Biochemical characterization and epididymal localization of the maturation-dependent ram sperm surface antigen ESA152.

We examine here the biochemical properties and epididymal localization of a maturation dependent ram sperm surface antigen. A monoclonal antibody, ESA152, identifies an antigen that is present on the surface of ejaculated sperm, but is absent from testicular sperm. Crosslinking of the ESA152 antigen with bivalent antibodies induces the acrosome reaction, redistributing the antigen into the anterior region of the sperm head where it associates with the fusion product of the plasma membrane and the outer acrosomal membrane. The ESA152 antigen appears as a polypeptide of 18 kDa on immunoblots of SDS-polyacrylamide gels. The ESA152 epitope includes the sialic acid termini of N-linked oligosaccharides, as shown by its sensitivity to neuraminidase and endoglycosidase F. The ESA152 antigen is a highly hydrophobic integral membrane protein that resists aqueous extraction, partitions into the detergent phase of Triton-X-114, and solubilizes in chloroform-methanol mixtures. The anchoring of ESA152 is unaffected by phosphtidylinositol specific phospholipase C. The antigen is absent from extracts of caput and corpus epididymidis but appears abruptly in the first segment of the cauda. Immunofluorescence reveals that the ESA152 epitope first appears in clusters of cells in the luminal epithelium of the proximal cauda, prior to or concurrent with its appearance on sperm.

Cross-linking a maturation-dependent ram sperm plasma membrane antigen induces the acrosome reaction.

ESA152 is a highly hydrophobic 18 kDa sialoglycoprotein, which becomes expressed on ram sperm in the proximal cauda epididymis. ESA 152 is expressed on all regions of the sperm surface, most strongly on the posterior region of the head, most weakly on the anterior region of the head. In this paper, we show that induction of the acrosome reaction with Ca2+ ionophore causes ESA152 to be redistributed from the posterior to the anterior region of the head plasma membrane. Cross-linking ESA152 with bivalent antibody causes similar redistribution and induces the acrosome reaction. Induction of the acrosome reaction with ESA152 antibody requires Ca2+ but is insensitive to (10 ng/ml) pertussis toxin.

Relationship of hemolysis buffer structure, pH and ionic strength to spontaneous contour smoothing of isolated erythrocyte membranes.

Isolated human erythrocyte membranes crenate when suspended in isotonic medium, but can use MgATP to reduce their net positive curvature, yielding smooth discs and cup forms that eventually undergo endocytosis. An earlier report from this laboratory (Patel, V.P. and Fairbanks, G. (1981) J. Cell Biol. 88, 430-440), has described a phenomenon of ATP-independent shape change in which ghosts prepared by hemolysis and washing in synthetic zwitterionic buffers crenated at 0 degree C, but underwent conversion to smooth discs and cups when warmed in the absence of MgATP. We have further explored the effect of the hemolysis condition on the requirement for ATP in ghost shape change. 25 hemolysis buffers were applied at 10 mM (pH 7.4, 0 degree C). Eight anionic buffers with relatively high ionic strength (e.g., phosphate and diethylmalonic acid (DMA] yielded ghosts requiring ATP for shape change, while two cationic buffers (Bistris and imidazole) and ten synthetic zwitterionic buffers (e.g., Tricine and Hepes) with lower ionic strength produced ghosts that smoothed spontaneously at 30 degrees C. Hemolysis at intermediate ionic strength yielded mixed populations in which spontaneous smoothing was expressed in all-or-none fashion. Maximal ATP-independent shape change was induced by hemolysis at pH 7.3-7.7, while ATP was required after hemolysis at pH less than or equal to 7.1 even when the ionic strength at hemolysis was low. Ghosts requiring ATP could be converted to ATP independence by washing at low ionic strength, but ATP independence could not be reversed readily by washing at high ionic strength. Exposure to low ionic strength at pH greater than 7.1 presumably changes membrane organization in a way that alters the temperature dependence of tensions within the bilayer or skeleton of the composite membrane.

Cation transport in oxidant-stressed human erythrocytes: heightened N-ethylmaleimide activation of passive K+ influx after mild peroxidation.

Normal and chronically dehydrated (hereditary xerocytosis) human red cells were subjected to mild peroxidative treatment (315 microM hydrogen peroxide (H2O2), 15 min) in the presence of azide. The subsequent expression of passive (ouabain-resistant) K+ transport activities was analyzed by measurement of 86Rb+ influx. Peroxidation of normal red cells did not affect basal K+ transport activity, but the increment in K+ influx elicited by 0.5 mM N-ethylmaleimide (NEM) was increased 3-fold. The enhanced K+ influx was chloride-dependent, but only partially inhibited by 0.1 mM furosemide. Stimulated activity declined progressively after NEM activation, but could be restored by a second NEM treatment. Prior conversion of hemoglobin to the carbonmonoxy form abolished the response to peroxide, while 200 microM butylated hydroxytoluene (BHT) exerted only partial inhibition, suggesting that the effect of H2O2 requires interaction of activated, unstable hemoglobin species with the membrane, but that lipid peroxidation is not sufficient. Peroxidation following NEM treatment also enhanced NEM activation, indicating that enhancement does not require altered NEM reactions with stimulatory or inhibitory sites. Passive K+ transport in hereditary xerocytosis red cells was not activated by NEM, with or without H2O2 pretreatment. The results demonstrate that modest peroxidative damage to red cells can heighten the activation of a transport system that is thought to be capable of mediating net K+ efflux and volume reduction in cells that express it. Models are proposed in which the effects of NEM, H2O2, cell swelling and other factors are mediated by conformational changes in a postulated subpopulation of anion channel (Band 3) molecules that bind the K+ transporter.

Lateral regionalization and diffusion of a maturation-dependent antigen in the ram sperm plasma membrane.

We have used a monoclonal antibody ESA 152 in fluorescence recovery after photobleaching (FPR) studies of a maturation-dependent surface antigen of ram sperm. The antibody is an immunoglobulin G secreted by a hybridoma derived from NS1 mouse myeloma cells. The ESA 152 antigen is not detectable in testicular sperm. It is localized on the surface of ejaculated sperm where it is present on all regions of the surface, but tends to be concentrated on the posterior region of the head. The ESA 152 antigen can be extracted by detergents or chloroform-methanol. The extracted antigen is sensitive to proteases and migrates with an apparent Mr approximately 30,000 in SDS-containing 10-20% polyacrylamide gradient gels. FPR measurements of ESA 152 lateral mobility in the membrane yield diffusion coefficients in the range 10(-9)-10(-8) cm2/s, values typical of lipids but observed for proteins only at the fluid dynamic limit where diffusion is controlled by lipid fluidity. Immobile fractions, typical of membrane proteins, are observed on all regions. When the antigen is stained by a fluoresceinated Fab fragment of the ESA 152 antibody, the diffusibility is highly regionalized, with particularly low, but rapid, recovery on the midpiece. Cross-linking of the antigen with the intact ESA 152 antibody induces a redistribution in which the antigen is excluded from the posterior head region. This cross-linking is accompanied by increases in ESA 152 diffusibility on both the anterior head and the midpiece.

Relationship of major phosphorylation reactions and MgATPase activities to ATP-dependent shape change of human erythrocyte membranes.

Human erythrocyte ghosts prepared by hemolysis and washing in hypotonic Tris are crenated by salt and divalent cations, but undergo shape change to smooth biconcave discs and stomatocytic forms when incubated with MgATP at 37 degrees C. This is normally accompanied by protein and lipid phosphorylations in which the major phosphate acceptors are the spectrin beta-chain and inositol phospholipids, respectively. The system was manipulated in several ways to demonstrate the independence of ATP-dependent shape change from the major phosphorylation reactions. Salt-extracted membranes incubated with adenosine, an inhibitor of spectrin and phosphatidylinositol kinases, underwent normal shape change despite reductions of greater than 90% in spectrin and phospholipid labeling by [gamma-32P]ATP. ATP-dependent shape change was blocked by vanadate at micromolar concentrations (half-maximal inhibition at less than 1 microM), but vanadate did not inhibit membrane autophosphorylation reactions or turnover of spectrin- or lipid-bound phosphate. Vanadate inhibited part of the ATP hydrolysis that accompanies shape change and is expressed in the presence of ouabain and EGTA. The vanadate-sensitive MgATPase activity was approximately 3 nmol Pi X min-1 X mg of protein-1. The results implicate it in ATP-dependent shape change.

Properties and characterization of vesicles released by young and old human red cells.

We have presently demonstrated morphologic differences between young and senescent red cells following 18 h of metabolic depletion in vitro. Young and old red cells both form echinocytes, whereas only young cells demonstrated myelin forms or microspheres. Furthermore, vesicles were released in greater quantities into the cell-free supernatant from young cells. Isolated vesicles from both young and old red cells contained lipids, intrinsic membrane proteins (especially band 3), and haemoglobin, and they were also enriched in acetylcholinesterase (AChE). Young cells produced more vesicles than old cells but the composition of the low density vesicles was similar except that haemoglobin-spectrin complex was found exclusively in vesicles from young cells. Oxidation of young red cells prior to metabolic depletion prevented both myelin formation and vesicle release.

Inhibition of erythrocyte membrane shape change by band 3 cytoplasmic fragment.

The ATP-dependent transformation of crenated white human erythrocyte ghosts into smoothed disc and cup forms is inhibited by the soluble 40-45-kilodalton (kDa) cytoplasmic portion of the major transmembrane protein, band 3. The band 3 fragment was prepared by chymotryptic treatment of inverted vesicles stripped of peripheral proteins. When present at greater than or equal to 0.2 mg per mg membrane protein (ie, greater than or equal to 2 mol fragment per mol endogenous band 3), the fragment significantly reduced the rate of shape change but did not alter the proportion of membranes that were ultimately converted into smoothed forms (greater than 90%). The inhibitory activity of the fragment could not be attributed to contamination of the fragment preparation by actin or proteolytic enzymes. ATP-independent shape transformation was not inhibited. The band 3 fragment may compete with endogenous, intact band 3 for an association with the spectrin-actin network required for ATP-dependent smoothing of crenated membranes.

Surface glycoprotein changes in ram spermatozoa during epididymal maturation.

Three radiolabeling procedures were applied to ram spermatozoa obtained at three different stages of posttesticular development: on leaving the testis (testicular sperm); after epididymal transit (cauda epididymal sperm); and after exposure to accessory sex gland secretions (ejaculated sperm). The washed spermatozoa were subjected to three radiolabeling treatments: 1) galactose oxidase and sodium boro [3H]hydride (galactosyl and galactosaminyl residues); 2) sodium metaperiodate and NaBH4 (sialyl residues); and 3) chloroglycoluril and Na125 I (tyrosyl residues). High resolution sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoretic analysis of the surface radiolabeling patterns confirms earlier studies in demonstrating an overall shift in the predominant labeled glycoproteins from the zone 78-115 kd in testicular spermatozoa to relatively low molecular weights of between 15 and 95 kd in cauda epididymal or ejaculated spermatozoa. Labeling procedures specific for glycoproteins and sialoglycoproteins revealed additional complexities in the surface transformation patterns of ram spermatozoa and suggest that cauda epididymal spermatozoa exposed to accessory sex gland secretions adsorb or produce a component of high molecular weight (approx. 350 kd).

Cocaine uses and abuses.

Cocaine is a time-honored topical anesthetic for intranasal surgery. It combines superb anesthesia with constriction of nasal vasculature and patient euphoria, which facilitates surgery and enhances its tolerability to the patient. Dosages of 200 mg (2 ml of 10% solution) are considered safe and effective for surgical anesthesia. Central nervous system excitability is the predominant toxic reaction followed by convulsions and apnea, which require respiratory support. Intravenous diazepam effectively averts this reaction. Cardiovascular toxicity appears at higher dosages (such as with ingestion for concealment during smuggling) or when epinephrine is concurrently used with cocaine. Cocaine is also damaging to nasal membranes and the nasal septal cartilage, due to its vasoconstrictive effect and the irritative effects of its diluting contaminants. Its major psychological effect is stimulation similar to that of the amphetamines. Prolonged recreational use may lead to paranoia and violent, antisocial behavior, including homicide and suicide.

Protein kinases and membrane protein phosphorylation in normal and abnormal human erythrocytes: variation related to mean cell age.

Protein kinase activities and membrane autophosphorylation reactions of normal and abnormal human erythrocytes were analyzed. Erythrocytes from patients with high reticulocytosis due to sickle cell anemia and other disorders (n = 13) exhibited elevated activities of total and membrane-bound cAMP-independent casein kinase and cAMP-stimulated histone kinase. Relative to normal controls (n = 10), the average total activities in these abnormal cells were increased 50% and 81%, respectively. The casein and histone kinase activities of normal and abnormal erythrocytes declined significantly with increasing age and buoyant density in Stractan density gradients. Casein kinase activity was highly correlated (r = 0.88; n = 23) with the percentage of reticulocytes in the fraction, consistent with either a progressive loss of activity in mature erythrocytes or an abrupt decline during reticulocyte maturation. The cAMP-independent and cAMP-stimulated autophosphorylation activities of isolated membranes also declined with increasing erythrocyte age. On average, the initial rate of spectrin labeling was 36% lower in ghosts from Stractan gradient bottom fractions, relative to ghosts from top fractions similarly incubated with gamma-32P-ATP. Incorporation into the "band 4.5 zone" (primarily labeling bands 4.8 and 4.9, mol wt 47,800 and 44,600) was also age-dependent. In membranes of unfractionated sickle cells, spectrin autophosphorylation was within normal limits, while 4.5 zone autophosphorylation was increased. Membranes from high reticulocytosis controls (vitamin B-12 deficiency) exhibited similar autophosphorylation patterns, suggesting that the altered autophosphorylation pattern of sickle cell membranes may be attributed to the predominance of very young cells.

Altered red blood cell surface area in hereditary xerocytosis.

Hereditary xerocytes appear larger than normal red cells in scanning electron micrographs and exhibit a higher ghost packing volume. The major chemical components--protein, phosphorus, cholesterol and sialic acid--are increased uniformly, as are all polypeptides visible on gel electrophoresis patterns of xerocyte membranes. These data are consistent with a xerocyte surface area 15 to 25% above normal. Certain clinical anomalies common to this disorder, including unexpectedly low reticulocyte count and 2,3-diphosphoglycerate level, are discussed in the light of the present findings.

Spectrin phosphorylation and shape change of human erythrocyte ghosts.

Human erthrocyte membranes in isotonic medium change shape from crenated spheres to biconcave disks and cup-forms when incubated at 37 degrees C in the presence of MgATP (M. P. Sheetz and S. J. Singer, 1977, J. Cell Biol. 73:638-646). The postulated relationship between spectrin phosphorylation and shape change (W. Birchmeier and S. J. Singer, 1977, J. Cell Biol. 73:647-659) is examined in this report. Salt extraction of white ghosts reduced spectrin phosphorylation during shape changes by 85-95%. Salt extraction did not alter crenation, rate of MgATP-dependent shape change, or the fraction (greater than 80%) ultimately converted to disks and cup-forms after 1 h. Spectrin was partially dephosphorylated in intact cells by subjection to metabolic depletion in vitro. Membranes from depleted cells exhibited normal shape-change behavior. Shape-change behavior was influenced by the hemolysis buffer and temperature and by the time required for membrane preparation. Tris and phosphate ghosts lost the capacity to change shape after standing for 1-2 h at 0 degrees C. Hemolysis in HEPES or N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid yielded ghosts that were converted rapidly to disks in the absence of ATP and did not undergo further conversion to cup-forms. These effects could not be attributed to differential dephsphorylation of spectrin, because dephosphorylation during ghost preparation and incubation was negligible. These results suggest that spectrin phosphorylation is not required for MgATP-dependent shape change. It is proposed that other biochemical events induce membrane curvature changes and that the role of spectrin is passive.

Biochemistry of ATP-dependent red cell membrane shape change.

Red cell membranes prepared by hemolysis and washing in hypotonic Tris buffer crenate when suspended at 0 degrees C in isotonic medium. At 37 degrees C, in the presence of 1 mM MgATP, the crenated membranes are progressively converted to smooth-contoured discs and cup-forms. The phosphorylation of proteins and lipids during shape transformation in the presence of [gamma-32P]ATP has been studied. Spectrin phosphorylation and shape change could be dissociated in several ways, demonstrating that spectrin phosphorylation is neither necessary nor sufficient for the membrane smoothing reaction. Adenosine markedly inhibited phosphoinositide regeneration without altering shape change. Phosphatidic acid synthesis from endogenous diacylglycerol was not affected by adenosine and comparison of sheep, human and rabbit ghosts, which vary greatly in shape change capacity, demonstrated a direct correlation between phosphatidic acid synthesis and shape change rate. The results suggest that membrane curvature may be induced by diacyglycerol phosphorylation at the inner surface of the membrane bilayer, while the membrane skeleton limits the curvature and determines the shape ultimately assumed.

Nasal septal perforation: prevention and management.

Thirty-five patients with nasal septal perforations were treated over a ten-year period. Prior nasal septal surgery was the most common cause of perforation. A combined flap and graft technique was employed for surgical repair which produced a success rate of over 90% for relief of symptoms and closure of the perforation. Etiology, prevention, and medical and surgical management of septal perforation are detailed.

Increased erythrocyte lipid peroxidation in hereditary xerocytosis.

Xerocytosis is a chronic hemolytic anemia with abnormal membrane function manifested by an increase in passive potassium permeability. Xerocytes demonstrate a greater susceptibility to hydrogen peroxide manifested by the production of malondialdehyde (MDA). Xerocyte membrane phospholipid and fatty acid analysis is normal except for a slight increase in phosphatidyl choline, a commensurate decrease in sphingomyelin, as well as a decrease in linoleic acid. Metabolism and glutathione stability are normal as well as plasma vitamin E levels in patients with xerocytosis. The increased susceptibility to oxidant stress is exaggerated in the "older aged" xerocyte population and correlated well with decreased intracellular potassium concentration.