Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.32 (bromelain)
 1,025 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human germ cell alkaline phosphatase (GCAP), which shares 98% amino acid sequence identity with the placental AP (PLAP), is expressed by malignant trophoblasts. Protein sequence analysis suggests that the Ser residue at position 92 is the putative active site of GCAP which contains two recognition sequences (Asn122-Thr-Thr124 and Asn249-Arg-Thr251) for asparagine-linked glycosylation. To examine the roles of the Ser residue and glycan moieties on GCAP activity and processing, we altered the GCAP cDNA by site-directed mutagenesis and expressed the GCAP mutants in COS-1 cells. Substitution of Ser-92 with either a Thr (S92T) or an Ala (S92A) residue yielded a GCAP devoid of catalytic activity, suggesting that the Ser codon 92 is the active site of GCAP. Six GCAP mutants that lack one or both glycosylation sites were constructed by substituting either Asn-122 or Asn-249 with an Asp residue or either Thr-124 or Thr-251 with an Ala residue. The mature GCAP migrated as a 65-kDa product, but GCAP mutants lacking one or both glycosylation sites migrated as 62- or 58-kDa polypeptides, respectively, indicating that both sites were glycosylated. All six glycosylated mutants were active enzymatically and, in addition, were equally sensitive to heat, L-leucine, and EDTA inhibition as the parental enzyme. GCAP as well as its two active-site and six glycosylation mutants could be released from the plasma membrane of transfected COS-1 cells by the proteinase bromelain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structural and functional analysis of human germ cell alkaline phosphatase by site-specific mutagenesis. 155 93

A431 human epidermoid carcinoma cells monophenotypically express the placental alkaline phosphatase (PLAP)-like enzyme shown by its catalytic and antigenic characteristics, properties which are shared by the Nagao isozyme. More specifically, it is L-leucine sensitive just as is the rare placental D-variant of PLAP and the testicular heat-stable enzyme. Collectively, these are all referred to as PLAP-like enzymes. The enzyme was localized to the surface of the plasma membrane since it was released in an active form by bromelain treatment of cells. The number of molecules per A431 cell was estimated by radioimmunoassay at 7.5 X 10(5), a value significantly higher than that observed for HeLa TCRC-1 cells (5 X 10(4) which express the S-variant of PLAP, also referred to as the Regan isozyme. The quantity of the enzyme was increased significantly (10-fold) by treating the cells with modulating agents including sodium butyrate, prednisolone, and hyperosmolar sodium chloride. The identification of a cell line such as A431 with enhanced expression in the amount of the PLAP-like enzyme and which can be further enhanced by modulating agents will facilitate studies of the differences and the similarities between this protein and other variants of PLAP. The A431 cell line now takes its place with other cell lines which are phenotypically restricted in their expression of alkaline phosphatase. Finally, the A431 cell line is also shown here to be a suitable model system for in vivo tumor studies such as immunolocalization.
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PMID:Characterization of the placental alkaline phosphatase-like (Nagao) isozyme on the surface of A431 human epidermoid carcinoma cells. 257 98

L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA)--a convenient chromogenic substrate for assay of thiol proteinases papain, ficin, and bromelain--was prepared by enzymatic synthesis with chymotrypsin as a catalyst. The thiol proteinases hydrolyze PFLNA with the liberation of p-nitroaniline, estimated spectrophotometrically by its absorbance at 410 nm. The phenylalanine residue in the P2 position of PFLNA meets the specificity demands of thiol proteinases. The following values of Km were found for PFLNA hydrolysis: by papain, 0.34 mM; by ficin, 0.43 mM; by bromelain, 0.30 mM. This substrate was successfully applied to monitor thiol proteinase affinity chromatography on bacitracin-Sepharose, which resulted in a 2- to 4-fold purification from commercial preparations.
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PMID:L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide--a chromogenic substrate for thiol proteinase assay. 639 97

Two component random copolypeptide membranes, consisting of N-hydroxyalkyl L-glutamine and L-alanine or L-leucine were prepared by carrying out aminolysis reactions with 2-amino-1-ethanol (E) or 5-amino-1-pentanol (Pe), together with a cross-linking reaction with 1,8-octamethylenediamine (OMDA) on membranes of the starting copolymers consisting of gamma-benzyl L-glutamate (B) and L-alanine (A) or L-leucine (L). The relationships between their bulk structure and membrane properties were investigated, such as the swelling ratio in water, aqueous vapour permeability, tensile properties and enzymatic degradation behaviour of the membranes in a pseudo-extracellular fluid (PECF). The tensile properties of the hydrophilic membranes were highly dependent on the swelling ratio of PECF, and the hydrophobicity of the side chains, whose behaviour was typical of an elastomer. We showed that a common relation was obtained between the rate of water vapour permeability and the swelling ratio of membranes in PECF despite the difference of the nature of the side chains. Biodegradation of these membranes in vitro by bromelain indicated that the degradation was a bulk rather than a surface phenomenon, and that the rate of degradation was also highly dependent on the swelling ratio of samples and on the hydrophobicity of the side chains of samples.
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PMID:Biodegradation of random copolypeptide membranes consisting of N-hydroxyalkyl L-glutamine as one component. 832 21

Two component random copolypeptide hydrogels consisting of N-hydroxyalkyl L-glutamine and L-leucine were prepared by carrying out aminolysis reactions with 3-amino-1-propanol(P) together with cross-linking reactors with 1,8-octamethylenediamine (OMDA) on hydrogels of the starting copolymers consisting of gamma-methyl-L-glutamate(M) and L-leucine(L). The relation between their bulk structure and properties was investigated with regard to the swelling ration in water, aqueous vapor permeability, tensile properties, and enzymatic degradation behavior in a pseudoextracellular fluid (PECF). The tensile property of the hydrogels was highly dependent on the swelling ratio in PECF, and on the hydrophobicity of the side chains, whose behavior was typical of an elastomer. It was shown that a common relation was obtained between the rate of water vapor permeabilities and the swelling ratio of hydrogels in PECF regardless of the difference of the nature of side chains. Biodegradation of the hydrogels in vitro by bromelain indicated that the degradation took place in bulk rather than on surface, and that the rate of degradation was also highly dependent on the swelling ratio of samples as well as on the hydrophobicity of the side chains of samples.
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PMID:Enzymatic hydrolysis of copoly(N-hydroxypropyl-L-glutamine/L-leucine) hydrogels in vitro. 877 84