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M E Lowe

Publications and source records attributed to M E Lowe.

14 recordsLinked to original sources
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↗
PubMed1984

A method of purification of partially methylated alditol acetates in the methylation analysis of glycoproteins and glycopeptides.

A method for methylation analysis of intact glycoproteins is described. Starting with intact glycoprotein, the oligosaccharides are methylated, hydrolyzed, reduced, and acetylated. The partially methylated alditol acetates are then separated from noncarbohydrate contaminants on a silica gel G column. Partially methylated hexitol acetates are eluted from the column with petroleum ether:ethyl acetate (1:1, v/v) and partially methylated N-acetylhexosaminitol acetates are subsequently eluted with methanol. Analysis by gas-liquid chromatography/mass spectrometry of the partially methylated alditol acetates shows no interfering contaminants. This method circumvents the need to make pronase glycopeptides and avoids the pitfalls of other methylation procedures.

Animals↗
PubMed1982

Radiation inactivation of the glycoprotein, invertase.

The radiation target size for invertase activity has been determined for the Saccharomyces cerevisiae glycoprotein which contains 50% carbohydrate. Identical inactivation curves were observed for the native enzyme as well as samples depleted of carbohydrate by incubation with Endo-beta-N-acetylglucosaminidase H. The functional unit of 120,000 daltons was unaltered by the per cent of oligosaccharide cleaved by the enzyme, or by the presence or absence of the released sugars. The irradiated samples showed no change in hexose content even after radiation exposures which grossly destroyed enzymatic activity. Reducing sugars appeared in the irradiated samples, indicating radiation damage to the oligosaccharides. These results unequivocally identify the enzymatically functional portion of the invertase molecule as the polypeptide homodimer, independent of the covalently-bound carbohydrates, and indicate that transfer of radiation energy from protein to oligosaccharide or vice versa is inefficient.

Carbohydrates↗
PubMed1978

Effects of phorbol-12-myristate-13-acetate on the phenotypic program of cultured chondroblasts and fibroblasts.

Phorbol-12-myristate-13-acetate (PMA) has a prompt, differential, and partially reversible effect on cultured chick chondroblasts. Within 36 hr PMA transforms sessile, polygonal, epithelioid chondroblasts into motile, multilayered, fibroblastic cells. In PMA chick chondroblasts rapidly cease to synthesize two of their terminal luxury molecules, the type IV sulfated proteoglycan that characterizes the extracell matrix and a glycosylated protein with an apparent molecular weight of 180,000. This glycosylated protein constitutes approximately 5% of the total protein in normal chondroblasts. If returned to normal medium after 4 days in PMA, virtually 100% of the cells reinitiate the synthesis of their type IV sulfated proteoglycan, of the 180,000-dalton protein, and reacquire their polygonal, epithelioid morphology. If returned to normal medium after 12 days in PMA, the cells fail to synthesize their two characteristic luxury molecules, and 100% of the cells remain fibroblastic. PMA alters the morphology of chick fibroblasts but does not block synthesis of their characteristic type III sulfated proteoglycan. PMA proves to be a mitogen for chondroblasts but not for fibroblasts, in spite of the phenotypic similarities of these two cell types.

Animals↗
PubMed1968

Isolation of crustecdysone (20R-hydroxyecdysone) from a crayfish (Jasus lalandei H. Milne-Edwards).

1. A small amount (2mg.) of crustecdysone, a moulting hormone of crustaceans, was isolated from 1 ton of crayfish waste. 2. The purification procedure used was developed with the aid of crustacean and insect bioassays. 3. CM-Sephadex was found to be superior to Sephadex and very effective for the chromatographic separation of crustecdysone from other non-ionic compounds. The higher efficiency of CM-Sephadex is attributed to the greater number of carboxyl groups available for hydrogen-bonding. 4. Reversed-phase chromatography, with butan-1-ol-cyclohexane mixtures as the stationary phase and water as the flowing phase, proved superior to countercurrent distribution with these solvents for the fractionation of purified extracts. 5. A second moulting hormone, deoxycrustecdysone, and the red-concentrating hormone were obtained in a partially purified form.

Animals↗
PubMed1967

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