C-TERMINAL MODIFICATIONS OF PERTUSSIS-TOXIN-SENSITIVE G-PROTEIN ALPHA-SUBUNITS DIFFERENTIALLY AFFECT IMMUNOREACTIVITY - EVIDENCE AGAINST ENDOGENOUS ADP-RIBOSYLATION IN HUMAN HEART, LUNG, THROMBOCYTES AND ADIPOSE-TISSUE

Immunochemical detection of pertussis toxin-sensitive guanine-nucleotide binding proteins has been suggested to represent the most direct approach to quantitate the protein than pertussis toxin catalysed [P-32]ADP-ribosylation. The latter technique is potentially hampered by pre-existing covalent modification of the C-terminus. However, limited data exist as to whether and in what way modifications of the C-terminus affect immunoreactivity of Gi alpha (alpha-subunit of the inhibitory G-protein of adenylyl cyclase). Membranes from human myocardium, thrombocytes, adipose tissue and Lung were treated with pertussis toxin or N-ethylmaleimide. Both, conditions prevented high affinity agonist binding to m-cholinoceptors and inhibited [P-32]ADP-ribosylation by pertussis toxin consistent with the notion that the modifications took place at the C-terminus. Pertussis toxin treatment increased immunoreactivity to different antisera raised against the C-terminal decapeptide of transducin alpha(KENLKDCGLF, DS 1-4, AS). N-Ethylmaleimide reduced immunoreactivity towards all antisera studied. Pertussis toxin reduced the mobility of Gi alpha on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) depending on the presence of the toxin and sensitivity to inhibition of ADP-ribosylation by nicotinamide. In native membranes from none of the tissues studied, immunoreactive material comigrating with pertussis toxin-modified form of Gi alpha was detected. It is concluded that modification of the C-terminus by pertussis toxin or N-ethylmaleimide resulting in the same functional consequence, i.e. prevention of high affinity agonist receptor binding, is capable of producing opposite changes of immunoreactivity. Pertussis toxin treatment reduces the electrophoretic mobility on SDS-PAGE. Separation of the native and pertussis toxin-modified form of Gi alpha on SDS-PAGE demonstrates that endogenously ADP-ribosylated Gi alpha is lacking in membranes from human myocardium, thrombocytes, lung and adipose tissue.