The Effect of pH on the Binding of Calcium to Pea Epicotyl Cell Walls and its Implications for the Control of Cell Extension

Cell walls were prepared from the epicotyls of dark-grown pea(Pisum sativum L.) seedlings. The walls were found to bind externally-added⁴⁵Ca²⁺, with a binding constant of 4 ? 10^–4 mol dm^–3and a maximum capacity of 1.5 ? 10^–8 g-ions of Ca²⁺ perg fresh weight of epicotyl. The binding capacity decreased asthe pH of the medium was decreased below 6.0, suggesting thatthe calcium was bound by an anionic group with an apparent pKof 4.7. More than half the calcium binding was due to polygalacturonicacid in the wall, since up to 60% of the calcium binding capacitywas removed by pre-incubation of the cell walls with polygalacturonase(E.C.3.2.1.15). Only small decreases in calcium binding wereseen following pre-incubation with protease, nucleases, phospholipaseand hemicellulase. These results indicate that calcium willbe displaced from the cell wall at hydrogen ion concentrationswhich are known to occur in the wall during wall extension.They are consistent with a mechanism by which calcium inhibitswall extension by forming ionic bridges between polygalacturonicacid molecules, and also with the hypothesis that calcium andhydrogen ions exert opposing influences on cell wall extensionby competing for the same binding sites on the polygalacturonicacid. Key words: Pea epicotyl, Cell wall, Calcium, pH     

The Role of Cell Calcium in the Contraction of Single Cannulated Muscle Fibers

Intracellular injections of the calcium-binding agent, EGTA,into single cannulated fibers of Balanus and Maia were ableto suppress, almost completely, the contractions induced byvarious contractile agents. The amount oE EGTA required in Maia fibers for the suppressionof the contractile response produced by caffeine and high-Ksaline, as has already been reported, was similar to the meanfiber calcium level. In Balanus fibers, however, although theamount of EGTA needed for the suppression of the caffeine-salineresponse was similar to the estimated level of fiber calcium,the amount required in the raised-K-saline experiments was considerablygreater. It has been suggested that membrane depolarizationunder these conditions allows calcium to enter the fiber fromthe external saline, the amount entering being related, at leastin part, to a large effective sarcolemmal surface area and thepresence of binding agent internally. The results of intracellularand plate-electrode stimulation of Belanus fibers also suggestedthat the fiber under certain conditions could utilize externalcalcium ions, while the results of plate-electrode stimulationof Maia fibers could be explained most easily in terms of mobilizationof mainly intracellular calcium for the process of contraction. Efflux of Sr⁸⁹ ions from Balanus fibers under various conditionssuggested that this ion is bound and mobilized internally ina manner similar to calcium. The results are seen not to contradict a chanelled-current theoryfor e-c coupling of the type proposed for crayfish fibers.     

Selective impairment of the synthesis of basic fibroblast growth factor binding domains of heparan sulphate in a COS cell mutant defective in N-sulphotransferase

N-Sulphation is a key step in the overall sulphation of heparansulphate. We have isolated a COS cell-derived mutant, CM-15,that is impaired in its ability to bind to basic fibroblastgrowth factor (bFGF) and has a 2- to 3-fold reduction in N-sulphotransferaseactivity [Ishihara et al., (1992a) Anal. Biochem., 206, 400–407].We now provide structural evidence that CM-15 is selectivelyimpaired in the synthesis of highly sulphated regions or ‘blocks’that display high-affinity binding to bFGF; these are completelyN-sulphated blocks of decasaccharide or greater length thatare enriched in O-sulphate groups. The synthesis of sulphatedblocks that did not show high affinity to the growth factorwas relatively unimpaired in the mutant cells; this includedfully N-sulphated octamer (or smaller) blocks and, unexpectedly,decasaccharide or larger blocks that were poorly O-sulphated.In the latter fraction, the failure to form high-affinity bindingregions was the result of a failure to stimulate O-sulphationrather than N-sulphation in CM-15 cells. In agreement with otherstudies, disaccharide analysis of the wild-type-derived sulphatedblocks suggested that 2-O-sulphation of iduronate residues inthe polymer was a necessary element to produce a high-affinitybinding sequence once N-sulphation was completed in the decasaccharideor larger fraction. These results suggest that a selective reductionin both N- and O-sulphation in the larger blocks produced byCM-15 cells is a consequence of the reduction of N-sulphotransferaseactivity. These data provide a potential mechanism for regulatingthe synthesis of high-affinity bFGF binding domains in the heparansulphate of mammalian cells. basic fibroblast growth factor COS cell mutant heparan sulfate N-sulphotransferase     

Role of calcium binding by sarcoplasmic reticulum in the contraction and relaxation of uterine smooth muscle

The binding of calcium by isolated sarcoplasmic reticulum from cow uterus was studied. Sarcoplasmic reticulum was prepared by differential centrifugation. Three fractions were obtained: I, sedimented between 2,500–15,000 x g; II at 40,000 x g; and III, at 150,000 x g. Fraction II was further purified on a sucrose density gradient. All three fractions contained considerable amounts of intrinsic calcium, mostly in fraction I. Calcium binding in the presence of ATP¹ and Mg also was greatest in fraction I, followed by fraction II, with less in fraction III. Without ATP no calcium was taken up. 5 and 10 mM sodium azide partially inhibited calcium binding in fraction I, but not in fraction II, suggesting the presence of some mitochondria or mitochondrial fragments in fraction I. Calcium binding in fraction II was completely inhibited by 3 mM salyrgan; this fraction thus appears to be sarcoplasmic reticulum. ATPase activity was found in all three fractions, highest in fraction II. It is computed that calcium binding in fractions I and II, on the basis of a 50% yield of protein, is sufficient to elicit contraction by supplying calcium to the contractile proteins of the smooth muscle cell and to regulate relaxation and contraction.     

Properties and Possible Physiological Significance of Cell Wall Calcium Binding in Etiolated Pea Epicotyls

Baydoun, E. A-H. and Brett, C. T. 1988. Properties and possiblephysiological significance of cell wall calcium binding in etiolatedpea epicotyls.—J. exp. Bot. 39: 199–208. The binding of ⁴⁵Ca²⁺ ions to cell walls prepared from pea epicotylswas examined in young and old parts of the epicotyl, and wasfound to be considerably greater, on a carbohydrate basis, inthe older, non-growing cells. A similar comparison between light-and dark-grown stems showed greater binding in the dark-grownstems. The polygalacturonase-insensitive component of the bindingcontained at least three types of binding with different affinities,and had an apparent pK of 4.3. The specificity of the bindingfor calcium ions was examined and a considerable degree of specificitywas observed. The specificity of inhibition by calcium of epicotylelongation was similar to the specificity of calcium binding.A specific calcium chelator, EGTA, when present at a concentrationof above 10 mol m^–3, promoted the extension of matureregions of the epicotyl, while inhibiting extension of youngertissue. Key words: Cell wall, calcium, pea epicotyl     

Heterogeneity of calcium channel agonist binding sites in the coronary artery

The binding properties (3H) BAY k 8644 a 1,4-dihydropyridine calcium channel agonist were studied in the subcellular membrane fraction isolated from the coronary artery by differential centrifugation. The specific binding of (3H) BAY k 8644 to microsomal membranes of the coronary smooth muscle was rapid, saturable, reversible and of both high and low affinity. The dissociation constants obtained from Scatchard analysis with (3H) BAY k 8644 and nitrendipine were 0.60 +/- 0.02 nmol.l-1 and 9.1 +/- 0.1 nmol.l-1 for the high and low affinity binding site respectively and the estimated maximal numbers of binding sites in the plasma membrane fraction were 0.76 +/- 0.02 and 3.15 +/- 0.18 pmol.mg-1 of protein respectively. The substituted dihydropyridine calcium channel antagonists nitrendipine and nifedipine competitively inhibited specific (3H)BAY k 8644 binding suggesting a common high affinity 1,4-dihydropyridine binding site in the coronary microsomal fraction for calcium channel activator and antagonists. The low affinity agonist binding sites were significantly inhibited by adding nucleoside carrier inhibitors, 2-deoxyadenosine and dipyridamole, and by -SH alkylating agent N-ethylmaleimide. The results suggests that the coronary artery contains both high and low affinity calcium channel binding sites (in a 1:5 ratio) with the low affinity calcium channel agonist binding sites being associated with nucleoside carrier and/or with-SH groups.     

Prostaglandins and oxytocin: Their effects on uterine smooth muscle

A subcellular fraction was isolated from uteri of non-pregnant and pregnant cows. ATP-dependent calcium binding was shown to take place in this fraction. This calcium binding was inhibited in a dose related fashion when increasing amounts of prostaglandin (PG) E₂ or F_2α were added to the in vitro experimental medium. The physiologically inactive PGF_1β had no inhibitory effect. Oxytocin caused inhibition of calcium binding in preparations from both pregnant and non-pregnant cows. The response to PGE₂ and PGF_2α was somewhat greater in preparations from pregnant uteri than from non-pregnant uteri. The response to oxytocin was very much greater in pregnant uteri. Because of the high PG sensitivity of calcium binding in preparations from the non-pregnant uterus, it is concluded that the PGs may be the more suitable agent in the control of reproduction.     

In vitro binding of IAA to plasma membrane-rich fractions containing Mg++-activated ATPase from mung bean hypocotyls

Cell homogenates of dark-grown mung bean hypocotyls were fractionatedinto six fractions (L-0, L-l to L-5) by stepwise sucrose density-gradientcentrifugation. The majority (ca. 84%) of Mg⁺⁺-activated ATPase activity ofthe 10,000 x g pellet was localized in the L-0 (1.03 d 1.14)and L-l (1.14 d 1.16) fractions. Over 40% of the vesicularmembrane in the L-0 fraction and 60% of the L-l fraction couldbe stained with phosphotungstic acid (PTA)-chromic acid, a selectivestaining for the plant plasma membrane. In vitro binding of ¹⁴C-IAA to the fraction components was thegreatest in the L-l fraction among the six. The binding of ¹⁴C-IAAto the L-l fraction in vitro was markedly interfered with bythe presence of a high concentration of cold IAA (2 x 10^–4M).However, it was not affected by the IAA analogues IPA, IBA andIAN. This indicates that IAA highly specifically binds to theL-l fraction. In vitro specific binding of ¹⁴C-IAA to L-l andL-0 was decreased with an increasing acidity from pH 8.0 to5.0. In vitro binding of ¹⁴C-IAA to L-l and L-5 was furtherenhanced when these fractions were isolated from sections pretreatedwith 10^–5M cold IAA for 60 min ¹Present address: Institute for Plant Virus Research, 959 Aobacho,Chiba 280, Japan. (Received August 14, 1975; )     

Do ice nucleating lipoproteins protect frozen insects against toxic chemical agents?

As the body fluid of freeze-tolerant organisms freezes, solutes become concentrated in the gradually smaller unfrozen fluid fraction, and dissolved trace metals may reach toxic levels. A dialysis technique was used to investigate the metal binding capacity of the low density fraction of the hemolymph from the freeze tolerant beetle Phyto depressus. The low density fraction, assumed to contain the ice nucleating lipoproteins, showed approximately 100 times greater capacity to bind metals (Cd ²⁺, Cu ²⁺ and Zn ²⁺) than the proteins albumin, hemoglobin and similar to metallothionein. The high metal binding capacity in the low density fraction raises the question if the ice nucleating lipoproteins might assist in detoxification of potentially toxic concentrations of metals that may occur when a large fraction of the bodyfluids of freeze tolerant insects freeze. This hypotheis is consistent with the fact that the lipoprotein ice nucleators are present in far greater amounts than required for ice nucleation, and also with the fact that the lipoprotein ice nucleators have a remarkably high content of amino acids with negatively charged residues that may act as metal binding sites.     

Properties of Alkaline Phosphatase in Cucumber Roots Induced by Calcium Starvation

Calcium deficiency caused an increase in alkaline phosphataseactivity in cucumber roots [Matsumoto and Yamaya (1981) Plant& Cell Physiol. 22: 1137]. The activities of other hydrolasesincluding acid phosphatase, nucleases and proteases, however,were much less affected by the removal of calcium. Nucleosidedi- and triphosphates and inorganic pyrophosphate were effectivelyhydrolyzed by the induced alkaline phosphatase, whereas nucleosidemonophosphate-hydrolyzing activity was basically equal in theroots grown with either complete medium or a medium lackingcalcium. The alkaline phosphatase in cucumber roots was foundin fractions pelleting at 3,000 x g and in the 100,000 x g supernatant.The calcium-starved roots increased their alkaline phosphataseactivity in both fractions. Four isozyme bands of the alkalinephosphatase in the soluble fraction were separated by polyacrylamidegel electrophoresis. One of the isozyme bands showed a prominentincrease with the calcium deficiency, but not in the presenceof cycloheximide. (Received June 24, 1981; Accepted September 3, 1981)     

Herbivory and Calcium Concentrations Affect Calcium Oxalate Crystal Formation in Leaves ofSida (Malvaceae)

Calcium oxalate crystals have potential roles in plants as partof a defence mechanism against herbivores and/or in accumulatingexcess calcium. To date, these potential roles have been studiedindependently. In this experimental study the effects of calciumlevels and herbivory on the production of calcium oxalate crystals(i.e. druse, spherical crystal aggregates) were examined inseedlings of Sida rhombifolia. Seedlings were subjected to threecalcium levels (low, normal or high) and an artificial herbivorytreatment. Calcium levels and herbivory both affected densityof crystals in leaves. Leaves from seedlings grown in low calciumhad a greater crystal density than those grown in high calcium.Leaves from seedlings subjected to herbivory had a greater crystaldensity than those from seedlings not subjected to herbivory.This study provides additional evidence that calcium oxalatecrystal production depends not only on calcium levels but canalso be influenced by external pressures such as herbivory.In addition to their physiological role in plants, these resultssuggest that calcium oxalate crystals can also act as a defencemechanism against herbivores. Copyright 2001 Annals of BotanyCompany Calcium concentrations, calcium oxalate crystals, herbivory, Malvaceae, Sida rhombifolia     

Effect of cholesterol nucleation-promoting activity on cholesterol solubilization in model bile

Human bile contains a factor with cholesterol nucleation-promoting activity that binds to concanavalin A-Sepharose. In this study we have investigated the effect of this activity on the dynamics of lipid solubilization in supersaturated model bile. A concanavalin A binding protein fraction of human bile was mixed with model bile and the effect on the distribution of cholesterol and phospholipid between mixed micelles and phospholipid/cholesterol vesicles was studied by means of density gradient ultracentrifugation. The nucleation-promoting activity containing fraction induced a transfer of cholesterol and phospholipid from the micellar to the vesicular phase. This led to a decrease in the density of the vesicular fraction. We have also studied the effect of promoting activity on the nucleation time of an isolated vesicle fraction. A decrease of the nucleation time of 10.7 +/- 1.3 to 2.3 +/- 0.3 days was observed. In conclusion, a concanavalin A binding protein fraction from human bile stimulated cholesterol nucleation via a double effect; it increased the amount of vesicular cholesterol and phospholipid, and it also directly induced nucleation of cholesterol from the vesicles.     

Calcineurin-NFATc signaling pathway regulates AQP2 expression in response to calcium signals and osmotic stress

The aquaporin (AQP)2 channel mediates the reabsorption of water in renal collecting ducts in response to arginine vasopressin (AVP) and hypertonicity. Here we show that AQP2 expression is induced not only by the tonicity-responsive enhancer binding protein (TonEBP)/nuclear factor of activated T cells (NFAT)5-mediated hypertonic stress response but also by the calcium-dependent calcineurin-NFATc pathway. The induction of AQP2 expression by the calcineurin-NFATc pathway can occur in the absence of TonEBP/NFAT5. Mutational and chromatin immunoprecipitation analyses revealed the existence of functional NFAT binding sites within the proximal AQP2 promoter responsible for regulation of AQP2 by NFATc proteins and TonEBP/NFAT5. Contrary to the notion that TonEBP/NFAT5 is the only Rel/NFAT family member regulated by tonicity, we found that hypertonicity promotes the nuclear translocation of NFATc proteins for the subsequent induction of AQP2 expression. Calcineurin activity was also found to be involved in the induction of TonEBP/NFAT5 expression by hypertonicity, thus further defining the signaling mechanisms that underlie the TonEBP/NFAT5 osmotic stress response pathway. The coordinate regulation of AQP2 expression by both osmotic stress and calcium signaling appears to provide a means to integrate diverse extracellular signals into optimal cellular responses. aquaporin; nuclear factor of activated T cells; tonicity-responsive enhancer binding protein; osmotic response     

Calcium-dependent protein interactions in MUC5B provide reversible cross-links in salivary mucus

The macromolecular organization within saliva was investigated by tracer diffusion measurements of fluorescent polystyrene microspheres by fluorescence recovery after photobleaching using a confocal microscope (confocal-FRAP). There was a concentration-dependent reduction in microsphere diffusion; this was much greater in the presence of calcium (10 mm) and was reduced by the addition of EGTA (10 mm). These effects on tracer diffusion showed that native saliva contained a macromolecular organization that was sensitive to free calcium concentrations. This was supported by a major increase in the weight average molecular weight of the high molecular weight mucin fraction in saliva (10-62 x 106) and an increase in intrinsic viscosity of saliva (733 to 1203 ml/g) both caused by calcium. Analysis of the change in tracer diffusion in saliva showed a 20-fold increase in the apparent pore size (from 130 nm in 10 mm CaCl2 to 2600 nm in 10 mm EGTA at physiological concentration). The effect was specific for calcium and was unaffected by up to 2 m NaCl. The calcium binding activity was contained in a high buoyant density fraction of saliva excluded from Sepharose CL-2B. Calcium binding to this fraction gave an approximate Kd of 7 x 10-6 m, and the binding was irreversibly destroyed by treatment with 6 m guanidinium chloride and by mild reduction, suggesting it to be to a protein site. This fraction of saliva was shown to contain MUC5B as the single major protein species by positive ion electrospray ionization-tandem mass spectrometry analysis. The results suggested that oligomeric MUC5B in saliva is assembled into much larger linear or branched assemblies through calcium-mediated protein cross-links.     

Selective interactions of lectins with amino acid taste receptor sites in the channel catfish

Five lectins of varying carbohydrate specificities, Dolichosbiflorus (DBA), jacalin, Phaseolus vulgaris (PHA), Pisum sativum(PSA) and Ricinus communis (RCA I), were used to extend characterizationof the glycoprotein nature of taste plasma membranes and todifferentially affect the binding of two taste stimuli, L-alanineand L-arginine, to their respective taste receptor sites inthe cutaneous taste system of the channel catfish (Ictaluruspunctatus). The binding of the taste stimulus L-arginine toa partial membrane fraction (P_–) from taste epitheliumwas inhibited by 68 and 74% by preincubation in the presenceof the unconjugated lectins PHA and RCA I respectively. A correspondinglevel of inhibition of L-alanine binding was seen in the presenceof RCA I (76%); however, PHA had little effect upon L-alaninebinding. DBA appeared to selectively inhibit L-alanine but notL-arginine binding (60 versus 8% respectively) while jacalinmoderately inhibited the binding of both stimuli to fractionP2. PSA had little effect upon the binding of either L-alanineor L-arginine (4 and 5% inhibition respectively). Inhibitionof taste receptor binding by all lectins was time- and dose-dependent,and was fully abolished by incubation in the presence of theappropriate hapten sugar. The biotinylated lectins DBA, jacalin,PHA, RCA I and concanavalin A (Con A) were used to identifythe glycoprotein components of the chemosensory plasma membranesafter polyacrylamide gel electrophoresis. As previously shown,numerous protein components were labeled by Con A. In contrast,only a few minor protein components were labeled by PHA, DBAand RCA I. This differential labeling of the taste membranesand the differential inhibition of receptor binding by lectinssuggest that they may prove useful as tools in the isolationand purification of taste receptor proteins.     

Isolation and Characterization of the Olfactory Epithelial Cells of the Catfish

A preparation enriched in olfactory receptor cells has beenobtained from the olfactory mucosa of the catfish (Ictaluruspunctatus). The tissue was treated successively with trypsin,DNase, trypsin inhibitor, EDTA in Ca^{+ +} , Mg^{+ +} free mediumaccording to a method derived from that of Cohen, et al.⁽¹⁾.After mechanical disruption of the isolated olfactory lamellae,the cells were isolated by centrifugation on a Ficoll gradient.Each type of cell was morphologically identified by comparingin situ and in vitro preparations by SEM. Small round cellswere collected on 10% Ficoll. The nature of these cells is notknown but part of them are certainly basal cells which havebeen shown⁽²⁾ to be the precursors of the constantly regeneratingolfactory neurons. Respiratory cells settled mainly on 20% Ficoll.A fraction containing 60% sustentacular cells was collectedon 33% Ficoll. Olfactory cells characterized by an axon, a dendriteand several cilia, were found on 37% Ficoll. This fraction alsocontains up to 40% sustentacular cells. A yield of 20% was measuredfor olfactory cell isolation. Vital staining and ability tosynthesize RNA indicate a viability of the final preparationof 70% to 80%. Further identification of the cells was performedby measuring the binding activity of a series of amino acidsto a preparation enriched in olfactory cells. A good correlationwas determined between the extent of the binding and the reportedelectrophysiological activities of these amino acids recordedin vivo. Although the final olfactory cell suspension is notpure, it constitutes the first step in the study of the olfactoryreceptor sites.     

Effects of enzymatically digested microsome fractions on red-light-enhanced pelletability of pea phytochrome in vitro in the presence of calcium ion

The 130,000 ?g supernatant of Sephadex G-50 filtrate and a 78,000?g microsomal fraction were prepared separately from homogenatesof etiolated pea (Pisum sativum L. cv. Alaska) shoots. The pelletabilityof phytochrome increased ca. 10-fold by exposure of the filtrateto red light and mixing the filtrate with the microsomal fractionin the dark at ca. 0?C. The increase of pelletable phytochromewas inhibited by 84% when the microsomal fraction digested withtrypsin was used. Phospholipase C (Clostridium welchii) digestionof the microsome inhibited no more than 13% of the pelletability.Although phospholipase A₂ (Crotalus terrificus terrificus) digestioninhibited 43% of the pelletability, addition of defatted albuminduring the enzymatic digestion completely restored the levelof the pelletability. The decrease of RNA content of the microsomalfractions by ribonuclease A digestion did not result in a proportionalinhibition of the pelletability. These results indicate thatproteinaceous component in the microsomal fraction is essentialfor phytochrome pelletability in vitro, that RNA and polar headsof phospholipids are unlikely to be the partner for the binding,and that products of phospholipase A₂ digestion inhibit thepelletability partially. (Received September 12, 1979; )     

Properties of the Respiratory NAD(P)H Dehydrogenase Isolated from the Cyanobacterium Synechocystis PCC6803

Activity staining with NADPH-nitroblue tetrazolium after native-PAGEof membrane proteins of Synechocystis PCC6803, solubilized with3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),revealed four NAD(P)H dehydrogenase (NDH) activities; an NDHcomplex of the respiratory chain, a ferredoxin NADP⁺ reductase(FNR), a drgA product which oxidized both NADH and NADPH, andan uncharacterized NADH-specific enzyme. The NDH complex waspurified with anion exchange and gel filtration chromatographies.The purified complex had a molecular mass of 376 kDa and wascomposed of 9 subunits. Western analysis showed that the complexcontained the NDH-H subunit, but not NDH-A or B. The enzymereduced ferricyanide much faster than plastoquinone and usedNADPH as its prefered electron donor rather than NADH. The enzymaticactivity was inhibited by diphenyleneiodonium chloride and salicylhydroxamicacid, but not by rotenone, p-chloromercuribenzoate, N-ethylmaleimide,flavon, dicumarol, or antimycin A. These results suggest thatthe purified complex is a hydrophilic subcomplex which containsan NADPH binding site and flavin, and is dissociated from ahydrophobic subcomplex, which contains quinone binding site. ¹Present address: Division of Applied Life Sciences, GraduateSchool of Agriculture, Kyoto University, Sakyo, Kyoto, 606-8502Japan ³Present address: Department of Biotechnology, Faculty of Engineering,Fukuyama University, 1 Gakuencho, Fukuyama, Hiroshima, 729-0292Japan     

Further characterization of boar sperm plasma membrane proteins with affinity for the porcine zona pellucida

Boar sperm plasma membrane proteins (PMPs) with affinity for the zona pellucida were partially purified from columns of dextran sulfate using a linear salt gradient and a buffered detergent that retained their ability to block directly the binding of uncapacitated and capacitated sperm to isolated porcine oocytes. PMPs that bound most strongly to dextran sulfate (fraction IV) were also most effective in blocking sperm binding to porcine oocytes. These tightly bound proteins also bound to isolated zonae to a greater extent than other fractions. Monovalent antibodies to fraction IV PMPs completely blocked sperm binding to isolated eggs. Fraction IV PMPs lost the ability to inhibit directly the binding to eggs when treated with chaotropic agents and trypsin; the fraction also displayed a tendency to aggregate in the absence of high salt. This property and the affinity of proteins in this fraction for sulfated polysaccharides indicate that specific hydrophilic interactions may play a significant role in sperm-zona attachments.     

Calcium binding by subcellular fractions of bovine adrenal medulla

Significantly more calcium per gram protein was found in a relatively pure granule fraction isolated from fresh bovine adrenal medulla than in predominantly mitochondrial fractions isolated from the same tissue. Sixty-four and 55% of the calcium associated with chromaffin granule and mitochondrial fractions, respectively, was released into the supernatant upon lowering the tonicity of the medium. The per cent calcium released by this procedure was significantly greater for granules than for mitochondria (p < 0.05). The amount of calcium per gram protein released into the supernatant also was greater in granule fractions than in mitochondrial fractions (p < 0.05). These data, coupled with a previous report that 10^?3 M EDTA does not markedly decrease the calcium content of whole granules, indicate that the excess calcium of the granule fractions relative to the mitochondrial fractions is maintained within the particles of that fraction. The functional significance of the relatively large amount of calcium in chromaffin granules is not clear. The presence of 150 mM sodium chloride or potassium chloride decreases calcium binding by granule or mitochondrial fragments incubated in 2.2 mM calcium chloride in 0.2 M Tris, pH 7, by about 50%. EDTA, 10^?3 M, removes all but a small residual of the calcium associated with the granule or mitochondrial fragments whereas lowering the concentration of Tris increases calcium binding to about the same extent in both these subcellular fractions. The calcium-binding properties of granule and mitochondrial fragments therefore appear to be quantitatively and qualitatively similar. Inhibition of catecholamine release by relatively high concentrations of sodium may be explained by competitive inhibition of calcium binding. Calcium binding by granule fragments decreases with an increase in hydrogen ion concentration.