Abstract
Subcellular fractions were prepared from human neutrophils by sucrose density gradient centrifugation and analyzed for Gi-like proteins by pertussis toxin-catalyzed [32P]ADP-ribosylation and by immunoblotting with rabbit antiserum AS/6 which recognizes purified transducin and Gi, but not Gs or Go alpha-subunits. In resting cells, approximately equal to 60% of pertussis toxin substrate retrieved from the sucrose density gradient localized to the plasma membrane-enriched fraction, approximately equal to 35% to the specific granule-enriched fraction, and approximately equal to 5% to cytosol. The azurophil granule-enriched fraction did not contain pertussis toxin substrate. In contrast to plasma membrane, the specific granule-enriched fraction demonstrated increased AS/6 immunoreactivity of a approximately equal to 41-kDa protein relative to a approximately equal to 40-kDa protein. Within the specific granule-enriched fraction, the peak of pertussis toxin substrate detected immunochemically or by [32P]ADP-ribosylation sedimented at a lighter density (rho = 1.6 g/ml) than did lactoferrin (rho = 1.19 g/ml), suggesting that the intracellular compartment bearing pertussis toxin substrate may not be the lactoferrin containing specific granule, per se. Furthermore, in neutrophils exposed to 10(-8) M N-formylmethionylleucylphenylalanine, a weak degranulating stimulus (7% lactoferrin degranulation), there was a 31-42% decline in pertussus toxin-catalyzed [32P]ADP-ribosylation of approximately equal to 40-41-kDa proteins in the specific granule-enriched fraction accompanied by a near-quantitative increase in labeling of plasma membrane. The pool of intracellular formyl peptide receptors localized to the specific granule-enriched fraction appeared functionally coupled to a cosedimenting G-protein in experiments demonstrating modulation of high affinity N-formylmethionylleucyl[3H]phenylalanine binding by guanosine 5'-(3-O-thio)triphosphate or pertussis toxin. The data indicate that neutrophils contain a surface translocatable pool of intracellular G-protein sedimenting in the specific granule-enriched fraction and support the view that mobilization of intracellular G-protein represents a mechanism by which cells can regulate receptor activity.
Highlights
G-proteins’ are a family of heterotrimeric molecules that neutrophils by sucrose density gradient centrifugatioarne important constituentosf certain receptor-mediated signal and analyzed for Gi-like proteins by pertussis toxin- transduction systems
Subcellular Localization of Pertussis Toxin Substrate by stratepresent in the gradient shown in Fig. 2 A, 59% was Sucrose Density GradientFractionation and Changes with retrieved in the plasma membrane-enriched fraction, 35% in Cellular Activation-Individual fractions obtained from su- the specific granule-enriched fraction, and 6% in cytosol
Fractions enriched for plasma membrane or specific granules were pooled and used assubstrate for pertussis toxin-catalyzed [32P]ADP-ribosylationand analyzed by SDS-PAGE and autoradiography
Summary
Subcellular Localization of Pertussis Toxin Substrate by stratepresent in the gradient shown in Fig. 2 A , 59% was Sucrose Density GradientFractionation and Changes with retrieved in the plasma membrane-enriched fraction, 35% in Cellular Activation-Individual fractions obtained from su- the specific granule-enriched fraction, and 6% in cytosol. Inthreeseparatepreparations giving (Fig. 2, A and B)despite an appreciable diminution inpertus- near-linear increases in densitometry relative to protein, the sis toxin substrate within the specific granule-enriched frac- specific immunoreactivity (arbitrarily defined as absorbance tion (Fig. 2B) To assess quantitatively these changes sucrose units perpg of protein) of the plasma membrane fractionwas density gradient fractions corresponding to cytosol To exclude the possibility that localization of alkaline phosphatase activity (and, asa consequence, demonstration of pertussis toxin substrate) at this position in the gradient reflects contamination of the specific granule-enrichedfraction by plasma membrane, we surface radiolabeled neutrophils with '*'I and measured specific activity of the label in subcellular fractions. Subcellular localization of pertussis toxin substrate in resting and activated neutroDhik
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