Abstract
The state of self-association of the apoprotein components of human high density lipoprotein have been studied by use of the cross-linking reagent dimethyl-suberimidate. Analusis of the cross-linked products was carried out by soduim dodecyl sulfate-gel electrophoresis and by agarose column chromatography in 6 M guanidine hydrochloride. Apo-A-I was found to exist as a monomer at low concentration, but associates to tetrameric and pentameric forms at concentrations of 0.5 mg/ml or higher. The self-association was found to be ionic strength-dependent, with association promoted by the presence of salt. Apo-A-II was also found to associate, but the major oligomeric form observed was dimeric (Mr = 34,000), and the association was less dependent on ionic strength than for apo-A-I. Cross-linking in the presence of various concentrations of guanidine hydrochloride showed that apo-A-II self-association persisted at higher concentrations of the denaturant than for apo-A-I. Studies of the effect of temperature demonstrated that the self-association of both proteins was diminished at temperatures above 30 degrees C. Recombination of apo-A-II with phospholipid resulted in the formation of particles which yielded primarily trimers upon cross-linking. This suggests that phospholipid binding causes major reorganization of the self-associated forms of apo-A-II.
Highlights
We have recently reported preliminary studiesof the quaternary structure of apo-A-I and apo-A-II by chemicalcrosslinking with the reagent dimethylsuberimidate [16], as contrasted to the physical methods used in other laboratories
We have identified the apo-A-I
The cross-linking approach to studies of apo-A-I and apoA-II self-association represents an alternative to the physical techniques which have been applied to this problem by others [3, 5, 6, 10, 11]
Summary
Prior to cross-linking, proteins were freshly renatured by the addition reports are in disagreementas to whether this apoprotein of a 6 M guanidine solution, followed by exhaustive dialysis against existsasan equilibrium mixture of monomersand dimers[3], 0.15 M NaCl or water. In studies of the effect of varying ionic strength on self-association, the stock protein solution was mixed with an equal volume of 0 to 4 M NaCl. Cross-linking was effected by the addition of 1 part DMS, 10 mg/ml in 0.3 M triethanolamine (to reduce the contribution to the ionic strength by the buffer), to 10 parts protein solution. Molecular weight standards (y-globulin, human serum albumin) were tagged with a small amount of [‘?]succinic anhydride and cross-linked with dimethylsuberimidate (2 mg/ml) at a protein concentration of 10 mg/ml using the method already described. The elution position of marker proteins was determined by counting radioactivity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
