Thiamine pyrophosphatase and nucleoside diphosphatase in guinea pig pinealocytes shown by a cerium-based method

Abstract

Thiamine pyrophosphatase (TPPase) and nucleoside diphosphatase (NDPase) both have a similar localization in the endoplasmic reticulum (ER), the perinuclear space (PNS) and the Golgi apparatus (GA) in hepatocytes and other cell types. Whether they are two separate enzymes or a single one has not been determined. Recently cerium has been reported as an excellent substitute for lead. The study described here compared the activities of TPPase and NDPase in pinealocytes by the cerium-based method.Guinea pig pineal glands were fixed by perfusion with a mixture of 1% glutaraldehyde and 2% paraformaldehyde followed by immersion in the same fixative for a total of 30 min. Sections cut at 100μm on a vibratome were incubated in substrate-free incubation medium for 30 min, then for 3 hrs at room temperature in the complete medium, which contained thiamine pyrophosphate (1 mg/ml) for TPPase and inosine diphosphate (1 mg/ml) for NDPase, cerium chloride (0.7 mg/ml), manganese chloride (1 mg/ml), and 5% sucrose in buffer.