The Core-membrane Linker Polypeptide Analysis Of The Phycobilisome From Polysiphonia Urceolata Grev

The marine red macroalga of Polysiphoia urceolata was used as experimental material to extract phycobilisomes?PBSs?in high salt solution,and then the intact PBSs were isolated and prepared by ultracentrifugation on a sucrose density gradient.After sub-ultracentrifugation,the PBS fractions from the extracted PBS solutions with and without Triton X-100 solubilization were situated in 0.2 mol/L sucrose band,and the 0.2mol/L PBS fractions of the two extracted PBSs were both proved to be intact PBSs on the basis of their absorption and fluoresce spectral properties.In contrast to the intact PBSs prepared from the extracted PBSs with Triton X-100 solubilization?NTM-PBSs?,those from the extracted PBSs without Triton X-100 solubilization?TM-PBSs?displayed the light absorption and fluorescence properties of chlorophyll a-protein complexes,demonstrating that TM-PBSs carried the Chl a-protein complexes of thylakoid membranes.By ultracentrifugation on a sucrose density gradient,TM-PBSs and NTM-PBSs from the sub-ultracentrifugation were both further isolated as the two relatively stable PBS fractions:one fraction with larger content and lower density was situated in 0.8 mol/L sucrose layer,TM-PBS 0.8L and NTM-PBS 0.8L;and the other with smaller content and higher density was in 1.2 mol/L sucrose layer,TM-PBS 1.2L and NTM-PBS 1.2L.The results from SDS-PAGE analysis of TM-PBSs and NTM-PBSs showed that two protein polypeptides which conform to the structural characteristics of the reported core-membrane linker polypeptide(L_CM)in phycobilisomes were identified,and the molecular mass of the two protein polypeptides were 86.0 kDa and 98.9 kDa,respectively.In addition,when the PBSs carrying thylakoid membranes?TM-PBSs?were dissociated,the two proteins of ^98.9L_CMM and ^86.0L_CMM showed different fate:^98.9L_CMM seemed to bind strongly to the thylakoid membrane and to tend relatively to enter the thylakoid membrane sample fraction,whereas^86.0L_CMM seemed to bind weakly to thylakoid membrane and to tend relatively to dissolve in the phycobiliprotein sample fraction.The results of 2-D SDS-PAGE analysis showed that ^98.9L_CMM and ^86.0L_CMM carried no other colored subunits of PBSs,demonstrating that they were two kinds of L_CMM with different molecular mass.The analysis of urea-denatured isoelectric focusing?Urea-IEF?showed that the isoelectric points?pIs?of ^86.0L_CMM and ^98.9L_CMM were within pH 6.0⁵.0 and pH 7.0⁷.5,respectively;furthermore the second dimensional SDS-PAGE also demonstrated that the phycocyanin-carried colored^98.9L_CMM with weak basic pI??7.25?matched mostly with the predominant structural properties of the reported core-membrane linker polypeptide L_CM?basic pI,carrying PCB and having molecular mass within 80 kDa¹20 kDa?,^98.9L_C⁷.² _M⁵.However,it needs to be demonstrated via further experiments whether the effects from the phycobiliprotein subunits with acidic pIs on ^86.0L_CMM make it exhibit weak acidic pI??5.35?.In the Blue-native PAGE and hrClear-native PAGE gel electrophoresis,protein bands that may be AP-L_CMM complexes were observed.However,whether the observed complexes are composed of ^98.9L_CM,^86.0L_CMM and the?and?subunits of AP needs to be further determined by 2-D SDS-PAGE.