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M E Lowe

Publications and source records attributed to M E Lowe.

At least 37 records · Page 2Linked to original sources
PubMed1996

Pancreatic lipase-related protein 2 but not classical pancreatic lipase hydrolyzes galactolipids.

The pancreatic lipase family contains three subfamilies, the 'classical' lipases and the pancreatic lipase-related proteins 1 (PLRP1) and 2 (PLRP2). Galactolipids are present in membranes of leaves and vegetables and consist of digalactosyldiacylglycerol (DGalDG) monogalactosyldiacylglycerol (MGalDG) and sulfoquinovosyldiacylglycerol (SQDG). These lipids were incubated with PLRP2 from guinea-pig (GPLRP2) and rat (RPLRP2). In the presence of bile salts DGalDG was efficiently hydrolyzed by GPLRP2 and, although less efficiently, by RPLRP2 to digalactosylmonoacylglycerol (DGalMG), free fatty acids and water-soluble galactose-containing compounds. Also, MGalDG and SQDG were hydrolyzed by GPLRP2 and RPLRP2. These data suggest a possible role of PLRP2 in the digestion of dietary galactolipids.

Animals↗
PubMed1996

Mutation of the catalytic site Asp177 to Glu177 in human pancreatic lipase produces an active lipase with increased sensitivity to proteases.

The catalytic mechanism for members of the lipase gene family incorporates a serine-histidine-acidic group triad. In general, the acidic group is an aspartate, Asp177 in human pancreatic lipase, but glutamate is found in some lipases. Previously, we demonstrated that site-specific mutagenesis of Asp177 to Glu177 produced a mutant human pancreatic lipase with near normal activity against triolein, thereby, raising questions about the role of Asp177 in the catalytic triad and about the evolutionary pressure which selected Asp over Glu in the catalytic mechanism. To address these questions, we constructed and expressed mutants of Asp177 and Asp206, another acidic residue that could participate in the catalytic triad. The Glu177 mutant had a substrate specificity, specific activity, pH profile, colipase dependance, and interfacial activation comparable to the native lipase, Asp177. Several mutants of Asp206 were normally active, thus, confirming the important role of Asp177 in pancreatic lipase function. Additionally, we found that the Glu177 mutant had increased susceptibility to proteases and to urea denaturation. These findings demonstrated decreased conformational stability of the mutant lipase and provided an explanation for the preference of aspartate in the catalytic triad of human pancreatic lipase.

Amino Acid Sequence↗
PubMed1996

Dexamethasone-regulated expression of pancreatic lipase and two related proteins in AR42J cells.

The pancreas makes two pancreatic proteins [pancreatic lipase-related protein-1 (PLRP-1) and PLRP-2] with marked homology to pancreatic lipase (PL). To determine if a pancreatic acinar cell line, AR42J, also expresses PLRP-1 and PLRP-2, we examined the cells for the presence of PL, PLRP-1, and PLRP-2. RNA blot analysis with specific probes and immunoblot analysis with antipeptide antibodies demonstrated the presence of mRNA and protein for all three homologues in AR42J cells. Additionally, we showed that dexamethasone decreased PLRP-1 mRNA levels twofold and increased PLRP-2 mRNA 20-fold but had little effect on PL or colipase mRNA. Extracellular PLRP-2 protein levels increased threefold, and intracellular PLRP-2 protein levels increased about fourfold. The characteristics of the dexamethasone-induced increase in PLRP-2 mRNA, a rapid change requiring new protein synthesis independent of mRNA turnover, suggested that dexamethasone regulated transcription. We conclude that AR42J cells synthesize and secrete PL, PLRP-1, and PLRP-2 and that dexamethasone discoordinately regulates the expression of the three genes.

Animals↗
PubMed1995

C-terminal domain of human pancreatic lipase is required for stability and maximal activity but not colipase reactivation.

Fungal lipases and human pancreatic lipase (hPL) share a common tertiary structure termed the alpha/beta hydrolase fold. In contrast, the region C-terminal to the common tertiary structure does not share any common structural features with fungal lipases, leading to the hypothesis that the divergent C-terminal domain confers specific properties to hPL. To study the role of the C-terminal domain in hPL function, we made substitution and deletion mutations in the C-terminal domain. The mutant proteins were expressed in transfected COS-1 cells and the secreted proteins were analyzed by immunoblot and for lipase activity. Substitution mutants in multiple lysine residues, in aspartate 390, or in tyrosine 404 did not affect secretion or lipase activity of the mutants. Significantly, the mutants still required colipase for maximal activity. Deletion of the C-terminal domain decreased the amount of truncated, mutant protein in the medium of transfected cells and decreased the specific activity of the mutants. Still, maximal activity required colipase, indicating that the deletion mutants interacted with colipase. Interfacial binding of the truncated deletion mutants was decreased relative to wild-type hPL. The newly synthesized deletion mutants were not as efficiently secreted from the transfected cells as wild-type hPL, and the mutant proteins that appeared in the medium were less stable than the wild-type hPL. These findings suggest that the C-terminal domain is required for proper folding or processing of hPL, confers stability, and increases activity, but is not absolutely required for colipase reactivation of the bile salt-inhibited enzyme.

Amino Acid Sequence↗
PubMed1995

Recording committee meetings: keeping it simple.

Using ingenuity, and a word processor, a format for recording committee meetings meets standards and requirements in a concise, easy manner. This step-by-step process provides a method to record the minutes proper, the attendance log and the work status. All information is stored in the computer for easy reference.

Documentation↗
PubMed1995

Rat GP-3 is a pancreatic lipase with kinetic properties that differ from colipase-dependent pancreatic lipase.

The pancreas contains three homologous proteins, colipase-dependent pancreatic lipase (PL) and two recently described pancreatic lipase-related proteins, PLRP1 and PLRP2. Rat (r) PLRP2 was first identified as a zymogen granule membrane protein, GP-3. Subsequently, we showed that rPLRP2 could cleave fatty acids from triglycerides, but the kinetic properties of rPLRP2 have not been further investigated. To further characterize rPLRP2, we expressed the recombinant enzyme in a baculovirus system, purified the secreted protein, and measured its kinetic properties. rPLRP2 had a broad pH optimum and the curve was similar to that of rPL. At pH 7.5, rPLRP2 cleaved short, medium, and long chain triglycerides by a kinetic mechanism that did not include interfacial activation. The activity against these substrates was not affected by bile salts. In particular, rPLRP2 did not show the bile salt inhibition typical of PL. Although colipase increased rPLRP2 activity in the presence of bile salts, the increase was only 2- to 5-fold compared to the absolute requirement for colipase that rPL had under these conditions. Finally, rPLRP2 could hydrolyze phospholipids, a substrate poorly hydrolyzed by PL. Our characterization of rPLRP2 demonstrates clear differences among the kinetic properties of rPLRP2 and rPL, rPLRP2, and PLRP2 homologues isolated from guinea pig and coypu pancreas. These findings have important implications for the physiological function of rPLRP2.

Amino Acid Sequence↗
PubMed1994

A surface loop covering the active site of human pancreatic lipase influences interfacial activation and lipid binding.

The distinguishing feature of lipases is their increased activity at an oil-water interface, termed interfacial activation. X-ray crystallography of lipases suggested a mechanism for interfacial activation by revealing conformational changes in several surface loops that cover the active site. In one conformation, these loops prevented substrate from entering the active site, and, in the other conformation, movement of the loops opened the active site. We tested the role of the major surface loop, the lid domain, in human pancreatic lipase (hPL) function by creating deletions in this region and expressing the mutant proteins in baculovirus-infected insect cells. the mutants were tested for activity against tributyrin and triolein, colipase interaction, interfacial activation, and binding to tributyrin. The purified mutants had decreased activity against both tributyrin and triolein compared to wild-type hPL and did not show a preference for either substrate. Although colipase was required for maximum activity in the presence of bile salts, the mutants had significant activity against tributyrin, but not triolein, in the absence of colipase. Both mutants were active against monomers of tributyrin demonstrating that they did not require an interface for activity. Finally, both mutants had decreased binding to tributyrin particles. These results suggest that the lid domain in hPL mediates interfacial activation and influences interfacial binding.

Animals↗
PubMed1994

Human pancreatic procolipase expressed in insect cells: purification and characterization.

A cDNA clone encoding human pancreatic procolipase was incorporated into a recombinant baculovirus. Spodoptera frugiperda insect cells infected with the recombinant baculovirus secreted procolipase into the medium, which could be isolated in a single step by immunoaffinity chromatography. The highly purified protein reactivated human pancreatic lipase in a concentration-dependent fashion and was efficiently converted to colipase by limited trypsin digestion. This expression system is suitable for producing amounts of procolipase adequate for biophysical studies.

Animals↗
PubMed1994

Pancreatic triglyceride lipase and colipase: insights into dietary fat digestion.

Dietary fats have an impact on health and disease. A pancreatic exocrine protein, pancreatic triglyceride lipase, is essential for the efficient digestion of dietary fats. This enzyme requires another pancreatic exocrine protein, colipase, for full activity in the gut lumen. In addition to its importance in fat digestion, pancreatic triglyceride lipase has potential applications in medical therapy, medical diagnostics, and industry. This potential stimulated interest in lipases; radiograph during the last few years, studies applying the technologies of molecular biology and radiograph crystallography greatly increased our knowledge about pancreatic triglyceride lipase and colipase protein structure, enzyme mechanism, and gene structure. This review focuses on these recent advances and discusses models for the kinetic properties of pancreatic triglyceride lipase and for the interaction of pancreatic triglyceride lipase with colipase.

Amino Acid Sequence↗
PubMed1994

Rat pancreatic lipase and two related proteins: enzymatic properties and mRNA expression during development.

We report the cDNA sequences of rat colipase, rat pancreatic lipase (rPL), and a rat pancreatic lipase-related protein (rPLRP). Comparison to the human PLRP cDNA suggests that the isolated clone encodes rPLRP-2. Both cDNA and a third cDNA encoding rPLRP-1 are secreted from Sf9 cells infected with recombinant baculovirus. rPL and rPLRP-2 hydrolyze triolein, 8.0 and 4.4 mumol.min-1.microgram-1, respectively. They are inhibited by bile salts, and activity is restored by (pro)colipase. PLRP-1 has barely detectable activity against triolein, even with (pro)colipase present. The pattern of mRNA expression during development in the rat reveals that all mRNA are low in the fetal rat pancreas. Both PLRP mRNA rise just before birth to a maximum 12 h after birth. They fall to low levels in the adult. In contrast, the PL mRNA is low at birth and rises rapidly during the suckling-weanling transition. In conclusion, the rat has at least three genes encoding different lipases, and these related genes have separate regulatory controls.

Aging↗
PubMed1992

The catalytic site residues and interfacial binding of human pancreatic lipase.

In this study, the essential serine residue and 2 other amino acids in human pancreatic triglyceride lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) were tested for their contribution to the enzyme's catalytic site or interfacial binding site. By site-specific mutagenesis of the cDNA for human pancreatic lipase, amino acid substitutions were made at Ser153, His264, and Asp177. The mutant cDNAs were expressed in transfected COS-1 cells. Both the medium and the cells were examined for the presence of pancreatic lipase by Western blot analysis. The activity of the expressed proteins against triolein and the interfacial binding was measured. Proteins with mutations in Ser153 were secreted by the cells and bound to interfaces but had no detectable activity. Changing His264 to a leucine or Asp177 to an asparagine also produced inactive lipase. Substituting glutamic acid for Asp177 produced an active protein. These results demonstrate that Ser153 is involved in the catalytic site of pancreatic lipase and is not crucial for interfacial binding. Moreover, the essential roles of His264 and Asp177 in catalysis were demonstrated. A Ser-His-Asp catalytic triad similar to that present in serine proteases is present in human pancreatic lipase.

Animals↗
PubMed1992

The human colipase gene: isolation, chromosomal location, and tissue-specific expression.

The digestion of dietary triglycerides occurs in the duodenum through the action of triglyceride lipase, a pancreatic exocrine protein. The activity of pancreatic lipase is inhibited by the bile salts normally found in the gut lumen. Another pancreatic exocrine protein, colipase, restores the lipolytic activity of triglyceride lipase. The synthesis and secretion of both triglyceride lipase and colipase is increased by dietary fats and secretin. An increase in mRNA accompanies the increased activity, suggesting that the genes for triglyceride lipase and colipase contain nucleotide elements responsive to dietary fats or secretin or both. To study the regulation of colipase expression, we have first isolated the gene for human colipase from a cosmid library with a cDNA probe. The gene was localized to chromosome 6 and is organized into three exons contained in a single 3.3-kb BamHI fragment. The 5'-flanking region of the gene contains a TATA box, a GC box, and a 28-bp region with homology to the rat pancreatic-specific enhancer. This region directs the tissue-specific expression of the chloramphenicol acetyltransferase gene in a transfected rat pancreatic acinar cell line, AR42-J. The same construct is inactive in HEPG2, C2C12, and COS-1 cells. These results demonstrate that the isolated gene for human colipase contains tissue-specific promoter activity in the 5'-flanking DNA. The 28-bp region specifically binds to a factor in nuclear extracts.

Amino Acid Sequence↗
PubMed1992

Site-specific mutations in the COOH-terminus of placental alkaline phosphatase: a single amino acid change converts a phosphatidylinositol-glycan-anchored protein to a secreted protein.

Placental alkaline phosphatase (PLAP) is anchored in the plasma membrane by a phosphatidylinositol-glycan moiety (PI-glycan). PI-glycan is added posttranslationally to the nascent peptide chain after the removal of 29 amino acids from the COOH-terminus. The contribution of selected COOH-terminal amino acids to the signal for PI-glycan addition was tested by creating a fusion protein with the COOH-terminus of PLAP and a secreted protein and by mutagenesis of specific PLAP COOH-terminal amino acids. The cDNA encoding the COOH-terminus of PLAP was fused in frame to the cDNA for human clotting Factor X and expressed in transfected COS-1 cells. Fusion proteins containing 32 amino acids of the PLAP COOH-terminus were modified by PI-glycan addition. Thus, the signal for PI-glycan modification must reside in these amino acids. Next, the region between the hydrophobic domain and the cleavage site was examined for additional determinants. Mutations of the hydrophilic residues in the spacer region demonstrated that these amino acids do not contribute to the signal for PI-glycan addition. Deletion of amino acids in the spacer region prevented the addition of PI-glycan suggesting that the length of the spacer domain or the amino acids around the cleavage site are important determinants. Finally, we demonstrated that interruption of the hydrophobic domain by a charged residue prevents PI-glycan addition and results in a protein that is secreted into the medium. The finding that a single Leu to Arg substitution in the hydrophobic domain converts a PI-glycan anchored, membrane protein to a secreted protein suggests that an essential signal for the correct sorting of PI-glycan anchored proteins versus secreted proteins resides in the hydrophobic domain. Substitution of a charged amino acid for a hydrophobic amino acid may be a mechanism for producing membrane bound and secreted forms of the same protein.

Alkaline Phosphatase↗
PubMed1990

Expression of a Nagao-type, phosphatidylinositol-glycan anchored alkaline phosphatase in human choriocarcinomas.

The alkaline phosphatase (AP) synthesized by human tumor cells closely resembles human placental AP (PLAP). Little is known about the molecular events that lead to the expression of a placenta-like AP in tumor cells. The complementary DNA encoding the AP expressed by a choriocarcinoma cell line, BeWo, was isolated and characterized. The complementary DNA is the product of the germ cell AP (Nagao isozyme) gene and not of the term PLAP gene. Like placental AP, the tumor AP can be released from the cell membrane by a phosphaditylinositol-specific phospholipase C and has a phosphaditylinositol-glycan (PI-glycan) moiety at the COOH terminus. Immunoprecipitation of phosphaditylinositol-specific phospholipase C-treated AP and analysis by polyacrylamide gel electrophoresis or isoelectric focusing demonstrates that at least 95% of the AP contains PI-glycan. Two-dimensional gel electrophoresis reveals two precursors of the mature AP. One of these does not bind an antibody against the Trypanosoma variable surface glycoprotein cross-reacting determinant and probably does not contain PI-glycan. This precursor had a shorter half-life than the more prominent PI-glycan-containing precursor in pulse-chase experiments, suggesting a precursor-product relationship between the two proteins. These data demonstrate that BeWo AP is the product of a gene normally expressed in testis, thymus, and germ cells, but not in placenta. Thus, the expression of BeWo AP results from the repression of the PLAP gene and derepression of the germ cell AP gene and, as such, the expression is ectopic. The BeWo AP (Nagao isozyme) is modified with PI-glycan that is added soon after translation, not cotranslationally.

Alkaline Phosphatase↗
PubMed1990

Cloning and characterization of the human colipase cDNA.

Pancreatic lipase hydrolyzes dietary triglycerides to monoglycerides and fatty acids. In the presence of bile salts, the activity of pancreatic lipase is markedly decreased. The activity can be restored by the addition of colipase, a low molecular weight protein secreted by the pancreas. The action of pancreatic lipase in the gut lumen is dependent upon its interaction with colipase. As a first step in elucidating the molecular events governing the interaction of lipase and colipase with each other and with fatty acids, a cDNA encoding human colipase was isolated from a lambda gt11 cDNA library with a rabbit polyclonal anti-human colipase antibody. The full-length 525 bp cDNA contained an open reading frame encoding 112 amino acids, including a 17 amino acid signal peptide. The predicted protein sequence contains 100% of the published protein sequence for human colipase determined by chemical methods, but predicts the presence of five additional NH2-terminal amino acids and four additional COOH-terminal amino acids. Comparison of the predicted protein sequence with the known sequences of colipase from other species reveals regions of extensive identity. In vitro translation of mRNA transcribed from the cDNA gave a protein of the expected molecular size that was processed by pancreatic microsomal membranes. Sequence analysis of the in vitro translation product after processing demonstrated signal peptide cleavage and the presence of a human procolipase, as exists in the pig and horse colipases. DNA blot analysis was consistent with the presence of a single gene for colipase. RNA blot analysis demonstrated tissue-specific expression of colipase mRNA in the pancreas. Thus, we report, for the first time, a cDNA for colipase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of the gene from a human genome encoding 17 beta-estradiol dehydrogenase: a comparison of Jar and BeWo choriocarcinoma cell lines.

17-beta-Estradiol dehydrogenase is required for the enzymatic interconversion of estradiol and its weaker related sex steroid, estrone. We isolated and sequenced a complementary deoxyribonucleic acid clone for 17 beta-estradiol dehydrogenase from the BeWo choriocarcinoma cell line. Comparison of the BeWo complementary deoxyribonucleic acid sequence to a previously derived placental complementary deoxyribonucleic acid sequence yields greater than 98% homology. We also isolated the gene for 17 beta-estradiol dehydrogenase from the Jar human choriocarcinoma cell line and elucidated its primary nucleic acid structure. Significant differences in the Jar-deduced complementary deoxyribonucleic acid sequence clearly differentiate it from both the human placental and BeWo forms of 17 beta-estradiol dehydrogenase, indicating the existence of two genes for 17 beta-estradiol dehydrogenase in the human genome. Evaluation of 17 beta-estradiol dehydrogenase gene expression in BeWo and Jar cells was compared with expression in luteinized granulosa cells. Messenger ribonucleic acid for human placental 17 beta-estradiol dehydrogenase was identified in all three cell types as a 1.3 kilobase band on Northern blot analysis. A second messenger ribonucleic acid species measuring 2.1 kilobase was abundantly present in the granulosa cells. Whether these two species of messenger ribonucleic acid are involved in the regulation of the estradiol dehydrogenase genes is yet to be determined.

Base Sequence↗
PubMed1989

Cloning and characterization of human pancreatic lipase cDNA.

Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) hydrolyzes dietary long chain triacylglycerol to free fatty acids and monoacylglycerols in the intestinal lumen. In the presence of bile acids, the activity of lipase is stimulated by colipase. As a prelude to studying the relationship of the protein structures to the functional properties of lipase and colipase, a cDNA encoding human pancreatic lipase was isolated from a lambda gt11 cDNA library screened with a rabbit polyclonal anti-human pancreatic lipase antibody. The full length cDNA clone of 1477 base pairs contained an open reading frame encoding a 465-amino acid protein, including a 16-amino acid signal peptide. The nucleotide sequence was 69% identical to the dog pancreatic lipase cDNA. The predicted NH2-terminal protein sequence agreed with the published NH2-terminal sequence of human pancreatic lipase and the predicted protein sequence was 85 and 70% identical to the protein sequences of pig and dog pancreatic lipase, respectively. A region of homology around Ser-153 is conserved in a number of lipid-binding proteins. Human hepatic lipase and lipoprotein lipase share extensive homology with pancreatic lipase, suggesting that the three proteins are members of a small gene family. In vitro translation of mRNA transcribed from the cDNA resulted in a protein of the expected molecular size that could be processed by microsomal membranes to yield a glycolated protein with proper signal peptide cleavage. RNA blot analysis demonstrated tissue specificity for pancreatic lipase. Thus, for the first time, a full length human pancreatic lipase cDNA has been isolated and characterized. The demonstrated regions of homology with other lipases will aid definition of interactions with substrate and colipase through site-specific mutagenesis.

Amino Acid Sequence↗