scholarly journals Structural features of the low-molecular-mass human salivary mucin

1992 ◽  
Vol 287 (2) ◽  
pp. 639-643 ◽  
Author(s):  
M S Reddy ◽  
L A Bobek ◽  
G G Haraszthy ◽  
A R Biesbrock ◽  
M J Levine
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Structural Features ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Mrna Species ◽  
Salivary Mucin

The low-molecular-mass human salivary mucin has at least two isoforms, MG2a and MG2b, that differ primarily in their sialic acid and fucose content. In this study, we characterize further these isoforms, particularly their peptide moieties. Trypsin digests of MG2a and MG2b yielded high- and low-molecular-mass glycopeptides following gel filtration on Sephacryl S-300. The larger glycopeptides from MG2a and MG2b had similar amino acid compositions and identical N-terminal sequences, suggesting common structural features between their peptides. An oligonucleotide probe generated from the amino acid sequence of the smaller glycopeptide from MG2a was employed in Northern-blot analysis. This probe specifically hybridized to two mRNA species from human submandibular and sublingual glands. A cDNA clone selected from a human submandibular gland cDNA expression library with antibody generated against deglycosylated MG2a also hybridized to these two mRNA species. In both cases, the larger mRNA was polydisperse, and the hybridization signal was more intense in the sublingual gland. In addition, the N-terminal amino acid sequence of the larger glycopeptide was found to be part of one of the selected MG2 cDNA clones.

scholarly journals Dictyostelium discoideum myosin: isolation and characterization of cDNAs encoding the essential light chain.

1988 ◽  
Vol 8 (2) ◽  
pp. 794-801 ◽  
Author(s):  
R L Chisholm ◽  
A M Rushforth ◽  
R S Pollenz ◽  
E R Kuczmarski ◽  
S R Tafuri
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Dictyostelium Discoideum ◽  
Light Chain ◽  
Single Gene ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Essential Light Chain ◽  
Isolation And Characterization

We used an antibody specific for Dictyostelium discoideum myosin to screen a lambda gt11 cDNA expression library to obtain cDNA clones which encode the Dictyostelium essential myosin light chain (EMLC). The amino acid sequence predicted from the sequence of the cDNA clone showed 31.5% identity with the amino acid sequence of the chicken EMLC. Comparisons of the Dictyostelium EMLC, a nonmuscle cell type, with EMLC sequences from similar MLCs of skeletal- and smooth-muscle origin, showed distinct regions of homology. Much of the observed homology was localized to regions corresponding to consensus Ca2+-binding of E-F hand domains. Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the EMLC. Examination of the pattern of EMLC mRNA expression showed that a significant increase in EMLC message levels occurred during the first few hours of development, coinciding with increased actin expression and immediately preceding the period of maximal chemotactic activity.

Dictyostelium discoideum myosin: isolation and characterization of cDNAs encoding the essential light chain

1988 ◽  
Vol 8 (2) ◽  
pp. 794-801
Author(s):  
R L Chisholm ◽  
A M Rushforth ◽  
R S Pollenz ◽  
E R Kuczmarski ◽  
S R Tafuri
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Dictyostelium Discoideum ◽  
Light Chain ◽  
Single Gene ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Essential Light Chain ◽  
Isolation And Characterization

We used an antibody specific for Dictyostelium discoideum myosin to screen a lambda gt11 cDNA expression library to obtain cDNA clones which encode the Dictyostelium essential myosin light chain (EMLC). The amino acid sequence predicted from the sequence of the cDNA clone showed 31.5% identity with the amino acid sequence of the chicken EMLC. Comparisons of the Dictyostelium EMLC, a nonmuscle cell type, with EMLC sequences from similar MLCs of skeletal- and smooth-muscle origin, showed distinct regions of homology. Much of the observed homology was localized to regions corresponding to consensus Ca2+-binding of E-F hand domains. Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the EMLC. Examination of the pattern of EMLC mRNA expression showed that a significant increase in EMLC message levels occurred during the first few hours of development, coinciding with increased actin expression and immediately preceding the period of maximal chemotactic activity.

Physicochemical properties of a novel α-L-arabinofuranosidase fromRhizomucor pusillusHHT-1

2001 ◽  
Vol 47 (8) ◽  
pp. 767-772 ◽  
Author(s):  
A KM Shofiqur Rahman ◽  
Shinya Kawamura ◽  
Masahiro Hatsu ◽  
M M Hoq ◽  
Kazuhiro Takamizawa
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Rhizomucor Pusillus ◽  
Terminal Amino ◽  
Metal Cofactors

The zygomycete fungus Rhizomucor pusillus HHT-1, cultured on L(+)arabinose as a sole carbon source, produced extracellular α-L-arabinofuranosidase. The enzyme was purified by (NH4)2SO4fractionation, gel filtration, and ion exchange chromatography. The molecular mass of this monomeric enzyme was 88 kDa. The native enzyme had a pI of 4.2 and displayed a pH optimum and stability of 4.0 and 7.0–10.0, respectively. The temperature optimum was 65°C, and it was stable up to 70°C. The Kmand Vmaxfor p-nitrophenyl α-L-arabinofuranoside were 0.59 mM and 387 µmol·min–1·mg–1protein, respectively. Activity was not stimulated by metal cofactors. The N-terminal amino acid sequence did not show any similarity to other arabinofuranosidases. Higher hydrolytic activity was recorded with p-nitrophenyl α-L-arabinofuranoside, arabinotriose, and sugar beet arabinan; lower hydrolytic activity was recorded with oat–spelt xylan and arabinogalactan, indicating specificity for the low molecular mass L(+)-arabinose containing oligosaccharides with furanoside configuration.Key words: α-L-arabinofuranosidase, enzyme purification, amino acid sequence, Rhizomucor pusillus.

scholarly journals Purification, characterization and cDNA cloning of human lung surfactant protein D

1992 ◽  
Vol 284 (3) ◽  
pp. 795-802 ◽  
Author(s):  
J Lu ◽  
A C Willis ◽  
K B M Reid
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Human Lung ◽  
Solid Phase ◽  
Surfactant Protein ◽  
Amino Acid Sequences ◽  
Surfactant Protein D ◽  
Cdna Clones ◽  
Oligonucleotide Probes

Human pulmonary surfactant protein D (SP-D) was identified in lung lavage by its similarity to rat SP-D in both its molecular mass and its Ca(2+)-dependent-binding affinity for maltose [Persson, Chang & Crouch (1990) J. Biol. Chem. 265, 5755-5760]. For structural studies, human SP-D was isolated from amniotic fluid by affinity chromatography on maltose-Sepharose followed by f.p.l.c. on Superose 6, which showed it to have a molecular mass of approx. 620 kDa in non-dissociating conditions. On SDS/PAGE the human SP-D behaved as a single band of 150 kDa or 43 kDa in non-reducing or reducing conditions respectively. The presence of a high concentration of glycine (22%), hydroxyproline and hydroxylysine in the amino acid composition of human SP-D indicated that it contained collagen-like structure. Collagenase digestion yielded a 20 kDa collagenase-resistant globular fragment which retained affinity for maltose. Use of maltosyl-BSA as a neoglycoprotein ligand in a solid-phase binding assay showed that human SP-D has a similar carbohydrate-binding specificity to rat SP-D, but a clearly distinct specificity from that of other lectins, such as conglutinin, for a range of simple saccharides. Amino acid sequence analysis established the presence of collagen-like Gly-Xaa-Yaa triplets in human SP-D and also provided sequence data from the globular region of the molecule which was used in the synthesis of oligonucleotide probes. Screening of a human lung cDNA library with the oligonucleotide probes, and also with rabbit anti-(human SP-D), allowed the isolation of two cDNA clones which overlap to give the full coding sequence of human SP-D. The derived amino acid sequence indicates that the mature human SP-D polypeptide chain is 355 residues long, having a short non-collagen-like N-terminal section of 25 residues, followed by a collagen-like region of 177 residues and a C-terminal C-type lectin domain of 153 residues. Comparison of the human SP-D and bovine serum conglutinin amino acid sequences indicated that they showed 66% identity despite their marked differences in carbohydrate specificity.

Molecular Cloning of Bovine Amelogenin cDna

1987 ◽  
Vol 1 (2) ◽  
pp. 293-297 ◽  
Author(s):  
H. Shimokawa ◽  
Y. Ogata ◽  
S. Sasaki ◽  
M.E. Sobel ◽  
C.I. Mcquillan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Cloning ◽  
Peptide Sequence ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Coding Region ◽  
Expression Library ◽  
Amino Acid Homology ◽  
Signal Peptide Sequence ◽  
Cloned Cdna

Molecular cloning of a bovine amelogenin cDNA was accomplished by construction of a cDNA expression library (λgt11 cDNA library) from the bovine ameloblast mRNA and then screening of the library with antibodies to bovine amelogenins. The complete primary structure of an amelogenin was deduced from cloned cDNA. One of the cDNA clones isolated from a bovine ameloblast phage λgt11 library had an 864-base-pair-long insert that encoded a protein with 216 amino acid residues. This cDNA clone appears to represent the complete coding region of amelogenin mRNA, including a putative AUG initiation codon and a signal peptide sequence. The predicted bovine amelogenin sequence has 87% amino acid homology with murine amelogenin.

scholarly journals The amino acid sequences of the myelin-associated glycoproteins: homology to the immunoglobulin gene superfamily

1987 ◽  
Vol 104 (4) ◽  
pp. 957-965 ◽  
Author(s):  
JL Salzer ◽  
WP Holmes ◽  
DR Colman
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Growth Factor Receptor ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Immunoglobulin Gene ◽  
Expression Library ◽  
Extracellular Region ◽  
Immunoglobulin Gene Superfamily ◽  
Membrane Spanning

The myelin associated glycoproteins (MAG) are integral plasma membrane proteins which are found in oligodendrocytes and Schwann cells and are believed to mediate the axonal-glial interactions of myelination. In this paper we demonstrate the existence in central nervous system myelin of two MAG polypeptides with Mrs of 67,000 and 72,000 that we have designated small MAG (S-MAG) and large MAG (L-MAG), respectively. The complete amino acid sequence of L-MAG and a partial amino acid sequence of S-MAG have been deduced from the nucleotide sequences of corresponding cDNA clones isolated from a lambda gt11 rat brain expression library. Based on their amino acid sequences, we predict that both proteins have an identical membrane spanning segment and a large extracellular domain. The putative extracellular region contains an Arg-Gly-Asp sequence that may be involved in the interaction of these proteins with the axon. The extracellular portion of L-MAG also contains five segments of internal homology that resemble immunoglobulin domains, and are strikingly homologous to similar domains of the neural cell adhesion molecule and other members of the immunoglobulin gene superfamily. In addition, the two MAG proteins differ in the extent of their cytoplasmically disposed segments and appear to be the products of alternatively spliced mRNAs. Of considerable interest is the finding that the cytoplasmic domain of L-MAG, but not of S-MAG, contains an amino acid sequence that resembles the autophosphorylation site of the epidermal growth factor receptor.

scholarly journals Characterization of cDNA clones for human myeloperoxidase: Predicted amino acid sequence and evidence for multiple mRNA species

1987 ◽  
Vol 15 (5) ◽  
pp. 2013-2028 ◽  
Author(s):  
Keith R. Johnson ◽  
William M. Nauseef ◽  
Alessandra Care ◽  
Margaret J. Wheelock ◽  
Sara Shane ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Mrna Species

scholarly journals 2-Ketocyclohexanecarboxyl Coenzyme A Hydrolase, the Ring Cleavage Enzyme Required for Anaerobic Benzoate Degradation byRhodopseudomonas palustris

1998 ◽  
Vol 180 (9) ◽  
pp. 2330-2336 ◽  
Author(s):  
Dale A. Pelletier ◽  
Caroline S. Harwood
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Coenzyme A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ring Cleavage ◽  
Benzoate Degradation ◽  
Coa Hydrolase

ABSTRACT 2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium Rhodopseudomonas palustris by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography. The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the badI gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of badI indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins. Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration. This indicates that the native form of the enzyme is a homotetramer. The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 μmol min−1 mg of protein−1. Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate. An R. palustris mutant, carrying a chromosomal disruption of badI, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate. These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by R. palustris.

scholarly journals Molecular cloning of cDNA species for rat and mouse liver α-methylacyl-CoA racemases

1997 ◽  
Vol 326 (3) ◽  
pp. 883-889 ◽  
Author(s):  
Werner SCHMITZ ◽  
Heli M. HELANDER ◽  
J. Kalervo HILTUNEN ◽  
Ernst CONZELMANN
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Rat Liver ◽  
Mouse Liver ◽  
Amino Acid Sequences ◽  
Cdna Libraries ◽  
Cdna Expression Library ◽  
Coding Region ◽  
Expression Library ◽  
Cdna Expression

cDNA species coding for α-methylacyl-CoA racemase were cloned from rat and mouse liver cDNA libraries and characterized. The rat liver λgt11 cDNA expression library was screened with anti-racemase IgG [Schmitz, Albers, Fingerhut and Conzelmann (1995) Eur. J. Biochem. 231, 815–822]. Several full-length clones were obtained that contained an open reading frame of 1083 bp, coding for a protein of 361 amino acid residues with a predicted molecular mass of 39679 Da. The sequences of three peptides that were isolated by HPLC from a tryptic digest of purified rat liver racemase fully matched the cDNA-derived amino acid sequence. The cDNA coding for mouse racemase was cloned from a mouse liver λZAP cDNA expression library and sequenced. The coding region of 1080 bp codes for a 360-residue protein (molecular mass 39558 Da) that shares 89.7% similarity with the rat protein. Expression of the rat racemase as a recombinant protein in Escherichia coli with the pTrcHisB-expression vector yielded enzymically active protein. The amino acid sequences of α-methylacyl-CoA racemases do not resemble any known sequence of β-oxidation or auxiliary enzymes, supporting the view of a highly diverse evolutionary origin of enzymes acting on fatty acyl-CoA S-esters.

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