A cold-clamping technique for the rapid sampling of rat liver for studies on enzymes in separate cell fractions. Suitability for the study of enzymes regulated by reversible phosphorylation-dephosphorylation.
A technique for the rapid sampling, cooling and homogenization of rat liver is described. Its effectiveness in preserving the activity status of pyruvate kinase (soluble) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) (microsomal) during sampling is assessed in comparison with that of the freeze-clamping technique and of simple excision and mincing of liver tissue before homogenization. The results suggest that cold-clamping is equally effective as freeze-clamping in preserving the activity status of pyruvate kinase in liver samples obtained in situ, but in addition allows the subsequent separation of subcellular fractions, notably microsomes (microsomal fractions) and mitochondria. It is suggested that this property makes the technique useful in studying the activity status of enzymes (e.g. HMG-CoA reductase) the assay of which is subject to interference from the activity of other enzymes which are released from damaged organelles in crude homogenates of freeze-clamped liver samples. This suggestion was tested directly; the cold-clamping technique was found to preserve a substantially higher initial/total HMG-CoA reductase activity ratio [Easom & Zammit (1984) Biochem. J. 220, 739-745] in subsequently isolated microsomes compared with that obtained in microsomes prepared from liver samples processed in the conventional manner. The integrity of mitochondria isolated from homogenates of cold-clamped liver samples was preserved, as judged by the latency of intramitochondrial enzymes and by good respiratory control of the mitochondria. Possible further areas of metabolic studies to which the cold-clamping technique could be applied are suggested.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D003080:Cold Temperature; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D025782:Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D010101:Oxygen Consumption; D010766:Phosphorylation; D011770:Pyruvate Kinase / Q000378:metabolism; D051381:Rats
D006903:Hydroxymethylglutaryl CoA Reductases; D025782:Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; D011770:Pyruvate Kinase