Inhibition of Alkaline Phosphatase from Pearl Oyster Pinctada fucata by o-Phthalaldehyde： Involvement of Lysine and Histidine Residues at the Active Site

Alkaline phosphatase from Pinctada fucata was inactivated by o-phthalaldehyde （OPA）. The inactivation followed pseudo first-order kinetics with a second rate constant of 0.167 （mmol/L）^-1·min^-1 at pH 7.5 and 25℃. A Tsou＇s plot analysis showed that inactivation occurred upon formation of one isoindole group. The OPA-modified enzyme lost the ability to bind with the specific affinity column and the presence of substrates or competitive inhibitors protected the enzyme from inactivation. The results revealed that the OPA-reaction site was at the enzyme substrate binding site. Prior modification of the enzyme by lysine or histidine specific reagent abolished formation of the isoindole derivatives, suggesting that lysine and histidine residues were involved in the OPA-induced inactivation. Taken together, OPA inactivated the alkaline phosphatase from Pinctada fucata by cross-linking lysine and histidine residues at the active site and formed an isoindole group at the substrate binding site of the enzyme.     

Cloning, Characterization, and Expression Analysis of Calreticulin from Pearl Oyster Pinctada fucata

Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sar-coplasmic/endoplasmic reticulum.A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pead oyster.This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata,namely PCRT.PCRT encodes a deduced 414-amino acid protein,which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL.The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species.Semi-quantitative RT-PCR indicates that PCRT is ubiquitously ex-pressed in all tissues tested with the highest expression in the hemolymph and the mantle.In situ hybridiza-tion analysis of PCRT in the mantle showed strong signals in the inner fold,the inner side of middle fold,and the inner side of outer fold of the mantle epithelium.All these results suggest PCRT might be involved in Ca2+ transport and storage during oyster biomineralization.     

Localization of calmodulin and calmodulin-like protein and their functions in biomineralization in P. fucata

Calmodulin (CaM) and calmodulin-like protein (CaLP) are two proteins involved in biomineralization. Their localizations in Pinctada fucata mantle epithelia were studied by Western blot (WB) analysis of the nuclear/cytosol fraction of primary cultured Pinctada fucata mantle cells and immunogold electron microscopy. The results showed a completely different distribution of these two proteins at the subcellular level. CaM was distributed throughout both the nucleus and cytoplasm of the mantle epithelium but CaLP was distributed only in the cytoplasm. The functions of these two proteins in biomineralization were investigated by shell regeneration. During this process, the expressions of CaM and CaLP were greatly enhanced in different organelles of the mantle epithelium. Overexpression of these two proteins and a mutant of calmodulin-like protein (M-CaLP) that lacks an extra C-terminal tail in MC3T3-E1 promoted the mRNA expression of osteopontin, a biomineralization marker for osteoblasts. All of the results indicated that CaM and CaLP have completely different distributions in the mantle epithelium and affect the biomineralization process at different levels. The extra C-terminal tail of CaLP is important for its functions in biomineralization in Pinctada fucata.     

Extraction and Purification of Matrix Protein from the Nacre of Pearl Oyster Pinctada fucata

A soluble matrix protein P14 with an apparent molecular mass of 14.5 kDa was isolated from fragmented nacre of pearl oysters （Pinctada fucata） treated with 10% NaOH solution to investigate the nacre matrix proteins and their effect on the CaCO3 crystal. The protein was characterized by gel exclusion chromatography and reversed-phase high performance liquid chromatography after demineralization by 10% acetic acid. The X-ray diffraction pattern of P14 crystals indicates that P14 plays an important role in nacre biomineralization. P14 can induce aragonite formation, stimulate CaCO3 crystal formation, and accelerate aragonite precipitation. Heating of the acid insoluble nacre residue, which was named conchiolin, in 10% sodium dodecyl sulfate solution supplemented with 10% β-mercaptoethanol solution for 10-20 min at about 100℃ gave two other soluble proteins having molecular masses of 19.4 kDa and 25.0 kDa. The present study suggests that these two proteins are linked to the insoluble organic matrix by disulfide bridges because the extraction yield increases when β-mercaptoethanol is added to the medium.     

Partial Purification and Properties of an Acid Phosphatase from Pearl Oyster Pinctada Fucata

IntroductionAcid phosphatase ( ACP,EC 3.1 .3.2 ) andalkaline phosphatase ( ALP,EC 3.1 .3.1 ) havebeen found to exist widely in the biosphere frombacteria to plants,animals and humans[18] .Thetwo enzymes catalyze the hydrolysis of variousphosphate containing compounds and act astransphosphorylases at acid- and alkaline- p Hvalue.In our lab,an alkaline phosphatase fromthe pearl oyster Pinctada fucata has been purifiedand some of its kinetics has been studied[9] .　Acid phosphatases actas ma…     

EDTA对合浦珠母贝碱性磷酸酶活性的影响

以合浦珠母贝(Pinctada fucata)为实验材料,从其外套膜中分离提取碱性磷酸酶(ALP),分别用不同浓度的EDTA不同时间处理ALP,发现随着EDTA浓度的增大,酶的活力指数下降至完全消失,可见酶分子在脱除金属辅基后,活力完全丧失,它是一种金属酶,对酶活性中心金属离子替换实验中。发现分别加入Mg^2 、Mn^2 、Co^2 、Zn^2 后,酶活力可得到一定程度的恢复,而同时加入Mg^2 、Zn^2 ,可使酶活力恢复到92％．     

Localization of calmodulin and calmodulin-like protein and their functions in biomineralization in P. fucata

Calmodulin （CAM） and calmodulin-like protein （CaLP） are two proteins involved in biomineralization. Their localizations in Pinctada fucata mantle epithelia were studied by Western blot （WB） analysis of the nuclear/cytosol fraction of primary cultured P. fucata mantle cells and immunogold electron microscopy. The results showed a completely different distribution of these two proteins at the subcellular level. CaM was distributed throughout both the nucleus and cytoplasm of the mantle epithelium but CaLP was distributed only in the cytoplasm. The functions of these two proteins in biomineralization were investigated by shell regeneration. During this pro- cess, the expressions of CaM and CaLP were greatly enhanced in different organelles of the mantle epithelium. Overexpression of these two proteins and a mutant of calmodulin-like protein （M-CaLP） that lacks an extra C-terminal tail in MC3T3-E1 promoted the mRNA expression of osteopontin, a biomineralization marker for osteoblasts. All of the results indicated that CaM and CaLP have completely different distributions in the mantle epithelium and affect the biomineralization process at different levels. The extra C-terminal tail of CaLP is important for its functions in biomineralization in P. fucata.