Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.1 (asparaginase)
 2,695 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glycosylasparaginase (EC 3.5.1.26) from Sf9 cells (Spodoptera frugiperda) was purified to homogeneity with a specific activity of 2.1 unit/mg. The enzyme is composed of two non-identical alpha/beta subunits joined by strong non-covalent forces and has one glycosylation site located in the alpha subunit. Molecular masses of the subunits were determined to be 28 kDa and 17 kDa by SDS-PAGE. Native enzyme existed in quaternary structures of either heterodimer (alpha beta) or heterotetramer (alpha 2 beta 2). These forms exhibited different ionic characteristics during DE52 anion exchange chromatography, and their molecular masses were determined to be 47 kDa and 101 kDa by gel filtration. The enzyme was thermostable, requiring 65-70 degrees C to be denatured, and it had a broad pH optimum from 4-10.5 with a pKa around 6. SDS easily inactivated the enzyme. The K(m) of glycosylasparaginase for its normal substrate GlcNAc-Asn was 0.88 mM. The enzyme also exhibited asparaginase activity with a K(m) of 3.0 mM for asparagine. N-terminal amino acids of the denatured subunits were sequenced and degenerate primers were designed for cloning its cDNA using PCR and 5' and 3' RACE. Glycosylasparaginase cDNAs from bovine and rat were also cloned using similar strategies, and primary structures of glycosylasparaginases from six species (human, bovine, rat, mouse, Sf9 cells and Flavobacterium) have been compared and related to a recent crystal structure of the human enzyme.
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PMID:Purification, biochemistry and molecular cloning of an insect glycosylasparaginase from Spodoptera frugiperda. 887 73

A novel asparaginase-like protein (ALP) of spermatozoa was cloned from rat and human testis cDNA libraries on the basis of reactivity with antibodies produced after vasectomy. Although obstruction of the male reproductive tract is known to cause an immunologic response, few of the sperm antigens responsible for the generation of autoantibodies have been characterized. We are identifying proteins of interest by coring autoantigenic protein spots from two-dimensional (2-D) gels of rat sperm extracts and microsequencing them by mass spectrometry. The peptide sequences from ALP, a 28 kDa, pI 5.7 protein, matched to a single partial length rat EST. These peptide sequences were used to clone a cDNA encoding a novel 333 amino acid open reading frame. The new protein had a similarity to portions of L-asparaginases of plants (43%) and to glycosylasparaginases in animal cells (32%). Human ALP cDNA was subsequently cloned. It showed 77% identity to the rat ALP sequence and the gene, ASRGL1 (asparaginase-like 1), mapped to chromosome locus 11q12.3. Purified recombinant rat ALP (rALP), expressed in E. coli, was used to raise polyclonal antiserum in guinea pigs. Two observations verified that the correct protein had been cloned: 1) the anti-rALP antibody reacted with both rALP and rat sperm; and 2) post-vasectomy sera bound rALP. Anti-rALP antibody stained the midpiece of rat and human sperm coincident with staining by MitoTracker Green FM, suggesting that ALP is associated with the mitochondria. Northern analysis revealed that rat ALP message was abundantly expressed in the testis but was also present in heart, brain, liver, skeletal muscle, and kidney.
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PMID:A novel asparaginase-like protein is a sperm autoantigen in rats. 1198 34