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A B S T R A C T Polymorphonuclear leukocytes (PMN)aggregate and avidly attach to endothelium in response to chemotactic agents. This response may be related in part to the release of the specific granule constituent lactoferrin (LF). We found by using immunohistology and biochemical and biophysical techniques that LF binds to the membrane and alters the surface properties of the PMN. Upon exposure of PMN treated with 5 gg/ml cytochalasin B to 2 X 10' M formyl-methionine-leucine-phenylalanine for 5 min, the PMN mobilized LF to their surface as observed by immunoperoxidase staining for LF. At added LF levels ranging from 4 to 15 tg/107 PMN there was a dosedependent reduction in PMN surface charge reaching 4 mV, when the partitioning into the membrane of a charged amphipathic nitroxide spin label was measured by electron spin resonance spectroscopy, whereas transferrin was without effect. When '25I-FeLF was added to human PMN in increasing amounts and the results corrected for the residual amount of free LF contaminating the cells, the PMN were saturated with LF at concentrations between 100 and 200 nM in the medium. Human PMN bound 1.35 X 106 molecules per cell and the calculated value for the association constant for these receptors was 5.2 X 106 M-'. Additionally, 6 ,ug/ml LF served as an opsonin for rabbit PMN to promote PMN uptake by rabbit macrophages, when assessed by electron microscopy, but lysozyme did not. These studies indicate that LF can bind to the surface of the PMN and reduce its surface charge.

The outer membrane of Neisseria gonorrhoeae contains approximately 15 proteins, with 2 or 3 accounting for over 75% of the total protein mass. Samples of outer membrane from strain 2686 T4 analyzed by electrophoresis in 2% polyacrylamide gels revealed a band with an apparent molecular weight of 800,000. The band was protein material, as indicated by trypsin and pronase sensitivity and by L-[3H]proline incorporation. Peptidoglycan, nucleic acids, and carbohydrate were not detected in the band. Dye binding, L-[3H]proline incorporation, and labeling of solubilized outer-membrane proteins with '25I-labeled Bolton-Hunter reagent indicated that the band made up 10 to 13% of the total protein mass of isolated outer membranes. The material in the band was purified by gel filtration and, after reduction and alkylation, quantitatively recovered as subunits with an apparent molecular weight of 76,000. The protein in complex form was exposed at the cell surface, as evidenced by labeling whole cells with 125I by using a lactoperoxidase-catalyzed reaction and with CNBr-activated dextran. Rabbit serum raised against whole 2686 T4 gonococci contained antibody which reacted with the protein complex. The protein complex was detected in all gonococcal strains tested, but its presence could not be demonstrated in several other gram-negative species.

SUMMARY1. The microviscosity of the axoplasm of cat sciatic nerve was determined by an in vitro electron spin resonance (e.s.r.) method using the spin label tempone. To identify the spin label signal as one arising only from within the axoplasm, Ni2+ was used as a line broadening agent. In one series of experiments in nerves with sheath intact the Ni2+ ion was shown to eliminate the tempone signal arising from the surface water, and in another series of experiments, with the sheath slit, to eliminate the signal from the extracellular space as well.2. A microviscosity of less than 5 centipoise (cP), i.e. 5 x that of water, was determined for'the axoplasm. Changes in the viscosity of the nerve axoplasm as a function of temperature over a range of 380 down to 2 C were seen to follow closely the viscosity change found for a water solution.3. The microviscosity ofnerve axopolasm and its changewithtemperature were related to axoplasmic transport of material in nerve fibres. The results were used to exclude a large increase in viscosity at lowtemperatures as the cause for the cold-block of fast axoplasmic transport.

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