Isolation of insect microsomal oxidases by rapid centrifugation

• Published in: Pesticide biochemistry and physiology Vol. 16; no. 3; pp. 187 - 198
• Main Authors: Brattsten, L.B., Gunderson, C.A.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.01.1981
• Subjects: animal physiology; arthropods; entomology; Insecta
• ISSN: 0048-3575; 1095-9939
• DOI: 10.1016/0048-3575(81)90052-3

Only about 60% of the total relative gravitational force conventionally used to sediment microsomes is needed to prepare highly active microsomes from the midgut tissues of an insect larva. A rapid preliminary centrifugation for 2 min at 39,000 g max effectively removed contaminating microorganisms, tissue debris, nuclei, and mitochondria. The supernatant was recentrifuged for 20 min to 210,000 g to sediment the microsomes. There were no losses of microsomal oxidase activities or degradation of cytochrome P-450 to the inactive form ( P-420) resulting from the application of the higher gravitational force. Incorporation of 1 m M EDTA in the buffer and washing the microsomes resulted in an improved yield of the cytochrome compared to that in microsomes prepared in sucrose. Yields of microsomal protein, cytochrome P-450, and NADPH-cytochrome c reductase in the rapidly isolated microsomes were as good as those in conventionally prepared microsomes. The apparent kinetic characteristics of several microsomal oxidation activities and optical difference spectra of Types 1 and 2 ligands were identical in the rapidly and conventionally prepared microsomes. The morphological appearance of the microsomes was examined by electron microscopy. Microsomal pellets prepared by either method were indistinguishable. The rapid procedure saves significant time in microsome preparation and yields microsomal oxidase activities as good or slightly better than those prepared by usual centrifuged procedures.