Kinetics of tryptic hydrolysis as a probe of the structure of human plasma apolipoprotein A-II.
As a model system to understand apolipoprotein structure-function and their relationships to proteolytic events, the kinetics of tryptic hydrolysis of apolipoprotein A-II (apo A-II) was investigated in solution and in association with phospholipid. The rates of appearance and identities of specific peptides were determined by reversed-phase high-performance liquid chromatography and amino acid analysis, respectively. For the kinetics of hydrolysis of apo A-II in solution, the carboxyl-terminal peptides of residues 55-77 and 56-77 appeared first, followed by peptides of residues 4-23, 29-39, 40-44 and 45-54, which appeared at nearly identical rates. The kinetics of hydrolysis of apo A-II associated with 1,2-dimyristoyl-sn-glycero-3-phosphocholine showed several differences. First, a 100-fold larger amount of trypsin was needed to obtain a similar rate of product formation; second, a new peptide appeared, eluting earlier than apo A-II but having a similar amino acid composition; and third, the relative rates of appearance of peptides were different. The secondary structure surrounding the bonds susceptible to trypsin cleavage was determined by several predictive algorithms. The lysine amino acid bonds were found to be in regions defined by a high helical amphipathic moment. The reduced susceptibility to tryptic hydrolysis of apo-II associated with phospholipid appears to be due to a higher free energy of stabilization of protein secondary structure. As a consequence, the lysine amino acid bonds are in folded regions of the protein where they are conformationally inaccessible to enzymatic hydrolysis. By use of structure-prediction methods, it is possible to designate which regions of apolipoproteins may be important in proteolysis.
Massey
J B
JB
Department of Medicine, Baylor College of Medicine, Houston, TX.
Hickson-Bick
D L
DL
Gotto
A M
AM
Pownall
H J
HJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D016633:Apolipoprotein A-II; D001054:Apolipoproteins A / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000097:blood*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism*
D000596:Amino Acids; D016633:Apolipoprotein A-II; D001054:Apolipoproteins A; D008075:Lipoproteins, HDL; D010446:Peptide Fragments; D014357:Trypsin