Difficulties in the assay of phosphatidate phosphohydrolase activity. Influence of ionic strength, detergent, and selection of substrate

Abstract

In the present paper, problems in connection with assay of the activity of magnesium-dependent rat liver phosphatidate phosphohydrolase (PAP) are discussed. PAP activity is usually measured by following the production of diacylglycerol or inorganic phosphate from the substrate phosphatidate. These two methods may give widely different results due to a number of factors that may affect the assay. One such factor is the composition of the substrate. Higher apparent enzyme activity was observed with dioleoyl-phosphatidate than with dipalmitoyl-phosphatidate. This substrate-dependent difference in apparent PAP activity was 2-2.5-fold in the absence and 10-fold in the presence of Triton X-100, respectively. Triton X-100 reduced the activity as measured with the dipalmitoyl-phosphatidate substrate. In contrast, the activity of PAP as measured with dioleoyl-phosphatidate was stimulated by Triton X-100. The stimulatory effect of Triton was reduced or abolished when the ionic strength in the assay mixture was increased. Assays based on 32P-labeled substrate are rapid and sensitive. It is shown here that 33P can be used as an alternative. This radionuclide has a longer half-life and also emits particles with lower energy, thus posing less potential health hazards for the user.

1‐O‐Alkyl‐2‐Acetyl‐sn‐Glycero‐3‐Phosphate: Phosphohydrolase Activity in Tetrahymena pyriformis

“…When non‐labeled alkyl‐acetyl‐G was included in the incubation mixture, the amount of [ 3 H]alkylacetyl‐G produced from the cleavage of alkyl‐acetyl‐GP was not reduced, in contrast to other phosphatidate phosphatases which are inhibited by dia‐ cylglycerols (Stark and Humble 1996).…”

“…Under our standard incubation conditions, low levels of chimyl alcohol were also formed in the absence of NaF, which is a lipase inhibitor (Stark and Humble 1996). Sodium fluoride caused a reversible inhibition of the phospholipase A 2 activity that catalyzed the further cleavage of alkyl‐acetyl‐G to chimyl alcohol.…”

Βιοσύνθεση του παράγοντα ενεργοποίησης αιμοπεταλίων (PAF) στο πρωτόζωο Tetrahymena pyriformis

“…Κάτω από τις σταθερές συνθήκες επώασης σχηματίσθηκε η αλκυλο-G (χιμυλική αλκοόλη), ένα δευτερεύον προϊόν κυρίως λιπολυτικής δράσης [223]. Επιπλέον, εξετάσθηκε η πιθανότητα σχηματισμού του αλκυλο-ακετυλο-ακυλο-G, ενός ουδέτερου λιπιδίου, από ενεργότητα ακυλοτρανσφεράσης, αλλά δεν ανιχνεύθηκε κατά τη διάρκεια όλων των πειραμάτων.…”