Monoclonal antibodies against 5‘-nucleotidase from chicken gizzard. Evidence for species and tissue specific differences of 5‘-nucleotidase.

J Dieckhoff,R Lietzke,H G Mannherz

doi:10.1016/s0021-9258(17)36184-7

Abstract

5'-Nucleotidase from chicken gizzard smooth muscle was purified to homogeneity and used as immunogen for generating monoclonal antibodies. From about 150 positive clones nine IgG producing hybridoma cell lines have been selected for further characterization and antibody preparation. The resulting antibodies bind 5'-nucleotidase from chicken smooth muscle, chicken skeletal muscle, and chicken heart muscle but not the enzyme from chicken liver or rat liver. It could clearly be demonstrated that the nine antibodies recognize different antigenic determinants. Four of these antibodies are strong inhibitors of the AMPase activity of 5'-nucleotidase. One antibody is a weak inhibitor and four other antibodies have no effect on its enzymic activity. One of the monoclonal antibodies was used for immunoaffinity purification of 5'-nucleotidase from chicken heart muscle and chicken skeletal muscle. Pure and active enzymes could be isolated from detergent extracts in one step with a 10 to 20-fold higher yield compared to classical purification procedures. The subcellular distribution of 5'-nucleotidase in chicken gizzard was investigated using indirect immunofluorescence. We found a staining of the plasma membrane of smooth muscle cells and endothelial cells by all of the nine antibodies with variations in the staining intensity.

Highlights

5’-Nucleotidase from chicken gizzard smooth muscle lectins or lectin-like proteins on the outside of the plasma was purified to homogeneity and used as immunogen membrane [12]
From about150 In order to study the interaction of 5“nucleotidase with positive clones nine IgG producinghybridomacell lines actin we have isolated the enzymefrom chicken gizzard have been selected for further characterization and antibody preparation
It couldclearly be demonstrated that the nine antibodies recognize different antigenic determinants. Four of these antibodies are strong inhibitors of the AMPase activity of smooth muscle, chicken skeletal and heart muscle, rat liver, rat bile and snake venom to homogeneity [9,10, 13, 14]. Their ability to interact with filamentous (F-)actin was compared by analysis of the ability of F-actin to inhibit their AMPase activity

Summary

DISCUSSION

Polyclonal antibodies against 5’-nucleotidase have proven to be veryuseful for (i) demonstrating tissusepecific diversity of 5’-nucleotidase in different organs [29], (ii) studying the subcellular distribution of this enzyme by immunofluorescence [29, 30] or immunoelectron microscopy [17], (iii) detecting traces of this enzyme in complex protein mixtures, and (iv) defining the transmembrane character of this glycoprotein [31]. The following results indicate that the nine antibodies are directed against different epitopes on the enzyme: (a) the antibodies inhibit the AMPase activity of 5“nucleotidase to different degrees; ( b ) antibodies whichhave only slightly different effects on 5’-nucleotidase activity show variations in the stainingof plasma membranes of smooth muscle and endothelial cells on cryostat sections; (c) if 5”nucleotidase is sequentially incubated with a non-inhibiting and an inhibiting antibody, insome cases either a partial ocromplete reduction of the inhibitory capacity of inhibiting antibodies was observed.

Methods

EXPERIMENTAL PROCEDURES

RESULTS

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