Purification and characterization of guinea pig gastric phospholipase A₂ of the pancreatic type

Abstract

Guinea pig gastric mucosa and juice contained exceptionally high phospholipase-A, activity, whereas the activity in the pancreas was only minimal. Phospholipases A, were purified to homogeneity from these three tissues. Structural evidence, including the sequence of the NH,-tenninal41 residues, the amino-acid composition and the molecular mass (1 3 902 % 3 Da) determined accurately by mass spectrometry, showed that the gastric mucosa enzyme belongs to the pancreatic type. An unique feature of the sequence is the substitution of Phe for the hitherto invariant Tyr28 in the calcium-binding loop of pancreatic phospholipases A,. The affinity of the guinea pig enzyme for Ca2+ in the presence of substrate was, however, identical to that of the rat enzyme with Tyr28, suggesting the interaction of a phenolic hydroxyl group of the Tyr with its neighboring residues is not significantly linked to the binding of Ca". The NH,-terminal sequences and immunochemical properties of the enzymes purified from the gastric juice and pancreas were identical to those of the gastric mucosa enzyme. The distribution of cells immunoreactive with anti-(gastric PLA , ) immuno-' globulin in the stomach was quite similar to that of the chief cells. Unlike in pancreas of other animals, the prophospholipase A, was not detectable in gastric mucosa or juice homogenates treated with diisopropyl fluorophosphate or in column effluents during purification under acidic conditions. An appreciable prophospholipase-A,-activating activity was not detectable in gastric mucosa extracts at low pH relevant to gastric juice, using rat prophospholipase A, as substrate. This opposes the activation of secreted proenzyme in the gastric juice.Phospholipase A, (PLA,) catalyzes the hydrolysis of fatty-acyl ester bond at the sn-2 position of glycerophospholipids and produces free fatty acids and lysophospholipids. Pancreatic PLA2 has been purified to homogeneity from pancreas of several animal species and its physicochemical properties and reaction mechanism have been extensively studied as a typical lipolytic enzyme [l]. It has been believed to be the major enzyme for digesting dietary and biliary glycerophospholipids in the small intestine, since the intestinal absorption of phospholipids requires their prior hydrolyses to lysophospholipids [2, 31. Fauvel et al., however, presented evidence for the lack of this important enzyme in guinea pig pancreas and proposed the compensatory action of basic lipases with high phospholipase-A, activity on intestinal phospholipid digestion [41.Correspondence to H.

Formation of Lysophosphatidic Acid, a Wound-Healing Lipid, during Digestion of Cabbage Leaves

“…However, pancreatictype PLA 2 activity with the neutral pH optimum has been reported to be abundant in guinea pig and rat gastric mucosa. 27,28) If PLA 2 exists in the mucous gel layer, where secreted bicarbonate ions neutralize gastric acid, 29) it may exert its enzymatic activity toward PA to form LPA within the gel. The concentration of LPA in the stomach and lower digestive tract may be increased as enzymatic digestion proceeds.…”

Human Group IIA Secretory Phospholipase A2Induces Neuronal Cell Death via Apoptosis

“…Four portions of the brain (right cortex, right striatum, left cortex, and left striatum) were weighed and homogenized with three times the weight of 7.7 mM EDTA containing 1.5 g/ml prostaglandin E 1 . PLA 2 activity in the supernatant of the brain homogenates was measured in the presence or absence of 1 M indoxam with a phosphatidyl glycerol/sodium cholate mixed micelle assay (Tojo et al, 1993).…”

Pancreatic phospholipase A2from the small intestine is a secretin-releasing factor in rats

“…mRNA for PLA 2 -I has been detected in the mucosa of human ileum (34) and guinea pig stomach (47). Moreover, luminal secretion of PLA 2 activity has been documented in the rat small intestine (1,2) and guinea pig gastric juice (45). It should be noted that PLA 2 -LI in the intestinal acid perfusate could not be derived from gastric juice as the pylorus was ligated in the present study.…”

“…This view is supported by the findings that pancreatic PLA 2 is widely distributed in the intestine and is released in the duodenal lumen upon acid perfusion during pancreatic juice diversion. It should be noted that the presence of pancreatic PLA 2 in nonpancreatic tissues, including the stomach and the intestine, has been documented previously (25,37,40,45). mRNA for PLA 2 -I has been detected in the mucosa of human ileum (34) and guinea pig stomach (47).…”