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.

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.

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.

Phosphorylation and dephosphorylation of spectrin.

The phosphorylation of spectrin polypeptide 2 is thought to be involved in the metabolically dependent regulation of red cell shape and deformability. Spectrin phosphorylation is not affected by cAMP. The reaction in isolated membranes resembles the cAMP-independent, salt-stimulated phosphorylation of an exogenous substrate, casein, by enzyme(s) present both in isolated membranes and cytoplasmic extracts. Spectrin kinase is selectively eluted from membranes by 0.5 M NaCl and co-fractionates with eluted casein kinase. Phosphorylation of band 3 in the membrane is inhibited by salt, but the band 3 kinase is otherwise indistinguishable operationally from spectrin kinase. The membrane-bound casein (spectrin) kinase is not eluted efficiently with spectrin at low ionic strength; about 80% of the activity is apparently bound at sites (perhaps on or near band 3) other than spectrin. Partitioning of casein kinase between cytoplasm and membrane is metabolically dependent; the proportion of casein kinase on the membrane can range from 25% to 75%, but for fresh cells is normally about 40%. Dephosphorylation of phosphorylated spectrin has not been studied intensively. Slow release of 32Pi from [32P] spectrin on the membrane can be demonstrated, but phosphatase activity measured against solubilized [32P] spectrin is concentrated in the cytoplasm. The crude cytoplasmic phosphospectrin phosphatase is inhibited by various anions--notably, ATP and 2,3-DPG at physiological concentrations. Regulation of spectrin phosphorylation in intact cells has not been studied. We speculate that spectrin phosphorylation state may be regulated 1) by metabolic intermediates and other internal chemical signals that modulate kinase and phosphatase activities per se or determine their intracellular localization and 2) by membrane deformation that alters enzyme-spectrin interaction locally. Progress in the isolation and characterization of spectrin kinase and phosphospectrin phosphatase should lead to the resolution of major questions raised by previous work: the relationships between membrane-bound and cytoplasmic forms of the enzymes, the nature of their physical interactions with the membrane, and the regulation of their activities in defined cell-free systems.