Purification and Characterization of a Catalytic Domain of Rat Intestinal Phospholipase B/Lipase Associated with Brush Border Membranes

Tojo, Hiromasa; Ichida, Tetsuichi; Okamoto, Mitsuhiro

doi:10.1074/jbc.273.4.2214

Cited by 36 publications

(37 citation statements)

References 53 publications

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“…1A), even though they absorbed similar levels of the fatty acyl moiety of dietary phospholipids as the Pla2g1b ϩ/ϩ mice. Thus, the compensatory enzyme for phospholipid digestion in the absence of Pla2g1b is likely to be phospholipase B, a distal intestinal enzyme that hydrolyzes phospholipids at both the sn-1 and sn-2 positions to produce nonesterified fatty acids and glycerophosphocholine (15,16).…”

Section: Discussionmentioning

confidence: 99%

Group 1B Phospholipase A2–Mediated Lysophospholipid Absorption Directly Contributes to Postprandial Hyperglycemia

et al. 2006

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Postprandial hyperglycemia is an early indicator of abnormality in glucose metabolism leading to type 2 diabetes. However, mechanisms that contribute to postprandial hyperglycemia have not been identified. This study showed that mice with targeted inactivation of the group 1B phospholipase A 2 (Pla2g1b) gene displayed lower postprandial glycemia than that observed in wild-type mice after being fed a glucose-rich meal. The difference was caused by enhanced postprandial glucose uptake by the liver, heart, and muscle tissues as well as altered postprandial hepatic glucose metabolism in the Pla2g1b ؊/؊ mice. These differences were attributed to a fivefold decrease in the amount of dietary phospholipids absorbed as lysophospholipids in Pla2g1b

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Section: Discussionmentioning

confidence: 99%

Group 1B Phospholipase A2–Mediated Lysophospholipid Absorption Directly Contributes to Postprandial Hyperglycemia

et al. 2006

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“…Essentially these enzymes display phospholipase A/lysophospholipase activity, catalysing the total deacylation of phospholipids. Phosphatidylcholine (PC)-hydrolysing PLB activities have been described in bacteria [1], fungi [2], Dictyostelium discoideum [3] and in mammalian cells [4][5][6][7][8][9][10][11], and three distinct gene families have been identified from bacteria, fungi and mammals. In the microorganism Moraxella bovis, the plb gene encodes a protein of 616 amino acids and the enzyme hydrolyses PC/LPC (lysoPC) to produce NEFAs (non-esterified fatty acids, also known as free fatty acids) and glycerophosphorylcholine (GPC) [1].…”

Section: Introductionmentioning

confidence: 99%

“…PLB has also been cloned from mammalian cells, but is not sequence related to the fungal PLBs or to bacterial PLB. The enzyme was initially characterized from the intestine and is now known to be present in the external face of the brushborder membranes of mature enterocytes where it is involved in digestion of dietary lipids [4,[7][8][9][10][11][17][18][19]. The enzyme is an ecto-phospholipase with a short C-terminal domain, inserted in the membrane by a hydrophobic segment connected to a larger, heavily glycosylated globular domain [8,19].…”

Section: Introductionmentioning

confidence: 99%

Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast

Morgan

Insall

Haynes

et al. 2004

Biochemical Journal

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The social amoeba Dictyostelium discoideum exhibits high activities of phospholipase and lysophospholipase [Ferber, Munder, Fischer and Gerisch (1970) Eur. J. Biochem. 14, 253-257]. We assayed Dictyostelium lysates to demonstrate the presence of a highly active phospholipase B (PLB) enzyme that removed both fatty-acid chains from phosphatidylcholine and produced the water-soluble glycerophosphorylcholine. We purified the PLB activity from Dictyostelium cytosol using standard agarose media (size exclusion and ion exchange), and combined this with an affinity purification step using myristoylated ARF1 (ADP-ribosylation factor 1), a protein which has a single fatty acid at its N-terminus. Two proteins co-purified (48 kDa and 65 kDa), and the 48 kDa protein was digested with trypsin, peptide fragments were separated by reverse-phase chromatography, and the resultant peptides were sequenced by Edman degradation. From the peptide sequences obtained, database searches revealed a gene which encodes a protein of 65 kDa with unknown function. The 48 kDa protein therefore appears to be a fragment of the fulllength 65 kDa product. Expression of the gene in Escherichia coli confirmed that it encodes a PLB. Characterization of its substrate specificity indicated that, in addition to phosphatidylcholine deacylation, the enzyme also hydrolysed phosphatidylinositol and phosphatidylethanolamine. The PLB identified in the present study is not related to existing PLBs found in bacteria, fungi or mammals. There are, however, genes similar to Dictyostelium PLB in mammals, flies, worms and Giardia, but not in yeast. We therefore have identified a novel family of intracellular PLBs.

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“…In the preceding paper, we detailed the purification and characterization of the catalytic domain of PLB/ LIP after its solubilization from the membrane by autolysis. The purified enzyme consisted of a 14-kDa peptide and a 21-kDa glycosylated peptide and catalyzed PLA 2 , lysophospholipase, and lipase reactions in a single active site (9). To understand the molecular basis of such a broad enzyme specificity, the structure of the nascent enzyme, the membrane anchoring mode, and its physiological significance, we cloned and sequenced the full-length PLB/LIP cDNA based on information of the NH 2 -terminal amino acid sequences of the two peptides derived from the purified enzyme.…”

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confidence: 99%

Identification of Functional Domains of Rat Intestinal Phospholipase B/Lipase

Takemori¹,

Zolotaryov²,

Lü³

et al. 1998

Journal of Biological Chemistry

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A cDNA encoding a rat intestinal Ca 2؉ -independent phospholipase B/lipase (PLB/LIP) was cloned from an ileac mucosa cDNA library using a probe amplified by polymerase chain reaction based on the purified enzyme's sequence. PLB/LIP consists of an NH 2 -terminal signal peptide, four tandem repeats of about 350 amino acids each, and a hydrophobic domain near the COOH terminus. The enzyme purified previously was found to be derived from the second repeat part. To examine the function of each domain, the full-length PLB/LIP, individual repeats, and a protein lacking the COOH-terminal hydrophobic stretch were expressed in COS-7 cells. The results showed that the second repeat, but not the other repeats, had all the activities (phospholipase A 2 , lysophospholipase, and lipase) found in the purified natural and expressed full-length enzymes, suggesting repeat 2 is a catalytic domain. The full-length enzyme was mainly present in membrane fractions and efficiently solubilized by treatment with 1% Triton X-100, but not with phosphatidylinositol-specific phospholipase C. Deletion of the COOH-terminal hydrophobic stretch caused the secretion of >90% of synthesized PLB/LIP into culture media. These results suggest the hydrophobic domain is not replaced by a glycosylphosphatidylinositol anchor but serves as a membrane anchor directly. A message of the full-length PLB/LIP was abundantly expressed in the ileum and also, in a smaller, but significant amount, in the esophagus and testis. Immunohistochemistry showed that PLB/LIP is localized in brush border membranes of the absorptive cells, Paneth cells, and acrosomes of spermatid, suggesting its roles related and unrelated to intestinal digestion.Digestion of phospholipids and triacylglycerol in gastrointestinal tract involves several hydrolysis reactions. A variety of lipases, including acid lipase in lingual gland or stomach, and pancreatic lipase (which is of primary importance in luminal digestion of fats), hydrolyze triacylglycerol to produce monoacylglycerols and free fatty acids (see Refs. 1-3 for reviews)).Pancreatic phospholipases A 2 (PLA 2 ) 1 hydrolyze ester bonds at the sn-2 position of glycerophospholipids and produce fatty acids and lysophospholipids. These steps are prerequisites for lipid absorption by intestinal epithelium cells (4). Lysophospholipid can be directly absorbed, or hydrolyzed by pancreatic lysophospholipase and converted to glycerol 3-phosphate esters and fatty acids.Until recently, all those processes were believed to proceed in the lumen of alimentary tracts by the action of secretory enzymes mentioned above. However, recent studies suggest the presence of a lipid-hydrolyzing enzyme associated with intestinal brush border membranes (5, 6), named phospholipase B/lipase (PLB/LIP) because this enzyme displayed broad lipolytic activities (PLA 2 , lysophospholipase, and lipase activities) (7). PLB/LIP might participate in terminal digestion, or membrane digestion, of dietary lipids and biliary phospholipids, like well established glycosidases and pe...

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Purification and Characterization of a Catalytic Domain of Rat Intestinal Phospholipase B/Lipase Associated with Brush Border Membranes

Cited by 36 publications

References 53 publications

Group 1B Phospholipase A2–Mediated Lysophospholipid Absorption Directly Contributes to Postprandial Hyperglycemia

Group 1B Phospholipase A2–Mediated Lysophospholipid Absorption Directly Contributes to Postprandial Hyperglycemia

Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast

Identification of Functional Domains of Rat Intestinal Phospholipase B/Lipase

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