Immunological studies on pancreatic phospholipase A2. Antigenic characterization of the NH2-terminal region.

H Meijer,M.J Meddens,R Dijkman,A.J Slotboom,G.H De Haas

doi:10.1016/s0021-9258(17)34329-6

Abstract

Rabbit antisera elicited against pure pig, horse, ox, and sheep pancreatic phospholipase A2 revealed considerable immunological differences when tested by double immunodiffusion and microcomplement fixation assays. Snake venom phospholipases did not show any detectable cross-reactions with the pancreatic enzymes. Microcomplement fixation also clearly demonstrated conformational differences between porcine phospholipase A2 and its zymogen. NH2 terminally modified analogs of porcine phospholipase A2 could be clearly distinguished using the same assay. Moreover, strong evidence was obtained that Ala1-Arg6 is a part of an antigenic determinant. Radioimmune assay, using monovalent phospholipase-specific Fab fragments revealed a maximum number of three antigenic sites of phospholipase that can simultaneously be occupied by antibody. The Fab fragments were separated into three fractions, using three immunoadsorbent columns in series. These Fab fractions showed different inhibitory properties toward micellar binding of phospholipase A2. They also exhibited different protective effects against active center modification.

Highlights

Microcomplement strated conformational fixation clearly demondifferences between porcine phospholipase A2 and its zymogen
The complement fixation of this protein is dramatically decreased. These results provide additional evidence that besides the presumed Ala, salt bridge, the other NHz-terminal residues contribute to the ultimate conformation of amidinated phospholipase AZ (AMPA) or phospholipase
The Difference between the Various Phospholipases-The results obtained with the immunological comparison of the phospholipases AS from horse, pig, cow, and sheep pancreas indicate a generally moderate rate of molecular evolution of these enzymes

Summary

Introduction

AZ revealed considerable immunological differences when tested by double immunodiffusion and microcomplement fixation assays. Microcomplement strated conformational fixation clearly demondifferences between porcine phospholipase A2 and its zymogen. F,b fragments revealed a maximum number of three antigenic sites of phospholipase that can simultaneously be occupied by antibody. The F,b fragments were separated into three fractions, using three immunoadsorbent columns in series. These Fab fractions showed different inhibitory properties toward micellar binding of phospholipase. Ca” is required as an absolute cofactor [2] and the activity of the enzyme is greatly enhanced when the substrate passes from a monomeric to an aggregated state [3,4,5]

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Conclusion