Competition between ligands of glycosyltransferases and horseradish peroxidase for binding sites on intracellular and plasma membranes of HeLa cells

Summary A micro-method for the semi-quantitation of surface-bound horseradish peroxidase (HRP) was developed and was applied to study the competition between ligands of glycosyltransferases and HRP for binding sites on the surface of HeLa cells. Dried coverslip cultures of HeLa cells, fixed in methanol, were placed on 0.3 ml of the incubation medium on parafilm and were incubated for 45 min at 37° C. The incubation medium contained HRP, lysozyme and Ca²⁺ in HEPES buffer, pH 7.2. After washing, the cells were incubated for 60 min at 37° C in HEPES buffer containing 20 mM Ca²⁺. After this treatment, the plasma membranes showed a strong cytochemical reaction for HRP. Most of the HRP was released into buffer solution during a 5 h incubation at 37° C in the absence of Ca²⁺, and was measured by spectrophotometry. The addition of 20 mM Ca²⁺ to the buffer solution prevented the release of most of the HRP from the plasma membranes thus showing that the binding of HRP required Ca²⁺. Ligands of glycosyltransferases were added to the incubation medium with HRP. The amount of HRP released from the cells decreased in relation to the competing potency and concentration of these ligands. The method was applied to estimate the concentration of some ligands of galactosyltransferase and sialyltransferase that caused a 50% decrease in the release of previously-bound HRP. CMP-neuraminic acid and gangliosides showed a higher competing potency to the surface binding of HRP than UDP-galactose and chitotriose. The spectrophotometric analysis was correlated (on duplicate samples) with cytochemical observations. When dried HeLa cells, fixed in methanol, were incubated with HRP, lysozyme and Ca²⁺, without being subsequently incubated with Ca²⁺-containing buffer solution, HRP was also bound to membranes of intracellular granules. Cytochemical observations showed that UDP-galactose and chitotriose competed with the binding of HRP to most of these intracellular membranes whereas CMP-neuraminic acid and gangliosides did not. The possible binding of HRP to galactosyltransferase or sialyltransferase on cellular membranes is discussed.     

Binding sites for horseradish peroxidase on the cell surface. Suppression of binding by gangliosides and effects of some bivalent cations

The cytochemical reaction for surface-bound horseradish peroxidase (HRP) on cultured HeLa cells, GH3 cells, and isolated rat liver cells was suppressed by 30 microM monosialoganglioside, by 30 microM trisialoganglioside, or by 5 mM CMP-neuraminic acid. The reaction was also suppressed by 10 mM chitotriose or by 10 mM UDP-galactose, a galactose acceptor and donor, respectively, for galactosyl-transferase. The addition of 2 mM Mn2+ to the incubation medium with HRP suppressed the reaction for surface-bound HRP, and the addition of 10-20 mM Ca2+ intensified the reaction. The addition of 2 mM Zn2+ caused less inhibition than that of 2 mM Mn2+, and the addition of 2 mM Co2+ caused either a slight inhibition, or no inhibition. These observations support the hypothesis that HRP may be bound to a glycosyltransferase at the cell surface.     

Vesicle release from rat red cell ghosts and increased association of cell membrane proteins with cytoskeletons induced by cadmium

When rat red cell ghosts were incubated with 0.1-0.5 mM CdCl2 in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, they became irregular in shape and released small vesicles. The release of vesicles was dependent on the incubation temperature and Cd2+ concentration. The maximum release occurred at 37 degrees C in the presence of 0.2 mM Cd2+. The protein composition of Cd2+-induced vesicles was similar to that of the vesicles released from ATP-depleted red cells. Upon incubation with 0.1-0.2 mM Cd2+, more than 90% of the Cd2+ added to the incubation buffer was recovered in ghosts and 15-20% of the ghost Cd2+ was located on the cytoskeletons prepared by washing ghosts with 0.5% Triton X-100 solution containing 0.1 M KCl and 10 mM Tris-HCl (pH 7.4). Moreover, the cytoskeletons prepared from Cd2+-treated ghosts markedly contained cell membrane proteins, bands 2.1, 3, 4.2 and 4.5, and glycophorins. The association of bands 3 and 4.2 with cytoskeletons increased with increasing concentrations of Cd2+ added to the incubation buffer and saturated at 0.2 mM Cd2+. The solubilization of cytoskeletal proteins, bands 1, 2 and 5, from ghosts at low ionic strength was almost completely suppressed by preincubation of ghosts with 0.1 mM Cd2+. HgCl2, PbCl2 and ZnCl2 at 0.2 mM each also produced an increased association of cell membrane proteins with cytoskeletons, whereas CaCl2 and MgCl2 did not.     

Cytochemical observations on mannose-specific binding sites for horseradish peroxidase in liver sinusoidal cells

Paraformaldehyde-fixed, frozen sections of the liver of rats were processed for the detection of mannose-specific binding sites of horseradish peroxidase (HRP) by a method reported previously, with some modifications resulting in a more intense binding reaction. Before staining for peroxidase activity, the sections were held in buffered solutions of physiological saline at different temperatures and pH's, and in the presence or absence of added Ca2+, mannose or galactose. The gradual decrease and final disappearance of the binding reaction were observed. The release of HRP from the binding sites as determined by the disappearance of the cytochemical reaction was 50-100 times faster at 22 degrees C than at 4 degrees C and was 5-10 times faster at 37 degrees C than at 22 degrees C. The release was approximately twice as fast at pH 7.0 than at pH 9.0 and 20-30 times faster at pH 6.0 than at pH 7.0. The release of HRP was 10-15 times faster in the absence of 1 mM Ca2+ in the buffer solution and was approximately 100 times faster in the presence of 0.1 M D-mannose as compared to 0.1 M D-galactose. Pretreatment of the sections with trypsin abolished the binding reaction whereas neuraminidase, phospholipases A2 and C, and chondroitinase ABC were without effect. An acidic isoenzyme of HRP, Sigma type VIII, was bound more intensely and more widely to liver sinusoidal cells than another acidic isoenzyme, Sigma type VII, a basic isoenzyme, Sigma type IX, and the routinely used preparation, Sigma type VI. The effect of the temperature on the binding reaction was re-examined with an improved procedure. In contradistinction to the previous finding, strong binding of HRP after 2-4 h incubation at 4 degrees C was observed.     

Involvement of cellular calcium incorporated from the medium in production of inositol trisphosphate in A-431 cells

The property of intensive 45Ca2+ uptake by A-431 human epidermoidal carcinoma cells was indicated to be an influx, not binding to the cell surface, since the two apparent dissociation constants (Kd) between 45Ca2+ and cells were almost the same when measured in either the presence or absence of 1 mM [ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA); these constants were approximately 5-10 x 10(-6) and 1 x 10(-4) M, respectively, which are much higher than the chelating constant of EGTA for Ca2+ (approximately 10(-11) M). Furthermore, addition of A23187, a calcium ionophore, rapidly released the 45Ca2+ incorporated into cells at both 37 degrees C and 0 degrees C. The 45Ca2+ associated with the cells was slowly released or exchanged when cells were incubated in medium depleted of Ca2+, or in that containing 1 mM non-radioactive Ca2+. The ability of A-431 cells to respond to extracellular ATP by elevating their level of intracellular calcium ions, as well as by producing inositol trisphosphate (InsP3), was suppressed in cells depleted of cellular calcium. These data suggest that calcium ions are extensively incorporated or exchanged with those outside the cells, maintained as stored calcium, and involved in production of InsP3, when A-431 cells are stimulated by ATP to trigger the signal transduction system.     

Noninvolvement of the heat-induced increase in the concentration of intracellular free Ca2+ in killing by heat and induction of thermotolerance

Mouse C3H 10T1/2 cells exhibited a two- to threefold increase in the concentration of free Ca2+ during heating at 45 degrees C. The increase was maximal for a heat dose which was still in the shoulder region of the survival curve. The increase was fully reversible in heat-sterilized cells. By changing the concentration of extracellular Ca2+, it was possible to modulate the concentration of intracellular free Ca2+ in heated cells. Lowering the extracellular concentration to 0.03 mM reduced the baseline concentration of intracellular free Ca2+, and prevented it from increasing in heated cells to a level exceeding that of nonheated cells incubated in medium containing 2.0 or 5.0 mM Ca2+. Raising the concentration of extracellular Ca2+ to 15.0 mM raised the baseline, and resulted in a heat-induced increase in free Ca2+ which was twofold higher than that of cells heated in medium containing 2.0 or 5.0 mM Ca2+. An elevated concentration of intracellular free Ca2+ during and after heating did not potentiate thermal killing, nor did a reduced concentration during and after heating mitigate killing. Furthermore, the data argue against a heat-induced increase in free Ca2+ to some threshold level, which potentiates cell killing by some other parameter. In addition, cells heat-shocked in either 0.03 or 5.0 mM extracellular Ca2+, and then incubated in the same concentration for 12 h at 37 degrees C, developed quantitatively similar amounts of tolerance to a second heating. The data suggest that the concentration of intracellular free Ca2+ does not play a critical role in thermal killing or the induction and development of thermotolerance.     

Bacterial degradation of EDTA

Degradation of EDTA (ethylenediaminetetraacetic acid) or metal-EDTA complexes by cell suspensions of the bacterial strain DSM 9103 was studied. The activity of EDTA degradation was the highest in the phase of active cell growth and decreased considerably in the stationary phase, after substrate depletion in the medium. Exponential-phase cells were incubated in HEPES buffer (pH 7.0) with 1 mM of uncomplexed EDTA or EDTA complexes with Mg2+, Ca2+, Mn2+, Pb2+, Co2+, Cd2+, Zn2+, Cu2+, or Fe3+. The metal-EDTA complexes (Me-EDTA) studied could be divided into three groups according to their degradability. EDTA complexes with stability constants K below 10(16) (lg K < 16), such as Mg-EDTA, Ca-EDTA, and Mn-EDTA, as well as uncomplexed EDTA, were degraded by the cell suspensions at a constant rate to completion within 5-10 h of incubation. Me-EDTA complexes with lg K above 16 (Zn-EDTA, Co-EDTA, Pb-EDTA, and Cu-EDTA) were not completely degraded during a 24-hour incubation, which was possibly due to the toxic effect of the metal ions released. No degradation of Cd-EDTA or Fe(III)-EDTA by cell suspensions of strain DSM 9103 was observed under the conditions studied.     

N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) does not modify the angiotensin II-stimulated calcium signal in cultured bovine glomerulosa cells

Studies were performed to determine if the sustained elevation in [Ca2+]c noted previously in glomerulosa cells in response to Ang II resulted from the presence of HEPES in the experimental medium. At confluence, primary cultures of bovine glomerulosa cells were maintained for 24-30 h in the presence of either 14 mM NaHCO3/5% CO2 or 25 mM HEPES/4 mM NaHCO3/air. During subsequent experimental periods, cells were incubated in the presence of the corresponding or reciprocal buffer, and the effects of Ang II on [Ca2+]c were monitored by fura 2 fluorescence. Increases in [Ca2+]c produced by Ang II in cells continuously maintained in either HCO3(-) - or HEPES-buffered media were similar, and with the same monolayer the nature of the Ang II-stimulated Ca2+ signal was independent of the buffer employed. Moreover, the Ang II-stimulated Ca2+ signal was not significantly affected by the removal of HCO3- from the superfusate. These results indicate that the sustained increase in [Ca2+]c is not an artifact introduced by the use of HEPES as an experimental buffer, but rather a normal component of the Ang II-stimulated Ca2+ signal.     

Production of proliferation inhibitors by mature granulocytes

The aim of the experiments was to find ways of increasing the yield of small molecular weight inhibitors of cell proliferation released by granulocytes. Almost pure populations of granulocytes from pig or human blood, or from sterile inflammatory exudates in rats were treated in various ways and then spun down. Molecules below approximately 10 000 dalton (Diaflo ultrafiltration or Sephadex G 25 filtration) in the supernatants were tested for inhibitory activity by measuring 3H-thymidine incorporation in 5 to 6-h coverslip cultures of rat bone marrow cells. The different granulocyte treatments were: Freeze-thawing, sonication, incubation (at +4 degrees -37 degrees C) in hypotonic media (0-200 mosm/kg), storage in vitro overnight (at +4 degrees C) before incubation, incubation at 37 degrees C in complete and buffered tissue culture medium (Fischer's with 10 mmol/1 HEPES), incubation in saline only (2-h periods, approximately 70 X 10(6) cells/ml), or with lidocaine added, with Ca++ and the Ca++ ionophore A-23187, with K+ and the K+ ionophore Valinomycin, with a high K+ concentration (50 mmol/1), with arachidonic acid, with a cAMP analogue, or with a protease inhibitor added during or at the end of the incubation. On a per cell basis rat peritonitis granulocytes released more inhibitor than pig blood granulocytes, whereas human blood granulocytes were not detectably inhibitory at all. Arachidonic acid was the most promising agent tested to increase inhibitor release above that occurring spontaneously from granulocytes incubated in saline.     

Reversible induction of actin rods in mouse C3H-2K cells by incubation in salt buffers and by treatment with non-ionic detergents

Incubating conditions which induced actin paracrystal-like intracellular structures (actin rods) were investigated by using several cell lines. We have found that an incubation of cells of a mouse fibroblastic cell line, C3H-2K, in an isotonic solution of NaCl containing 1 mM MgCl2, 1 mM CaCl2 and 10 mM MES, pH 6.5, induced disintegration of stress fibers and formation of actin rods in the cytoplasm. Actin rods were induced also by incubating in salt buffers in which Na+ of the above solution was substituted by most cations except K+ or Rb+. When the actin rod-forming cells were transferred back to DMEM containing 10% FBS, actin rods disappeared and stress fibers subsequently re-formed within 1 h at 37 degrees C. Although the induction was observed in NaCl buffer at a wide range of pH values (5.5-10), the optimal pH was 6.5. Formation of actin rods is dependent upon cellular metabolism, as it was inhibited at 4 degrees C, or by metabolic inhibitors. Incubation in NaCl buffer induced actin rods in HeLa, L, NRK, BALB/c 3T3 and Swiss 3T3 cells, but not in CEF or MEF cells. A decrease in cell volume was observed parallel with the induction of actin rods, except for CEF and MEF cells. Alterations in intracellular concentrations of Na, K or Ca were not correlated with the induction, however. Actin rods were also induced in C3H-2K cells by a brief treatment with non-ionic detergents. Tween 80 at concentrations as low as 0.003% was effective for the induction, but did not increase the passive membrane transport of p-nitrophenylphosphate. In contrast to the induction by NaCl buffer, treatment with Tween 80 induced numerous tiny actin rods at 4 degrees C, which became larger when further incubated at 37 degrees C. Double immunofluorescence staining with anti-actin antibody and anti-vinculin antibody showed that vinculin plaques remained at least in an early stage of the actin rod formation. We discuss the mechanism for the induction of actin rods based upon the present findings.     

Receptor-mediated internalization of high density lipoprotein by rat sinusoidal liver cells: identification of a nonlysosomal endocytic pathway by fluorescence-labeled ligand

Rat sinusoidal liver cells possess the surface receptor for high density lipoprotein (HDL) (Murakami, M., S. Horiuchi, K. Takata, and Y. Morino. 1987. J. Biochem. (Tokyo) 101: 729-741). The present study was undertaken to determine whether cell surface-bound HDL underwent subsequent endocytic internalization by using 125I-labeled HDL and fluorescein isothiocyanate-labeled HDL (FITC-HDL). The cell-associated radioactivity obtained by a 40-min incubation with 125I-labeled HDL at 37 degrees C was released into the medium as acid-precipitable forms upon further incubation at 37 degrees C. When further incubated at 0 degree C instead of 37 degrees C, however, this release was significantly reduced. A similar phenomenon was observed after the cell-associated ligands had been treated with trypsin. The cell-associated ligands obtained after a 1-hr incubation with 125I-labeled HDL at 0 degree C were largely counted for by those bound to the outer surface of the cells, thus suggesting that HDL is internalized into cells at 37 degrees C but not at 0 degree C. Moreover, when cells were incubated with FITC-HDL at 0 degree C, the cell-associated ligands were found in a pH 7.2 +/- 0.1 compartment, whereas when incubated at 37 degrees C, its microenvironmental pH became much more acidic, exhibiting pH 6.2 +/- 0.1. Furthermore, this value returned to 7.1 +/- 0.1 upon treatment with carbonylcyanide m-chlorophenylhydrazone known to dissipate the total protonomotive force. These results suggest, therefore, that the internalization process does follow receptor-mediated binding of HDL in rat sinusoidal liver cells. This notion was also supported by fluorescence microscopic observations.     

Inhibitory effect of MgATP on the release of regulatory light chain from scallop myosin and light chain composition of scallop myosin hybridized with abalone light chain 2 at 30 degrees C

The experimental conditions for release of the regulatory light chain (RLC) of scallop myosin at 30 degrees C were studied. Substantially all RLC was released from myosin by incubation for 5 min in medium containing buffer and KCl. This release of RLC was inhibited strongly by Ca2+, while the effect of Mg2+ was about 10,000 times weaker than that of Ca2+. Even in the absence of Ca2+, MgATP and MgADP inhibited the release of RLC, while the protective effect of AMPPNP was negligible. Other Mg nucleotides also showed some protective effect, though appreciably less than MgATP. The incubation of scallop myosin with abalone regulatory light chain (LC2) at 30 degrees C for 5 min produced a hybrid myosin. In the presence of 5 mM MgCl2, 1 of the 2 mol of RLC per mol of scallop myosin was exchanged with 1 mol of LC2. In the presence of Ca2+ or MgATP, myosin bound 1 extra mole of LC2 besides the 2 mol each of SH-LC and RLC.     

Internalization of lectins in neuronal GERL

Conjugates of ricin agglutinin and phytohemagglutinin with horseradish peroxidase (HRP) were used for a cytochemical study of internalization of their plasma membrane "receptors" in cultured isolated mouse dorsal root ganglion neurons. Labeling of cells with lectin-HRP was done at 4 degrees C, and internalization was performed at 37 degrees C in a culture medium free of lectin-HRP. 15-20 min after incubation at 37 degrees C, lectin-HRP receptor complexes were seen in vesicles or tubules located near the plasma membrane. After 1-3 h at 37 degrees C, lectin-HRP-receptor complexes accumulated in vesicles and tubules corresponding to acid phosphatase-rich vesicles and tubules (GERL) at the trans aspect of the Golgi apparatus. A few coated vesicles and probably some dense bodies contained HRP after 3-6 h of incubation at 37 degrees C. Soluble HRP was not endocytosed under the conditions of this experiment or when it was present in the incubation medium at 37 degrees C. Internalization of lectin-HRP-receptor conjugates was decreased or inhibited by mitochondrial respiration inhibitors but not by cytochalasin B or colchicine. These studies indicate that lectin- labeled plasma membrane moieties of neurons are endocytosed primarily in elements of GERL.     

Activation of potassium channels in erythrocytes of marine teleost Scorpaena porcus

To assess the possibility of stimulating Ca2+-activated K+ channels, marine fish erythrocytes were incubated at 20-22 degrees C in saline containing a Ca2+-ATPase inhibitor (orthovanadate), a Ca2+ ionophore (A23187), propranolol or Pb2+. Incubation of the cells for up to 2 h under control conditions or in the presence of 5 mM NH4VO3 and 1 mM Ca2+ did not affect the intracellular K+ and Na+ concentrations. About 50% cellular K+ was lost from erythrocytes incubated in the presence of 0.01 mM A23187, 1 mM EGTA and 0.4-1.0 mM Ca2+. There was a significant loss of cellular K+ after the addition of 0.05-0.2 mM propranolol to the incubation medium. The stimulatory effect of propranolol on the K+ efflux was independent of external Ca2+. Blockers of Ca2+ transport, verapamil and Co2+, caused only a small decrease in the K+ loss induced by propranolol. The treatment of erythrocytes with 1-2 microM Pb2+ led to a minor K+ loss, but at a Pb2+ concentration of 20-50 microM, about 70% cellular K+ was lost. The K+ efflux induced by propranolol or Pb2+ was completely blocked by 1 mM quinine. The induced K+ loss from the erythrocytes was accompanied by a slight increase in the intracellular Na+ concentration. These data indicate the possibility of inducing Ca2+- and Pb2+-activated potassium channels in erythrocytes of S. porcus. A distinctive feature of the cells is a high sensitivity to propranolol, which activates K+ channels in the absence of external Ca2+.     

Ca2+- and Mg-ATP-dependent shape change of human erythrocyte ghosts and triton shells

Human erythrocyte membranes (ghosts) prepared from fresh blood changed in shape from spherical to crenated, when suspended in 10(-7)-10(-6) M Ca2+-EGTA buffers. Although the ghosts from long-stored ACD blood (10 weeks) were less sensitive to 10(-7)-10(-6) M Ca2+, the ghosts obtained from this blood after it had been preincubated with adenine and inosine for 3 h at 37 degrees C were highly sensitive to Ca2+. When these highly sensitive ghosts were incubated in 10 mM Tris-Cl buffer (pH 7.4) or 1 mM MgCl2 (pH 7.4) at 0 degrees C, they gradually lost Ca2+ sensitivity within 60 min, but they recovered Ca2+ sensitivity again after re-incubation with 2 mM Mg-ATP for 20 min at 37 degrees C followed by washing with 1 mM MgCl2 (pH 7.4). The shape of these highly Ca2+-sensitive ghosts immediately changed from crenate to disc on addition of 1 mM Mg-ATP even at 6 degrees C in the presence of 10(-7)-10(-6) M Ca2+. A similar shape change was also observed when ghosts treated with 0.5% Triton X-100 (Triton shells) were used. Triton shells from fresh blood ghosts or from long-stored blood ghosts which had been preincubated with 2 mM Mg-ATP for 20 min at 37 degrees C shrank immediately in the presence of 10(-6) M Ca2+ and then swelled on addition of 1 mM Mg-ATP. The specificity to ATP and the dependency on ATP concentration are in agreement with those of the ghost shape change at step 2 (Jinbu, Y. et al., Biochem biophys res commun 112 (1983) 384-390) [18]. These results suggest that cytoskeletal protein phosphorylation enhances sensitivity to Ca2+ and induces erythrocyte shape change in the presence of physiological concentrations of ATP and Ca2+.     

IgE receptor-mediated phosphatidylinositol hydrolysis and exocytosis from rat basophilic leukemia cells are independent of extracellular Ca2+ in a hypotonic buffer containing a high concentration of K+

In isotonic buffer, IgE receptor-mediated exocytosis from rat basophilic leukemia cells is dependent on extracellular Ca2+, with half-maximal degranulation requiring 0.4 mM Ca2+. No significant exocytosis occurs in the absence of extracellular Ca2+. This absolute requirement for Ca2+ is eliminated by suspending the cells in a hypotonic buffer containing 60 to 80 mM K+; Na+ cannot substitute for K+. Optimal Ca2(+)-independent exocytosis occurs in a buffer containing 20 mM dipotassium Pipes, pH 7.1, 40 mM KCl, 5 mM glucose, 7 mM Mg acetate, 0.1% BSA, and 1 mM EGTA. The cells maintain this Ca2(+)-independent exocytosis even if they are preincubated with 1 mM EGTA for 40 min at 37 degrees C before triggering. Exocytosis is eliminated as isotonicity is approached by adding sucrose, NaCl, KCl, or potassium glutamate to the buffer. Quin 2 fluorescence measurements reveal only a very small rise in [Ca2+]i when the cells are triggered in hypotonic buffer in the absence of extracellular Ca2+ and the presence of 1 mM EGTA. In isotonic buffer, degranulation does not occur under conditions that lead to such a small rise in [Ca2+]i. Sustained IgE receptor-mediated phosphatidylinositol hydrolysis, which is also Ca2+ dependent in isotonic buffer, becomes independent of Ca2+ in the hypotonic buffer. In fact, the rate of phosphatidylinositol hydrolysis in hypotonic buffer in the absence of Ca2+ (and presence of 1 mM EGTA) is twice that observed in isotonic buffer in the presence of 1 mM Ca2+. These data show that in hypotonic buffer, the requirement of IgE receptor-mediated PI hydrolysis for extracellular Ca2+ is eliminated, and degranulation proceeds with a [Ca2+]i of 0.1 microM, the baseline level of [Ca2+]i found in resting cells. These results are consistent with the hypothesis that, in isotonic buffer, the Ca2+ requirement for mast cell degranulation is for the generation of second messengers via hydrolysis of membrane phosphatidylinositols.     

Characterization of Ca2+ uptake by guinea pig epididymal spermatozoa

Comparative studies of Ca2+-uptake by guinea pig spermatozoa were performed with fresh epididymal sperm and with cells preincubated in a chemically defined, Ca2+-free medium for capacitation. Calcium uptake was negligible in fresh spermatozoa, but increased dramatically after 20 min of incubation at 37 degrees C in the presence of pyruvate and lactate. Spermatozoa incubated in the absence of these substrates accumulated only 34% as much 45Ca2+ as was taken up by cells in complete medium. The monosaccharides glucose, fructose, and mannose and the nonmetabolizable sugars 2-deoxyglucose and sucrose inhibited the enhancement of Ca2+-permeability. In the presence of 6 mM sucrose 45Ca2+ uptake was not influenced by external sodium chloride concentration between 0 mM and 145 mM. The respiratory activity of the capacitated spermatozoa not only was higher than that of uncapacitated cells, but it was stimulated by Ca2+. No effect of Ca2+ on respiration of fresh spermatozoa was detected. An increase in calcium uptake was associated with increasing pH of the medium. It is possible that a regulatory mechanism through the calcium permeability of the plasma membrane of guinea pig spermatozoa exists and controls the development of physiological events related with the fertilization process. The sugar composition, the availability of the energy substrates lactate and pyruvate, and the pH of the reproductive tract fluids could play an important role in the accessibility of Ca2+ into the cells in vivo, as has been demonstrated in vitro. The enhancement of calcium permeability during the preincubation could be a useful indicator to verify if capacitation has occurred.     

Transport of Ca2+ by Yersinia pestis.

Low-calcium-response, or Lcr, plasmids of yersiniae are known to promote an in vitro nutritional requirement for 2.5 mM Ca2+ at 37 degrees C which, if not fulfilled, results in cessation of growth with induction of virulence functions (Lcr+). The mechanism whereby Ca2+ regulates this metabolic shift is unknown. Radioactive Ca2+ was not actively accumulated by yersiniae but was excluded by an exit reaction analogous to those described for other bacteria. Nevertheless, cultivation at 37 degrees C with 0.1 mM Ca2+, a level insufficient to prevent restriction of cell division, promoted significantly more binding of the cation by Lcr+ organisms than by plasmid-deficient Lcr- mutants. According, Lcr+ yersiniae may possess unique ligands capable of recognizing Ca2+.     

Stimulation of Ca2+ uptake and protein phosphorylation in tumor cells by fibronectin

Suspensions derived from attached HeLa cells transported 45Ca2+ considerably faster than those derived from spinner cultures grown in liquid medium. Incubation of spinner cells with fibronectin or cold-insoluble globulin in the presence of 5% calf serum at 37 degrees C for 1 to 2 h greatly increased the rate of Ca2+ flux into the cells. Suspensions of cells transformed by Rous sarcoma virus transported Ca2+ much more slowly than cell suspensions of the parent strain of normal rat kidney. Incubation of the transformed cells or Ehrlich ascites tumor cells with fibronectin increased the rate of Ca2+ uptake, while no effect was seen on Ca2+ transport by this treatment of normal kidney cells grown in tissue cultures. A 45,500-dalton protein was found to interact firmly with Ca2+ that entered into attached HeLa cells or fibronectin-treated spinner cells. This Ca2+-associated protein was detected by lithium dodecyl sulfate gel electrophoresis at 0 degrees C after 30 s of exposure to radioactive Ca2+. In tumor cells without fibronectin treatment, the radioactive band was not seen under the same conditions, even after 10 min incubation with 45Ca2+. In fibronectin-treated tumor cells, addition of Ca2+ to buffered solutions resulted in increased phosphorylation of a protein in the 45,000-dalton region. The phosphorylated protein band which appears to be associated with the cytoskeleton can be resolved by isoelectric focusing into four polypeptide chains. The relation of these observations to the cascade of protein kinases involved in the phosphorylation of the beta-subunit of the (Na+-K+)-ATPase is discussed.     

Effect of calcium and anaerobiosis on the thermostability of Bacillus stearothermophilus.

Bacillus stearothermophilus NCA 2184 lost viability and subsequently released cytoplasmic components when suspended in 0.1 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.2) and incubated at 60 degrees C. Cell lysis was prevented by the addition of 10 mM CaCl2 to the Tris-buffer suspension. Cells which were incubated under anaerobic conditions for 20 min in the growth medium before they were collected were stable in the Tris-buffer suspension without added calcium. Anaerobic incubation effected an increase in membrane cardiolipin which appeared to be related to the increase in the thermostability of the cells.