scholarly journals cDNA cloning and expression in Xenopus laevis oocytes of pig renal dipeptidase, a glycosyl-phosphatidylinositol-anchored ectoenzyme

1990 ◽  
Vol 271 (3) ◽  
pp. 755-760 ◽  
Author(s):  
E Rached ◽  
N M Hooper ◽  
P James ◽  
G Semenza ◽  
A J Turner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Xenopus Laevis ◽  
Signal Sequence ◽  
Polyclonal Antiserum ◽  
Cloning And Expression ◽  
Kidney Cortex ◽  
Xenopus Laevis Oocytes ◽  
Glycosyl Phosphatidylinositol ◽  
Pig Kidney ◽  
C Terminus

Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form.

scholarly journals Molecular cloning and expression in COS-1 cells of pig kidney aminopeptidase P

1996 ◽  
Vol 319 (1) ◽  
pp. 197-201 ◽  
Author(s):  
Ralph J HYDE ◽  
Nigel M HOOPER ◽  
Anthony J TURNER
Keyword(s):  
Amino Acids ◽  
Signal Sequence ◽  
Hydrolytic Enzymes ◽  
Cloning And Expression ◽  
Kidney Cortex ◽  
Binding Motif ◽  
Primary Sequence ◽  
Sequence Comparisons ◽  
Pig Kidney ◽  
Aminopeptidase P

Aminopeptidase P (AP-P; X-Pro aminopeptidase; EC 3.4.11.9), a key enzyme in the metabolism of the vasodilator bradykinin, has been cloned from a pig kidney cortex cDNA library following the use of the PCR to identify sub-libraries enriched in AP-P clones. The complete primary sequence of the enzyme has been deduced from a full-length cDNA clone. This predicts a protein of 673 amino acids with a cleavable N-terminal signal sequence and six potential N-linked glycosylation sites. A stretch of mainly hydrophobic amino acids at the C-terminus is predicted to co-ordinate the attachment of a glycosyl-phosphatidylinositol (GPI) membrane anchor. Although AP-P is a zinc metallopeptidase, the predicted primary sequence does not contain any recognizable zinc-binding motif. Transient expression of AP-P cDNA in COS-1 cells resulted in enzymic activity characteristic of AP-P, namely apstatin- and EDTA-sensitive hydrolysis of bradykinin and Gly-Pro-Hyp. The expressed protein was recognized as a polypeptide of Mr 91000 under reducing conditions, following immunoblotting of COS-1 membranes with a polyclonal antibody raised against purified pig kidney AP-P. The presence of a GPI anchor on expressed AP-P was established by demonstrating release of the enzyme from a membrane fraction following treatment with bacterial phosphatidylinositol-specific phospholipase C and its corresponding conversion from an amphipathic to a hydrophilic form, as assessed by phase separation in Triton X-114. Sequence comparisons confirm that AP-P is a member of the proline peptidase family of hydrolytic enzymes and is unrelated in sequence to other brush-border membrane peptidases.

scholarly journals Expression of Na+-independent amino acid transport in Xenopus laevis oocytes by injection of rabbit kidney cortex mRNA

1992 ◽  
Vol 281 (3) ◽  
pp. 717-723 ◽  
Author(s):  
J Bertran ◽  
A Werner ◽  
G Stange ◽  
D Markovich ◽  
J Biber ◽  
...  
Keyword(s):  
Amino Acids ◽  
Xenopus Laevis ◽  
Transport System ◽  
Sucrose Density Gradient ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Xenopus Laevis Oocytes ◽  
Transport Pathways ◽  
Arginine Uptake ◽  
Dose Dependent Increase

Poly(A)+ mRNA was isolated from rabbit kidney cortex and injected into Xenopus laevis oocytes. Injection of mRNA resulted in a time- and dose-dependent increase in Na(+)-independent uptake of L-[3H]alanine and L-[3H]arginine. L-Alanine uptake was stimulated about 3-fold and L-arginine uptake was stimulated about 8-fold after injection of mRNA (25-50 ng, after 3-6 days) as compared with water-injected oocytes. T.I.C. of oocyte extracts suggested that the increased uptake actually represented an increase in the oocyte content of labelled L-alanine and L-arginine. The expressed L-alanine uptake, obtained by subtracting the uptake in water-injected oocytes from that in mRNA-injected oocytes, showed saturability and was inhibited completely by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) and L-arginine. The expressed L-arginine uptake in mRNA-injected oocytes also showed saturability, being completely inhibited by L-dibasic amino acids) and partially inhibited by BCH. Expression of both L-alanine and L-arginine uptake showed clear cis-inhibition by cationic (e.g. L-arginine) and neutral (e.g. L-leucine) amino acids. In all, this points to the expression of a Na(+)-independent transport system with broad specificity (i.e. b degree, (+)-like). In addition, part of the expressed uptake of L-arginine could be due to a system y(+)-like transporter. After size fractionation through a sucrose density gradient, the mRNA species encoding these increased transport activities (Na(+)-independent transport of L-alanine and of L-arginine) were found in fractions of an average mRNA chain-length of 1.8-2.4 kb. On the basis of these results, we conclude that Na(+)-independent transport system(s) for L-alanine and L-arginine from rabbit renal cortical tissues, most likely proximal tubules, are expressed in Xenopus laevis oocytes. These observations may represent the first steps towards expression and cloning of these transport pathways.

scholarly journals Expression of the poly(A)-binding protein during development of Xenopus laevis.

1989 ◽  
Vol 9 (6) ◽  
pp. 2756-2760 ◽  
Author(s):  
B D Zelus ◽  
D H Giebelhaus ◽  
D W Eib ◽  
K A Kenner ◽  
R T Moon
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Xenopus Laevis ◽  
Fusion Protein ◽  
Outer Segment ◽  
Binding Protein ◽  
Polyclonal Antiserum ◽  
Xenopus Laevis Oocytes ◽  
Cdna Clones ◽  
Protein Encoding

We have isolated and sequenced cDNA clones encoding the poly(A)-binding protein of Xenopus laevis oocytes. Polyclonal antiserum was raised against a fusion protein encoding 185 amino acids of the Xenopus poly(A)-binding protein. This antiserum localizes the poly(A)-binding protein to subcellular sites associated with protein synthesis; in the retina, immunoreactive protein is detected in the synthetically active inner segment of the photoreceptor but not in the transductive outer segment. Transcripts encoding the poly(A)-binding protein are present in oocytes, although no protein is detected on protein blots. In contrast, the levels of both transcripts and protein increase in development, which correlates with the observed increase in total poly(A) during Xenopus embryogenesis (N. Sagata, K. Shiokawa, and K. Yamana, Dev. Biol. 77:431-448, 1980).

Expression of the poly(A)-binding protein during development of Xenopus laevis

1989 ◽  
Vol 9 (6) ◽  
pp. 2756-2760
Author(s):  
B D Zelus ◽  
D H Giebelhaus ◽  
D W Eib ◽  
K A Kenner ◽  
R T Moon
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Xenopus Laevis ◽  
Fusion Protein ◽  
Outer Segment ◽  
Binding Protein ◽  
Polyclonal Antiserum ◽  
Xenopus Laevis Oocytes ◽  
Cdna Clones ◽  
Protein Encoding

We have isolated and sequenced cDNA clones encoding the poly(A)-binding protein of Xenopus laevis oocytes. Polyclonal antiserum was raised against a fusion protein encoding 185 amino acids of the Xenopus poly(A)-binding protein. This antiserum localizes the poly(A)-binding protein to subcellular sites associated with protein synthesis; in the retina, immunoreactive protein is detected in the synthetically active inner segment of the photoreceptor but not in the transductive outer segment. Transcripts encoding the poly(A)-binding protein are present in oocytes, although no protein is detected on protein blots. In contrast, the levels of both transcripts and protein increase in development, which correlates with the observed increase in total poly(A) during Xenopus embryogenesis (N. Sagata, K. Shiokawa, and K. Yamana, Dev. Biol. 77:431-448, 1980).

Amino-acid-dependent modulation of amino acid transport in Xenopus laevis oocytes.

1996 ◽  
Vol 199 (4) ◽  
pp. 923-931 ◽  
Author(s):  
P M Taylor ◽  
S Kaur ◽  
B Mackenzie ◽  
G J Peter
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Xenopus Laevis ◽  
Amino Acid Transport ◽  
Acid Mixture ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Transporters ◽  
Amino Acid Supplementation ◽  
Acid Transport ◽  
Acid Supplementation

We have measured rates of uptake of arginine, glutamine, glutamate, serine, phenylalanine and glycine in Xenopus laevis oocytes cultured for periods of up to 24h in saline in the presence or absence of a mixture of 20 amino acids at concentrations approximating those in Xenopus plasma. Amino acid supplementation increased the total intracellular amino acid concentration from 8.2 to 18.4 nmol per oocyte. Specific Na(+)-dependent amino acid transporters (systems B0,+, Xag-) exhibit 'adaptive regulation' (up-regulation during amino acid deprivation and down-regulation during amino acid supplementation). Na(+)-independent transporters of glutamate, glutamine and glycine (including system asc) display an opposite modulation in activity, which may help to combat amino-acid-induced oxidative stress by increasing the supply of glutathione precursors. Single amino acids at physiological plasma concentrations (0.47 mmol l-1 L-alanine, 0.08 mmol l-1 L-glutamate) mimicked at least some effects of the amino acid mixture. The mechanisms of transport modulation do not appear to include trans-amino acid or membrane potential effects and, in the case of Na(+)-independent transport, are independent of protein or mRNA synthesis. Furthermore, activation of protein kinase C by phorbol 12-myristate 13-acetate did not significantly affect endogenous glutamine and glutamate transport. The Xenopus oocyte appears to possess endogenous signalling mechanisms for selectively modulating the activity of amino acid transport proteins expressed in its surface membranes, a factor for consideration when using oocytes as an expression system for structure-function studies of cloned amino acid transporters.

scholarly journals Characterization of the Collagen-Binding S-Layer Protein CbsA of Lactobacillus crispatus

2000 ◽  
Vol 182 (22) ◽  
pp. 6440-6450 ◽  
Author(s):  
Jouko Sillanpää ◽  
Beatriz Martínez ◽  
Jenni Antikainen ◽  
Takahiro Toba ◽  
Nisse Kalkkinen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Type I Collagen ◽  
Signal Sequence ◽  
Type I ◽  
Binding Region ◽  
Sequence Comparisons ◽  
Collagen Binding ◽  
Reading Frame ◽  
Lactobacillus Crispatus ◽  
C Terminus

The cbsA gene of Lactobacillus crispatusstrain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. ThecbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three otherLactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsAwhere deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.

Molecular cloning and expression of an adenylyl cyclase from Xenopus laevis oocytes 1

FEBS Letters ◽  
1997 ◽  
Vol 404 (1) ◽  
pp. 91-94 ◽  
Author(s):  
Marcela Torrejón ◽  
Valentina Echeverrı́a ◽  
Gabriel Retamales ◽  
Luisa Herrera ◽  
Marı́a V Hinrichs ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Adenylyl Cyclase ◽  
Molecular Cloning ◽  
Cloning And Expression ◽  
Xenopus Laevis Oocytes
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