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Indian Journal of Clinical Biochemistry logoLink to Indian Journal of Clinical Biochemistry
. 2009 Sep 16;24(3):275–279. doi: 10.1007/s12291-009-0052-8

A sensitive assay for ornithine amino transferase in rat brain mitochondria by ninhydrin method

H Ravi Kumar 1,2,4,7,✉, S Ananda 6, K S Devaraju 3, B M Prakash 5, S Sampath Kumar 4, S V Suresh Babu 1, N Ramachandraswamy 2, H P Puttaraju 4
PMCID: PMC3453300  PMID: 23105849

Abstract

To establish/develop an assay method for measuring Ornithine Aminotransferase (EC.2.6.1.13) activity using rat brain mitochondria as a source of enzyme in presence and absence of Pyridoxal Phosphate (PLP). The modified method, with the improved sensitivity, is adopted for the assay of ornithine amino transferase activity in rat brain mitochondria. The enzyme activity was measured at 620 nm, the study showed that reaction was optimum at 37°C for 30 minutes. The assay is sensitive enough to detect activity at the order of nanomoles pyrroline-5-carboxylate/mg protein/minute and can be compared as an alternative to the radio isotopic method which is more cumbersome and aminobenzaldehyde method which is less sensitive. The Km & Vmax shows maximum activity in the presence of Pyridoxal Phosphate (Coenzyme) concentration at 0.05mM when compared with absence of Pyridoxal Phosphate as higher the concentration of Pyridoxal Phosphate affects the affinity of the enzyme to substrate. The OAT activity in different tissues of the rat was also studied and highest activity was found in liver and kidney.

Key Words: Ornithine Aminotransferase, Pyridoxal Phosphate, Pyrroline-5-carboxylate-Ninhydrin complex, Transmission electron microscope

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