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Failure of [32P]ADP-ribosylation by pertussis toxin to determine Gi alpha content in membranes from various human tissues. Improved radioimmunological quantification using the 125I-labelled C-terminal decapeptide of retinal transducin.

ABSTRACT: The quantitative determination of pertussis-toxin-sensitive guanine-nucleotide-binding proteins (G-proteins) in cell membranes is still a problem. Pertussis-toxin-catalysed [32P]ADP-ribosylation strongly relies on the substrate quality of the alpha-subunits and is influenced by the concentration of nucleotides, beta gamma-subunits, the physicochemical properties of the membranes influencing the availability of Gi alpha for pertussis toxin, and covalent modification of Gi alpha. Quantification of immunoreactive material on Western blots can be only imprecisely performed by two-dimensional densitometry. In order to generate a method for quantification of pertussis-toxin-sensitive G-proteins in membranes we have developed a fast and sensitive radioimmunoassay. The C-terminal decapeptide of retinal transducin alpha (KENLKDCGLF) was 125I-labelled and used as tracer. Polyclonal antiserum (DS 4) was raised against this peptide. Gi alpha proteins were determined by competition of solubilized membranes for 125I-KENLKDCGLF binding to DS 4 using dilutions of retinal transducin alpha as standard. The interassay variation was less than 10%, with a sensitivity of 2.5 micrograms/ml. The density of Gi alpha was highest in human adipose tissue, followed by HL60 cells, lung, mononuclear leucocytes, thrombocytes and left ventricular myocardium. A striking difference was observed between the density of Gi alpha and the amount of incorporation of [32P]ADP-ribose into the 40 kDa membrane proteins by pertussis toxin in the same samples. This is also demonstrated by comparison of the weak [32P]ATP-ribosylation of pertussis toxin substrates with the density of immunoreactive Gi alpha on Western blots in tissues such as lung. This study shows that the Gi alpha content can be exactly determined by a sensitive and fast radioimmunoassay using iodinated synthetic peptide homologues of Gi alpha proteins. Radioimmunological quantification of Gi alpha might be able to detect the 'true' Gi alpha content of membranes without being hampered by influences on the [32P]ADP-ribosylation reaction. It is concluded that this newly developed method may become an important tool for studying expression of Gi alpha proteins in a variety of tissues or cell types, and for precisely quantifying the changes caused by pathological conditions.

SUBMITTER: Bohm M

PROVIDER: S-EPMC1151213 | biostudies-other | 1991 Jul

REPOSITORIES: biostudies-other

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Project description:1. The incorporation in vitro of [(32)P]phosphate into phospholipids and RNA and of [(125)I]iodide into protein-bound iodine by pig thyroid slices incubated for up to 6hr. was studied. The subcellular distribution of the labelled products formed after incubation with radioactive precursor in the nuclear, mitochondrial, smooth-microsomal, rough-microsomal and cell-sap fractions was also studied. 2. Pig thyroid slices actively took up [(32)P]phosphate from the medium during 6hr. of incubation; the rate of incorporation of (32)P into phospholipids was two to five times that into RNA. 3. The uptake of [(125)I]iodide by the slices from the medium was rapid for 4hr. of incubation, 6-10% of the label being incorporated into iodoprotein. 4. Much of the (32)P-labelled phospholipid accumulated in mitochondria and microsomes, whereas the nuclear fraction contained most of the (32)P-labelled RNA. After 2hr. of incubation most of the (32)P-labelled cytoplasmic RNA accumulated in the rough-microsomal fraction. The major site of localization of proteinbound (125)I was the smooth-microsomal fraction, and gradually increasing amounts appeared in the soluble cytoplasm fraction, suggesting a vectorial discharge of [(125)I]iodoprotein (presumably thyroglobulin) from smooth vesicles into the colloid. 5. The addition of 0.1-0.4 unit of thyrotrophic hormone/ml. of incubation medium markedly enhanced the accumulation of (32)P-labelled phospholipids in the microsomal fractions and to a much smaller extent that of (32)P-labelled RNA without any increase in the total uptake of the label. Almost simultaneously the hormone increased the uptake of [(125)I]iodide by the slices and enhanced the accumulation of protein-bound (125)I in the smooth-microsomal fraction. 6. As a function of time of incubation, thyrotrophic hormone had a biphasic effect on [(125)I]iodide uptake and protein-bound (125)I formation, the stimulatory effect being reversed after 4hr. of incubation. 7. 6-N-2'-O-Dibutyryl-3',5'-(cyclic)-AMP, but not 3',5'-(cyclic)-AMP or 5'-AMP, mimicked the action of thyrotrophic hormone on iodine uptake as well as on iodination of protein. On the other hand, the mimicry by 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP of the stimulatory effect of thyrotrophic hormone on the formation of labelled thyroid phospholipids and RNA was only an apparent one resulting from an enhanced uptake of [(32)P]phosphate. 8. It is concluded that thyrotrophic hormone causes a co-ordinated increase in the formation or accumulation of phospholipids, RNA and iodoprotein associated with the endoplasmic reticulum, and that 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP mimics the more rapid effects of thyrotrophic hormone on transport and metabolic functions of thyroid cells, but does not influence their slower biosynthetic responses to the hormone.

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Failure of [32P]ADP-ribosylation by pertussis toxin to determine Gi alpha content in membranes from various human tissues. Improved radioimmunological quantification using the 125I-labelled C-terminal decapeptide of retinal transducin.

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