scholarly journals Purification of feline lysosomal α-mannosidase, determination of its cDNA sequence and identification of a mutation causing α-mannosidosis in Persian cats

1997 ◽  
Vol 328 (3) ◽  
pp. 863-870 ◽  
Author(s):  
Thomas BERG ◽  
K. Ole TOLLERSRUD ◽  
U. Steven WALKLEY ◽  
Donald SIEGEL ◽  
Øivind NILSSEN
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Reverse Transcriptase ◽  
Cdna Sequence ◽  
Molecular Heterogeneity ◽  
Lysosomal Storage ◽  
Reverse Transcriptase Pcr ◽  
Pcr Products

α-Mannosidosis is a lysosomal storage disorder that is caused by the deficiency of lysosomal α-mannosidase. Feline α-mannosidosis is a well-characterized animal model used for studying pathological and therapeutic aspects of lysosomal storage disorders. We here report the purification of feline liver lysosomal α-mannosidase and determination of its cDNA sequence. The active enzyme consisted of three polypeptides, with molecular masses of 72, 41 and 12 kDa, joined by non-covalent forces. The cDNA sequence of feline lysosomal α-mannosidase was determined from reverse transcriptase PCR products obtained from skin fibroblast mRNA. The deduced amino acid sequence contained the N-terminal sequences of the 72 and 41 kDa peptides. This indicated that the enzyme is synthesized as a single-chain precursor with a putative signal peptide of 50 amino acids followed by a polypeptide chain of 957 amino acids, which is cleaved into the three polypeptides of the mature enzyme. The deduced amino acid sequence was 81.1 and 83.2% identical with the human and bovine lysosomal α-mannosidases sequences respectively. A 4 bp deletion was identified in an α-mannosidosis-affected Persian cat by DNA sequencing of reverse transcriptase PCR products. The deletion resulted in a frame shift from codon 583 and premature termination at codon 645. No lysosomal α-mannosidase activity could be detected in the liver of this cat. A domestic long-haired cat expressing a milder α-mannosidosis phenotype than the Persian cat had a lysosomal α-mannosidase activity of 2% of normal. This domestic long-haired cat did not possess the 4 bp deletion, proving molecular heterogeneity for feline α-mannosidosis.

Molecular Heterogeneity of the L-1 Metallo-β-Lactamase Family from Stenotrophomonas maltophilia

1998 ◽  
Vol 42 (5) ◽  
pp. 1245-1248 ◽  
Author(s):  
François Sanschagrin ◽  
Julien Dufresne ◽  
Roger C. Levesque
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Stenotrophomonas Maltophilia ◽  
Amino Acid Sequences ◽  
Molecular Heterogeneity ◽  
Gene Encoding ◽  
Class B

ABSTRACT We have determined the nucleotide sequence of the blaSgene encoding the carbapenem-hydrolyzing L-1 β-lactamase fromStenotrophomonas maltophilia GN12873. Analysis of the DNA and deduced amino acid sequences identified a product of 290 amino acids. Comparisons of the L-1 amino acid sequence with those of other zinc β-lactamases showed 88.6% identity with the L-1 enzyme fromS. maltophilia IID1275 and less than 20% identity with other class B metalloenzymes.

scholarly journals Determination of Amino Acid Composition of Ganirelix Acetate in an Injectable Formulation by Pre-column Derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate

2020 ◽  
Vol 58 (8) ◽  
pp. 687-694
Author(s):  
Kumarswamy Ummiti ◽  
J V Shanmukha Kumar
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Flash Chromatography ◽  
Molar Ratio ◽  
Acid Mixture ◽  
Injectable Formulation

Abstract Ganirelix is a synthetic decapeptide linked with nine different amino acids. To understand the peptide amino acid sequence or primary structure, the first step is to determine the amino acid composition of the peptide which can be a determining factor for the peptide immunogenicity. Edman degradation is not a suitable analytical technique to identify amino acid sequence present in Ganirelix due to the absence of uncharged N-terminal amino group. To address this challenge, a pre-column derivatization method was developed with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent. In the present work, the Ganirelix active pharmaceutical ingredient present in the injectable formulation was isolated by fraction collection and further purified by flash chromatography. The amino acid composition of Ganirelix is assayed by carrying out acid hydrolysis with 6 mol L−1 hydrochloric acid solution containing 1% phenol at 100°C for 24 h and derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent solution, followed by determination of individual amino acids by reverse-phase chromatography using a C18 column. High resolution was achieved for the nine amino acid mixture. The amino acid composition results of temperature-stressed Ganirelix generic product and reference listed drug are in good agreement with the theoretical molar ratio of label information.

scholarly journals Molecular cloning and characterization of the cDNA coding for C4b-binding protein, a regulatory protein of the classical pathway of the human complement system

1985 ◽  
Vol 230 (1) ◽  
pp. 133-141 ◽  
Author(s):  
L P Chung ◽  
D R Bentley ◽  
K B Reid
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Sequence ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Protein A ◽  
Classical Pathway ◽  
Amino Acid Residues ◽  
Cloned Cdna

By using synthetic oligonucleotides as probes, plasmid clones containing portions of cDNA coding for human C4b-binding protein were isolated from a liver cDNA library. The entire amino acid sequence of the C4b-binding protein can be predicted from this study of the cloned cDNA when allied to a previous sequence study at the protein level [Chung, Gagnon & Reid (1985) Mol. Immunol. 22, 427-435], in which over 55% of the amino acid sequence, including the N-terminal 62 residues, was obtained. The plasmid clones isolated allowed the unambiguous determination of 1717 nucleotides of cDNA sequence between the codon for the 32nd amino acid in the sequence of C4b-binding protein and the 164th nucleotide in the 3′ non-translated region. The sequence studies show that the secreted form of C4b-binding protein, found in plasma, is composed of chains of apparent Mr 70 000 that contains 549 amino acid residues. Examination of the protein and cDNA sequence results show that there are at least two polymorphic sites in the molecule. One is at position 44, which can be glutamine or threonine, and the other is at position 309, which can be tyrosine or histidine. Northern-blot analysis indicated that the mRNA for C4b-binding protein is approx. 2.5 kilobases long. The N-terminal 491 amino acids of C4b-binding protein can be divided into eight internal homologous regions, each approx. 60 amino acids long, which can be aligned by the presence in each region of four half-cystine, one tryptophan and several other conserved residues. These regions in C4b-binding protein are homologous with the three internal-homology regions that have been reported to be present within the Ba region of the complement enzyme factor B and also to the internal-homology regions found in the non-complement beta 2-glycoprotein I.

scholarly journals Phenotypic, Biochemical, and Genetic Analysis of KPC-41, a KPC-3 Variant Conferring Resistance to Ceftazidime-Avibactam and Exhibiting Reduced Carbapenemase Activity

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Linda Mueller ◽  
Amandine Masseron ◽  
Guy Prod’Hom ◽  
Tatiana Galperine ◽  
Gilbert Greub ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Genetic Analysis ◽  
Amino Acid Sequence ◽  
Cloning And Expression ◽  
Content Type ◽  
Amino Acid Insertion

ABSTRACT A novel KPC variant, KPC-41, was identified in a Klebsiella pneumoniae clinical isolate from Switzerland. This β-lactamase possessed a 3-amino-acid insertion (Pro-Asn-Lys) located between amino acids 269 and 270 compared to the KPC-3 amino acid sequence. Cloning and expression of the blaKPC-41 gene in Escherichia coli, followed by determination of MIC values and kinetic parameters, showed that KPC-41, compared to those of KPC-3, has an increased affinity to ceftazidime and a decreased sensitivity to avibactam, leading to resistance to ceftazidime-avibactam once produced in K. pneumoniae. Furthermore, KPC-41 exhibited a drastic decrease of its carbapenemase activity. This report highlights that a diversity of KPC variants conferring resistance to ceftazidime-avibactam already circulate in Europe.

scholarly journals Amino Acid Sequence of a Feather Keratin From Silver Gull (Larus novae-hollandiae) and Comparison with One from Emu (Dromaius novae-hollandiae)

1974 ◽  
Vol 27 (4) ◽  
pp. 369 ◽  
Author(s):  
IJ O'Donnell ◽  
AS Inglis
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Tryptic Digestion ◽  
Crystalline Region ◽  
Feather Keratin ◽  
Matrix Region ◽  
Polypeptide Chains ◽  
Selection Of

The amino acid sequence of the major components of the silver gull feather calamus has been determined and compared with that of the emu. The sequenator was used with a modified Edman-Begg program to facilitate determination of the sequence of the large hydrophobic fragment obtained on tryptic digestion. The main features of the comparison were: (1) the overall structures of the polypeptide chains were similar, having non-crystalline cystine-rich sections towards either end of the chain separated by a large crystalline region of 62 residues which contained the majority of the hydrophobic and serine and glycine residues; (2) approximately one-sixth of the residues were different in the two species, with the majority of changes occurring in the tails (i.e. non-crystalline or matrix region). The data argue for stringent demands in the selection of amino acids for the crystalline part of the feather molecule, a severity that is probably comparable to the strict requirements for the sequence of some of the enzymes.

Amino acid sequence and d/l-configuration determination of peptides utilizing liberated N-terminus phenylthiohydantoin amino acids

1998 ◽  
Vol 813 (2) ◽  
pp. 267-275 ◽  
Author(s):  
Takayuki Iida ◽  
Hirokazu Matsunaga ◽  
Tomofumi Santa ◽  
Takeshi Fukushima ◽  
Hiroshi Homma ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
N Terminus

Amino acid sequence of penicillopepsin. II. Isolation and characterization of thermolytic peptides

1976 ◽  
Vol 54 (10) ◽  
pp. 885-894 ◽  
Author(s):  
Leticia Rao ◽  
Theo Hofmann
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Amino Acid Sequences ◽  
Porcine Pepsin ◽  
Isolation And Characterization

The determination of the amino acid sequences of 70 peptides obtained from a thermoiytic digest of penicillopepsin (EC 3.4.23.7) is described. Fifty-six unique sequences ranging from 2 to 13 amino acids were compiled. Among these was a heptapeptide whose sequence is nearly identical with that of the epoxide-reactive active site peptide of porcine pepsin (EC 3.4.23.1). Considering unrecognized overlaps, a minimum of 272 and a maximum of 293 unique amino acids have been obtained. They account for about 90% of the amino acids of the enzyme.

Sign in / Sign up

Export Citation Format

Share Document