Specific Increase in Phosphatase Isozymes in Cucumber Roots Caused by Calcium Deficiency

Phosphatases in cucumber roots, whose production was inducedby Ca^2$ deficiency, were characterized chromatographically usingATP, 2'(3')-AMP and p-nitrophenyl-phosphate (PNPP) as substrates.Ca^2$ deficiency stimulated greater than 10-fold increases inthe activities with these substrates of the non-adsorbed fractionfrom a DEAE-cellulose column. Several fractions associated withthese phosphatase activities were eluted from the column withNaCl solution; their levels increased less with Ca^2$ starvation.When the non-adsorbed fraction from Ca^2$-straved roots was appliedto a Sephadex G-200 column, fractions associated with 2'(3')-AMPase(phosphatase I) and with both ATPase and PNPPase (phosphataseII) were separated. In the control roots, very weak activitiesof phosphatases I and II were observed at the same positionon the gel filtration. The phosphatase I isolated from boththe control and Ca^2$-starved roots was extremely specific tonucleoside 2'(3')-monophosphates, whereas phosphatase II fromboth types of roots had a relatively broad substrate specificity.When phosphatase I from Ca^2$-starved roots was stained with2'(3')-AMP in CaCl₂ after polyacrylamide gel electrophoresis,a single band was obtained. Phosphatase I from control rootsalso showed a single band, with the same Rf value. PhosphataseII from both types of roots contained two isozyme bands whenthe activities were stained with either ATP or PNPP. These resultsindicate that Ca^2$ starvation causes specific increases in thelevel of phosphatases I and II in cucumber roots. (Received October 28, 1981; Accepted January 19, 1982)     

Repression of Proton extrusion from Intact Cucumber Roots and the Proton Transport Rate of Microsomal Membrane Vesicles of the Roots due to Ca2+ Starvation

Proton extrusion from cucumber roots decreased markedly duringCa²⁺ starvation in the presence of KC1. Vesicles with ATP-dependentproton transport activity were prepared from the microsomalmembrane fraction of control and Ca²⁺-starved roots. The protontransport rate of the vesicles from Ca²⁺-starved roots was repressedto less than half of the vesicles prepared from the controlroots. K⁺-Mg²⁺-ATPase activity associated with the vesiclesprepared from Ca²⁺-starved roots was approximately one-thirdof the activity associated with those prepared from controlroots. K_m values of the proton transport rate and K⁺-Mg²⁺-ATPasefor ATP were much higher in vesicles prepared from Ca²⁺-starvedroots. The repression of proton extrusion linked with K⁺ uptake inthe Ca²⁺-starved roots could be largely caused by the reducedproton pumping activity associated with microsomal membranesin the roots. (Received May 25, 1987; Accepted October 14, 1987)     

Repression of the K+ Uptake and Cation-Stimulated ATPase Activity Associated with the Plasma Membrane-Enriched Fraction of Cucumber Roots Due to Ca2+ Starvation

The uptake of K⁺ by cucumber plants decreased markedly duringCa²⁺ starvation. A plasma membrane-enriched fraction, judgedfrom the distribution of marker enzymes, was prepared from controland Ca²⁺-starved roots. The Mg²⁺- and K⁺-Mg²⁺-ATPase activitiesassociated with the plasma membrane-enriched fraction of controlroots were maxima at pH 6.5. Various monovalent cations andpotassium salts of monovalent anions stimulated Mg²⁺-ATPaseactivity. Vanadate, DES and DCCD inhibited K⁺- Mg²⁺-ATPase activity.Of the divalent cations and phosphate esters tested, Mg²⁺ andATP were most effective for the stimulation of ATPase by K⁺,whereas Ca²⁺ was ineffective in replacing Mg²⁺. Mg²⁺- and K⁺-Mg²⁺-ATPase activities associated with the plasmamembrane enriched fraction of Ca²⁺-starved roots were much lowerthan those of control roots. K_m values of K⁺-Mg²⁺-ATPase forATP were comparable for control and Ca²⁺-starved roots. The K⁺-stimulated activity of Mg²⁺-ATPase in Ca²⁺-starved rootswas approximately one fourth that of the control, whereas therate of stimulation was only slightly lower in Ca²⁺-starvedroots. (Received May 9, 1984; Accepted September 17, 1984)     

Effects of Calmodulin Antagonists on the Mitochondrial and Microsomal Ca2$ Uptake in Apple Fruit

In apple fruit, active ATP-dependent microsomal Ca^2$ uptakeand respiration-dependent mitochondrial Ca^2$ uptake were observed. The mitochondrial Ca^2$ uptake was depressed by the calmodulinantagonists chlorpromazine hydrochloride (CPZ) and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamidehydrochloride (W-7). The Ca^2$-ATPase from apple mitochondriawas also inhibited by CPZ or W-7. The apparent K_m value forCa^2$ in mitochondrial Ca^2$ uptake (K_m=0.35 mM) was similar tothat of mitochondrial Ca^2$-ATPase (K_m=0.32 mM). The inhibitoryeffect of W-7 on the activity of the mitochondrial Ca^2$ uptakewas closely correlated with the inhibition by W-7 of mitochondrialCa^2$-ATPase (r=0.996). These findings indicate that the mitochondrialuptake of Ca^2$ in apple fruit depends on the calmodulin-mediatedactivation of Ca^2$-ATPase. The microsomal Ca^2$ uptake was depressed by CPZ, suggestingthat the microsomal Ca^2$ uptake may also be modulated by calmodulin. ¹ Contribution No. C-72, Fruit Tree Research Station. (Received June 7, 1982; Accepted October 19, 1982)     

Repression of Asolectin-Dependent Activation of Partially Lipid Depleted ATPase Prepared from the Plasma Membrane-Enriched Fraction of Cucumber Roots due to Ca2+ Starvation

The ATPase activity of the plasma membrane-enriched fractionwas severely inhibited by withdrawal of Ca²⁺ from the mediumfor 5 days, although the root system appeared to be unaffectedto visual inspection. Partially lipid-depleted ATPases withsimilar ratios of phospholipid to protein were prepared fromthe plasma membrane-enriched fraction of cucumber roots culturedwith control medium and one lacking Ca²⁺, and their propertieswere compared. SDS disc polyacrylamide gel electrophoresis showedthat the polypeptide components were essentially similar betweencontrol and Ca²⁺-starved roots. Partially lipid-depleted ATPasereassociated with asolectin, the lecithin from soybean, showedtypical characteristics of plasma membrane type ATPase; pH optimumat 6.5, high specificity for ATP as substrate and strong inhibitionby vanadate but not nitrate. The activity of reassociated ATPaseobtained from the control roots was apparently higher than theactivity obtained from Ca²⁺-starved roots. The amount of asolectinrequired for maximum activation of the partially lipid-depletedATPase prepared from control roots was much lower than thatprepared from Ca²⁺-starved roots. Reassociation of partiallylipid-depleted ATPase with asolectin produced higher ATPaseactivity than that with individual phospholipids. The activationof partially lipid-depleted ATPase prepared from control rootswith asolectin was not inhibited by addition of a sample preparedfrom Ca²⁺-starved roots. Thus, a decrease in the functionalassociation of ATPase with phospholipids might be one of thephysiological injuries in root cell membranes of cucumber causedby Ca²⁺ starvation. ¹Permanent address: Department of Horticulture, College of Agriculture,Chonnam National University, Chonnam 500, Korea. (Received February 23, 1988; Accepted August 18, 1988)     

Polar transport of 45Ca2+ across the elongation zone of gravistimulated roots

The movement of calcium across the elongation zone of gravistimulatedprimary roots of maize (Zea mays L.) was measured using ⁴⁵Ca^2$.Radioactive calcium was applied to one side of the elongationzone about 4 mm back from the root tip and the distributionof radioactivity across the root in the region of applicationwas determined using scintillation spectrometry. The movementof ⁴⁵Ca^2$ across the elongation zone was non-polar in verticallyoriented roots. In gravistimulated roots the movement of labelwas polarized with about twice as much label moving from topto bottom as from bottom to top. A variety of treatments whichinterfere with gravitropism was found to eliminate the polarmovement of ⁴⁵Ca^2$ across the elongation zone. In maize cultivarswhich require light for gravitropic competency, dark grown rootsexhibited neither gravitropism nor polar movement of ⁴⁵Ca^2$across the elongation zone. Upon illumination the roots developedboth gravitropic competency and gravity-induced polar movementof ⁴⁵Ca^2$ across the elongation zone. Similarly, roots of light-grownseedlings lost both gravitropic competency and ⁴⁵Ca^2$ transportpolarity upon transfer to the dark. The results indicate a closecorrelation between calcium movement and gravitropism in primaryroots of maize. (Received July 20, 1985; Accepted September 25, 1985)     

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     

Salinity Effects on the Activity of Plasma Membrane H+and Ca2+Transporters in Bean Leaf Mesophyll: Masking Role of the Cell Wall

Net fluxes of H⁺and Ca²⁺were measured in the mesophyll tissueof broad bean (Vicia faba L.) leaves and in protoplasts derivedfrom these cells. NaCl at 90 m M enhanced H⁺extrusion in bothprotoplasts and tissue, but in different ways. Proton extrusionwas inhibited by vanadate, suggesting the involvement of theplasma membrane H⁺-ATPase in cell responses to salinity. Therewas virtually no effect of NaCl on the net Ca²⁺flux in protoplasts,while in the tissue a large transient Ca²⁺efflux followed thesalt treatment. Salt-induced Ca²⁺efflux was essentially independentof external Ca²⁺concentrations in the range 0.1 to 10 m M. Also,Ca²⁺flux responses were ‘saturated’ above 50 m MNaCl. It is suggested that almost all the measured Ca²⁺fluxoriginates from Na⁺/Ca²⁺and H⁺/Ca²⁺ion exchange in the cellwall. This conclusion was supported by the results of modellingcation exchange in the cell wall. Copyright 2000 Annals of BotanyCompany Salinity, membrane transporters, wall ion exchange, proton, calcium, Vicia faba     

Effect of GA3 on the Molecular Mass of Polyuronides in the Cell Walls of Alaska Pea Roots

The role of cell wall matrix polysaccharides in gibberellin-regulatedroot growth is unknown. We examined pectic polysaccharides frompea roots treated with or without gibberellin A₃ (GA₃) in thepresence of ancymidol, an inhibitor of gibberellin biosynthesis.Pectic polymers solubilized by CDTA (trans-l,2-cyclohexanediamine-N,N,N',N'-tetraaceticacid) at 23°C and subjected to gel permeation analysis exhibitedhigh polydispersity with a molecular mass in excess of 500 kDa.Subsequent extraction of cell walls with CDTA at 100°C solubilizedpolymers with an average mol mass of 10 to 40 kDa. Subjectingthe high molecular mass pectic polymers extracted at 23°Cto 70–100°C for 2h generated 10 to 40 kDa fragments,similar in size distribution to those solubilized directly fromcell walls by CDTA solutions at 100°C. Pectic polymers from(GA₃+Anc)-treated roots were of higher average mol mass thanthose from Anc-treated roots in both the elongation zone andin the basal maturation zone. Since (GA₃+Anc)-treated rootselongate more quickly than Anc-treated roots [Tanimoto (1994)Plant Cell Physiol. 35:1019], the slender, GA₃-treated rootsmay produce and deposit highly integrated pectins more rapidlythan the thicker, Anc-treated roots in the elongating or elongatedcell walls. ²Present address: Horticultural Sciences Department, POB 110690IFAS, University of Florida, Gainesville, FL 32611-0690 U.S.A.     

Oriented structure of pectic polysaccharides in pea epidermal cell walls

Polarized infrared absorption spectra of film specimens of theepidermal cell wall of the third internode of pea stems wererecorded before and after treatment with endopolygalacturonase(endo-PG) and endo-pectin lyase (endo-PL). The spectra showedthat the pectic polysaccharides solubilized with endo-PG wereessentially the same as those solubilized with endo-PL. Thedegree of esterification of the pectic polysaccharides was about20%, and their major sugar components were uronic acids (32.8%),arabinose (48.1%) and galactose (19.2%). The polarized infraredspectra showed that pectic polysaccharides have an orientedstructure in cell walls with their molecular chains orientedpreferentially parallel to the direction of cell elongation. ¹Present address: Research and Development, Kanzaki Paper Mfg.Co., Ltd., Amagasaki, Hyogo 660, Japan. ²Present address: Wakayama Research Laboratories, Kao Soap Co.,Ltd., Wakayama 640-91, Japan. (Received June 28, 1980; )     

Ionic Diffusion Through the Nitella Cell Wall in the Presence of Calcium

Diffusion potentials (concentration and bi- or multi-ionic potentials)in KC1, NaCl, or LiCl solutions have been measured across anisolated cell wall of Nitella, with or without the same concentrationof CaCl² on either side of the cell wall. The absolute valueof the potentials decreases as the external Ca²⁺concentrationincreases and it may happen that an inversion of the sign ofthe concentration potentials results when the external Ca²⁺solution reaches 1 mM. Dosages of K⁺ and Ca²⁺ in the cell wallhave shown that Ca²⁺ easily displaces the monovalent ion fromthe exchange sites and tends to neutralize the cationic exchanger.However, in most cases, the measured potentials are still morenegative than the theoretical potentials which would be setup by a neutral-site membrane in the same conditions. Theseresults suggest that Ca²⁺ largely reduces the discriminationproperties of the cell wall between cations and anions.     

Acid-Induced Stomatal Opening in Commelina communis and Vicia faba

The effects of H^$ and fusicoccin (FC) on stomatal opening inthe dark were investigated using epidermal strips of Commelinacommunis and Vicia faba cv. Ryosai Issun. Citrate-phosphatebuffer induced maximal opening of stomata at pH 3.0 when testedover the range of 2.7 to 5.0. HCl at 1 mM also induced stomatalopening without appreciable accumulation of K^$ in the guardcells. After 4 hr treatment with 10 µM FC, stomata openedwith concomitant accumulation of K^$ in the guard cells, although1–2 hr treatment caused opening without concomitant K^$increase. These results suggest that stomatal opening can be caused bysalt accumulation and/or changes of the physicochemical conditionsin the cell wall of the guard cells due to high acidity. ¹ Present address: Biological Laboratory, Faculty of Education,Nagasaki University, Nagasski 852, Japan. (Received April 30, 1982; Accepted July 17, 1982)     

Effect of Phosphorus Deficiency on Phosphatase Activity of Cell Walls from Roots of Subterranean Clover

Clover (Trifolium subterraneum L. cv. Mt. Barker) was grownin solution culture with adequate (+P) or no phosphate (–P).Cell walls were extracted from roots in such a way that theywere uncontaminated by other cellular materials. Phosphataseactivity was assayed using p-nitro-phenylphosphate (NPP). Phosphatasebound to cell walls had a pH optimum between 5.0 and 6.0, irrespectiveof the P supply to the plants. Activity of phosphatase boundto cell walls increased with electrolyte concentration of theassay medium at pH 6.5 but not at pH 5.5. This increase in activitywas probably due to a higher degree of ionization of the cellwall at pH 6.5 than at pH 5.5, and to effects of high ionicstrength in decreasing the mutual repulsion of negatively chargedNPP from negative charges on the cell walls. Cell wall-boundphosphatase did not exhibit Michaelis-Menten kinetics: the concentrationof NPP at which activity was half the maximum rate (S_0.5) was0.7 mM for cell walls extracted from roots of both +P and –Pplants. Up to 30% of the phosphatase activity bound to cellwalls could be removed using buffer solutions of high pH andhigh ionic strength which contained Triton X100. Both soluble and cell wall-bound phosphatase(s) of roots increasedin activity with P deficiency. The phosphatase activity of cellwalls increased 1.5 fold as the P concentration in the rootsfell from 0.4–0.2% dry weight. Experiments with sterileroots of clover showed that increases in cell wall-bound phosphataseactivity associated with P deficiency were not due to microbialcontamination. It is argued that phosphatase(s) in cell wallsof roots could make a substantial contribution to the P nutritionof clover in soils deficient in inorganic phosphate by hydrolysingorganic phosphate compounds in the soil. Key words: Phosphatase, Clover, Roots, Phosphorus deficiency, Cell walls     

Changes of Some Membrane-Associated Enzyme Activities Degradation of Membrane Phospholipids in Cucumber Roots Due to Ca2+ Starvation

Deprivation of Ca²⁺ from a complete culture medium affectedthe enzyme activities associated with five membrane fractionsof cucmber roots obtained by discontinuous sucrose density gradientcentrifugation. The total activity of K⁺-ATPase, Cyt. c oxidaseand NADPH-Cyt. c reductase of Ca²⁺-deficient roots, starvedfor only 4 days, had decreased to 14, 38 and 60% of the activityof the control roots. In general, loss of enzyme activitieswas accompanied by a shift of activity distribution from theheavier density fractions to lighter ones. The amounts of Ca²⁺ associated with membranes from Ca²⁺-starvedroots decreased to 50–60% of those of the control roots.Both phospholipid and neutral lipid contents in the membranesdecreased markedly while the protein content was not changedby Ca²⁺ deficiency. Phospholipid analysis indicated a drasticdrop in the percent composition of phosphatidylinositol butan increase of phosphatidic acid. Also, phospholipase D activityincreased remarkably during Ca²⁺ starvation, paralleling theappearance of Ca²⁺-deficiency symptoms. Thus, the major effects of Ca²⁺ deficiency appear to be to stimulatephospholipase D activity and a reduction in membrane bound Ca²⁺.These effect may be involved in disorganization of the membranestructure and the changes of enzyme activities associated withthe altered membranes. ¹Rubber Research Institute of Sri Lanka, Dartonfield, Agalwatta,Sri Lanka. (Received July 15, 1985; Accepted November 21, 1985)     

Control of Glutamate Dehydrogenase from Green Tobacco Callus Mitochondria by Ca2$ and pH

Glutamate dehydrogenase (GDH) (EC 1.4.1.3 [EC] .) purified from greentobacco callus mitochondria was activated markedly by Ca^2$ inthe amination reaction. This activation was detectable evenat concentrations below 5 µM Ca^2$. Saturation curves for the three substrates of the aminationreaction showed normal Michaelis-Menten kinetics in the presenceof 1 mM of Ca^2$, but pronounced substrate inhibition occurredwithout Ca^2$. The effect of Ca^2$ was chiefly on the maximalvelocity. The saturation curve for NH₄Cl in the presence of Ca^2$ was modulatedby a change in pH. The apparent K_m value for NH₄Cl markedlydecreased whereas that for -ketoglutarate increased slightlywhen the pH was raised from 7.3 to 9.0. In contrast, the K_mfor NADH was little affected by raising the pH. The characteristicof GDH which increases its affinity for NH₄Cl when the pH israised may be compatible with the detoxification of ammonia. ¹ Present address: Mochida Pharmaceutical Co., Ltd. (Received August 24, 1981; Accepted November 28, 1981)     

Glucose 6-phosphate dehydrogenase from sweet potato: Effect of various ions and their ionic strength on enzyme activity

Activity of glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate:NADP oxidoreductase, EC 1.1.1.49 [EC] ) preparation from sweet potatoroot tissue was markedly altered in the presence of variousions. Cations or anions were effective in the following order:Na^$, K^$>Tris^$>NH₄^$>Mg^2$>Ca^2$, or Cl^–>NO₃^–,HPO₄^2–>SO₄^2–>HCO₃^–. Activity was inhibitedat high concentrations of Ca^2$, and HCO₃^–,. In an investigationon the dependence of the activity on pH, two activity peakswere clearly observed at low ionic strength. Ionic strength altered both the Km and Vmax for glucose 6-phosphate(G6P). A Lineweaver-Burk plot for the enzyme, with respect toG6P, showed a bimodal nature at low ionic strength; suggestingnegative cooperativity. Deviation from linearity of the plotwas less with an increase in the ionic strength. ¹ Present address: Institute of Applied Microbiology, Universityof Tokyo, Bunkyo-ku, Tokyo 113. (Received September 18, 1971; )     

Some Characteristics of Membrane-Associated Protein Kinase in Lemna paucicostata

A protein kinase which phosphorylates histone was isolated fromthe endoplasmic reticulum-rich fractions of Lemna paucicostata.The enzyme could be solubilized by sonication, and its molecularweight was estimated as 220,000 by Sephacryl S-300 gel filtration.The optimum pH for enzyme activity was 9.0–9.5 and theactivity was stimulated by Co^2$, Mg^2$ and Mn^2$. Substrate proteinswhich might be phosphorylated by this protein kinase were alsodetected in microsomal fractions of Lemna plants. ¹ Present address: Advanced Research Laboratory, HITACHI LTD.,Kokubunji, Tokyo 185, Japan.     

Studies on the Cell Wall of Chlorella III. Incorporation of Photosynthetically Fixed Carbon into Cell Walls of Synchronously Growing Cells of Chlorella ellipsoidea

The carbon metabolism in cell walls of Chlorella ellipsoideawas studied by following ¹⁴C incorporation into cell wall constituentsin photosynthesizing, synchronously growing cells. The rateof incorporation was higher at an early growth phase of thecell cycle. The ¹⁴C was incorporated into both the major cellwall constituents, hemicellulose and ‘rigid wall’,and the radioactivity in the latter was distributed into itstwo components, glucosamine and amino acids. In pulse-chaseexperiments, the ¹⁴C fixed photosynthetically in the precedingcell cycle was rapidly transferred into the cell wall constituentsat the early growth phase of the ongoing cell cycle, and thereafterwas gradually released from the cell walls, although the totalamount of ¹⁴C in the cells remained constant. It was concludedthat the cell wall constituents are turned over during the growthphase of the algal cell cycle, and that the cell wall metabolismin the ongoing cell cycle is closely connected with the carbonmetabolism in the preceding cell cycle. (Received February 3, 1982; Accepted June 21, 1982)     

Specific Response of Partially Purified Cell Wall-Bound ATPases to Fungal Suppressor

It was found that NTPases were bound to cell walls of pea andcowpea. The suppressor in pycnospore germination fluid of apea pathogen, Mycosphaerella pinodes, inhibited the ATPase activityin the fraction, which was solubilized from pea cell wall with0.5% Triton X-100, in a dose-dependent manner, but rather enhancedthat from cowpea cell wall even at the concentration of 1 µgml^-1. Inhibition by the suppressor of pea cell wall-bound ATPasewas a mixed type of competitive and noncompetitive. Triton X-100PAGE and active staining of ATPase indicated that both TritonX-100 solubilized fractions contained plural molecules thathydrolyze ATP. The M_rs of cell wall-bound ATPases seem to beconsiderably different from those of plasma membranes, and thenumber of cell wall-bound ATPase molecules were different betweenpea and cowpea. The electroeluted fractions corresponding tothe bands of active-stained ATPases were also able to hydrolyzeNTP and PP_i. The respective electroeluted ATPases also showedthe species-specific response to the suppressor. These resultsmay confirm our previous concept that putative receptors forthe suppressor might tightly bind to cell wall-bound ATPaseor that the ATPase might be the receptor itself. (Received September 8, 1995; Accepted March 9, 1996)     

Purification of Major Isozymes of Nuclease C and Production of Active Fragments by Trypsin

Most nucleases from gametes of Chlamydomonas reinhardtii needCa^2$ for full activation. They have been named nuclease C andat least six species of isozymes have been found in the femalegamete (Ogawa and Kuroiwa 1985a). Nuclease C1&2 and C3 were purified from the vegetative cellsof this organism. Nuclease C1&2 exhibited a sharp pH optimumat 9.5, while nuclease C3 preferred a more neutral pH at 7.0–8.5.Use of the Ca^2$-EGTA [ethylene-glycol-bis-(2-aminoethyl ether)-N,N,N',N'-tetraaceticacid] buffer in the reaction mixture made it possible to determinetheir activity at the physiological Ca^2$ concentration. NucleaseC3 was not activated at low Ca^2$ concentration and exhibiteda sharp optimum at 10^–3{small tilde}10^–4 M. NucleaseC1&2 were activated at a physiological concentration of10^–6 M; increasing the Ca^2$ concentration did not affectthe activity. Nuclease C gave active fragments upon trypsin digestion. Trypticfragments of nuclease C1&2 and C3 had molecular weightsof 21,000 (referred as C6T) and 16,000 (C4T), respectively.Upon regulating the digestion, a few fragments were identifiedas intermediates of nuclease C6T by the in situ nuclease assay.These tryptic fragments were similar in molecular size to theminor components of nuclease C found in the cell lysates ofgametes and early zygotes. This finding suggests that a minorspecies of nuclease C may be produced from the major nucleaseC during gametogenesis. (Received June 26, 1985; Accepted August 28, 1985)