ABSTRACT
Molting is an important physiological phenomenon of many metamorphosis insects, during which the old and new epidermis are separated by enzymes present in the molting fluid. Various proteomic studies have discovered the presence of Bombyx mori carboxypeptidase A (Bm-CPA) in the molting fluid of silkworm, but its function remains unclear. In order to better understand the role of Bm-CPA in the molting process of silkworm, Bm-CPA was analyzed by bioinformatics analysis, real-time fluorescence quantitative PCR, antibody preparation, immunofluorescence staining, and expression in Pichia pastoris. The results showed that Bm-CPA had a conserved M14 zinc carboxypeptidase domain and glycosylation site. Its expression was regulated by ecdysone 20E, and large expression was observed in the epidermis of the upper cluster stage. Immunofluorescence staining showed that Bm-CPA was enriched in the epidermis during the molting stage, and the inhibitor of Bm-CPA led to the larval death due to the inability to molt. We also successfully obtained a large number of recombinant Bm-CPA proteins by Pichia pastoris expression in vitro. These results may facilitate further understanding the molting development process of silkworm.
Subject(s)
Animals , Molting/genetics , Bombyx/genetics , Carboxypeptidases A/metabolism , Proteomics , Larva/metabolism , Fluorescent Antibody Technique , Insect Proteins/metabolismABSTRACT
Objective To investigate the diagnostic significance of basophil activation test (B A T) in ana-phylaxis to non-ionic contrast media through testing the content of CD 63, m ast cell-carboxypeptidase A 3 (M C-CPA 3), and term inal com plem ent com plex SC5b-9 of the individuals by testing their levels in the norm al im m une group and the anaphylaxis groups to β-lactam drugs and non-ionic contrast media. Methods The CD 63 expression of basophilic granulocyte in blood w as detected by flow cytom etry. The levels of M C-CPA 3 in blood serum and SC5b-9 in blood plasm a w ere detected by ELISA . Results The CD 63 expression of basophilic granulocyte in blood, the levels of M C-CPA 3 and SC5b-9 of anaphylaxis to non-ionic contrast media and β-lactam drugs w ere significantly higher than that in norm al im m une group (P<0.05). Conclusion There is activation of basophilic granulocytes, m ast cells and com plem ent system in anaphylaxis to non-ionic contrast media. B A Tcan be used to diagnose the anaphylaxis to non-ionic contrast media.
ABSTRACT
Objective: To clone human carboxypeptidase A1 and its active center gene as well as construct recombinant vector for expression before analysis their activities. Methods: CPA1 and CPAlactive center gene were amplified by RT-PCR from pancreas tissue, and then sequencing was carried out. The correct target genes were cloned into prokaryotic vector pGEX-4T-1 and transformed into E. coli BL21 before sequence analysis. After induced by IPTG, gene products were analyzed by SDS-PAGE. Target expressed proteins were denaturated, renaturated, purified and evaluated through MTT and agar colony form test. Results: Human carboxypeptidase Al and its active center gene were cloned successfully. New expected protein band of Mr 66,000 and 46,000 appeared on SDS-PAGE after inducement. Both of the expressed proteins have catalytic activity in vitro, but the activity of the latter is inferior when applied to tumor cells. Conclusions: Human carboxypeptidase A1 and its active center gene were cloned successfully. Their prokaryotic expression products were obtained too. The expressed proteins have catalytic activity in. vitro. A new prosperous beginning of further improvement for CPA1 therapy system has been established based on CPA1 and its active center gene in terms of ADEPT against prostate cancer to clinical application.
ABSTRACT
Studies with substrate analogues and the pH optimum indicated the involvement of carboxyl group in the active site of goat carboxypeptidase A. Chemical modification of the enzyme with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide methoI -p-toluene sulphonate, a carboxyl specific reagent, led to loss of both esterase and peptidase activities. Protection studies showed that this carboxyl group was in the active site and was protected by ßphenylpropionic acid and glycyl-L-tyrosine. Kinetic studies also confirmed the involvement of carboxylic group because the enzyme modification with water soluble carbodiimide was a two step reaction which excluded the possibility of tyrosine or lysine which are known to give a one step reaction with this reagent.
ABSTRACT
The two forms of buffalo carboxypeptidase A lose a part of their activity when incubated at their optimal temperature. Both the forms are protected from heat denaturation by L-phenylalanine, one of the reaction products while the other reaction product hippuric acid provides only limited protection. It has also been shown that L-phenylalanine and hippuric acid bind at the same site of the enzyme and that the release of L-phenylalanine follows that of hippuric acid. On this basis, a new mechanism for the hydrolysis of acyldipeptides by carboxypeptidase A has been proposed.
ABSTRACT
(Objective)To express the human carboxypeptidase A1 in soluble form in pichia pastoris.(Methods)Human carboxypeptidase A1 gene was amplified from pGEX-CPA1 recombinant plasmid and subcloned into pPIC9K yeast expression vector.Recombinant vector pPIC9K-CPA1 was transformed into pichia SMD1168 by electroporation.Positive recombinant yeast strains were screened through nutrition defect and G418 resistance.Expression of the integrated multicopy recombinant yeast strains were induced.Results The yeast recombinant expression vector pPIC9K-CPA1 was constructed.Recombinant yeast strains which integrated over 16 recombinant vector copies and soluble human carboxypeptidase A1 protein were obtained.(Conclusion)The human carboxypeptidase A1 can be expressed successfully in pichia pastoris,which will be useful for further research on ADEPT of prospate.