Richard G. Painter

Intracellular localization of N-formyl chemotactic receptor and Mg2+ dependent ATPase in human granulocytes☆

Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83000 X g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40-60% were achieved with a 20-70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g X cm-3. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimented with specific granules (p = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg2+-ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg2+-ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.

Centripetal myosin redistribution in thrombin-stimulated platelets: Relationship to platelet factor 4 secretion

We have examined the F-actin and myosin distribution in resting and thrombin-activated platelets by double label immunofluorescence microscopy. In resting, discoid platelets, F-actin and myosin staining was distributed in a diffuse pattern throughout the interior of the cell with slight accentuation at the cell periphery. In contrast, platelet factor 4 antigen (PF4) was more centrally localized in a fine punctate distribution which is consistent with its localization in alpha-granules. Within 5 sec after thrombin stimulation both F-actin and myosin staining were increased at the periphery of the now spherical platelets. Subsequently, a myosin-containing spherical structure decreased in diameter closely surrounding a phase-dense central zone. In contrast, F-actin staining continued to be accentuated at the cell periphery and was prominent in filopodia and blebs. As previously shown, PF4 staining was localized after 30 sec within large intracellular masses that corresponded to closed vacuolar structures at the ultrastructural level. Morphometric analysis of electron micrographs showed that formation of these vacuolar structures kinetically paralleled alpha-granule disappearance and preceded PF4 release. These PF4-containing structures translocated to the cell periphery after 1-3 min, where they appeared to fuse with the plasma membrane. Ultrastructural analysis of thin sections showed that the myosin-rich spherical structure spatially and temporally correlated with a band of microfilaments that closely surrounded the organelle-rich central zone of the cell. Morphometric analysis of these micrographs showed that the absolute volume of this central zone decreased with time after thrombin addition, showing a significant change after 15 sec and reaching a maximum value after 3-5 min. Changes in the volume of this compartment kinetically preceded PF4 release. On the basis of these data, we propose that an actomyosin contractile force is generated which centripetally redistributes the myosinrich structure and organelle zone. Conceivably this inward force may not only accelerate granule-granule fusion to form intracellular secretory vacuoles, but may also provide aid in their extrusion toward the platelet plasma membrane.

The Neutrophil N-Formyl Peptide Receptor: Dynamics of Ligand-Receptor Interactions and Their Relationship to Cellular Responses

The N-formyl peptides, as a class, evoke in neutrophils in vitro an array of responses that mimic the biological functions of these cells in the inflammatory process. Because these peptides can be prepared synthetically, it has been possible to correlate extensively the structure-function relationships of a variety of amino acid sequences in both stimulatory and inhibitory peptides. Moreover,.this diversity in available sequences has permitted the development not only of numerous radiolabeled ligands, but of photoaffinity and fluorescent molecules as well. During the short period since N-formyl-methionyl peptides were first identified as chemoattractants derived from bacteria, the N-formyl peptides receptor has been catapulted into a position of prominence as a model, not only for studies of neutrophil stimulation but for receptor-mediated cell stimulation in general.

Evidence for N-formyl chemotactic peptide-stimulated GTPase activity in human neutrophil homogenates

Neutropil homogenates contained a high affinity guanosine triphosphatase (GTPase) that was stimulatable (+ 27%) by the addition of 100 nM N‐formyl chemotactic peptide (CHO‐pep), but not by 1 μg·ml−1 phorbolmyristate acetate (PMA). Kinetic analysis of the stimulation demonstrated an apparent lagtime of 14.3 ± 6.9 s between the addition of CHO‐pep and the optimal GTPase stimulation. The GTPase activity (but not CHO‐pep‐stimulated GTPase activity) was preserved in a highly purified plasma membrane fraction of the homogenate. From these observations we suggest that both a high affinity guanine nucleotide binding protein and GTPase are closely associated with the plasma membrane CHO‐pep receptor. The possibility that GTPase activity may influence guanine nucleotide regulation of adenylate cyclase during CHO‐pep stimulation of neutrophils is discussed.