallRNA and miRNA yield. The 6 RNA extraction methods yielded considerably different total RNA extracts, with striking differences in low molecular weight RNA yield. The Phenol-free Total RNA Purification Kit (Amresco) showed the highest totalRNA yield, but the best miRNA/totalRNA ratio was obtained with the ZR MicroPrep Kit (Zymo Research). It was not possible to extract electrophoretically detectable miRNAs from Gyrodactylus salaris with the RNA Tissue Kit (GeneMole) or the Trizol Plus Kit (Invitrogen). CONCLUSIONS: We present an optimized extraction protocol for single and small numbers of Gyrodactylus salaris from infected Atlantic salmon that delivers a totalRNA yield suitable for downstream next generation sequencing analyses of miRNA. Two of the six tested totalRNA kits/methods were not suitable for the extraction of miRNAs from Gyrodactylus salaris.

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Purification of glutamate dehydrogenase from ox brain and liver. Evidence that commercially available preparations of the enzyme from ox liver have suffered proteolytic cleavage.

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