The essential role of Ca2+ in the activity of bovine pancreatic deoxyribonuclease.

DNase requires Ca2+ for activity against DNA with Mg2+. The Ca2+ selective chelating agent, ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid, (EGTA) inhibits DNase completely at pH 7 or 8, and subsequent addition of excess Ca2+ reverses inhibition in less than one second. DNase action can be stopped at any point by the addition of excess EGTA over Ca2+. Ca2+ is required for DNase to bind substrate. Gel filtration experiments fail to show DNase binding to 0.2 mg per ml of DNA at 5 mm Mg2+ and 10-4 M EGTA. The concentration of Ca2+ needed for half of maximum DNase activity decreases with increases DNA concentration, from 1.2 times 10-5 M Ca2+ at 2.3 times 10-5 M DNA-P to about 4 times 10-7 M Ca2+ at 2.3 DNA-P. Kinetic analysis by the titrametic assay of protons releases shows that V max is independent of Ca2+ concentration while Km increases from 7.7 times 10-5 M DNA-P at 5 times 10-4 M Ca2+ to 3.4 times 10-4 M DNA-P at 5 times 10-6 M Ca2+. Both of these results are predicted by a rate equation which is derived from the assumption that DNase must bind Ca2+ before it can bind DNA. The essential Ca2+ atom probably binds to the one of two high affinity Ca2+ binding sites on DNase which cannont bind Mg2+ or Mn2+. The only other divalent metal ions which can bind to this site, Sr2+ and Ba2+, are also the only metal ions which can substitute for Ca2+ in DNase action against DNA with Mg2+. Some DNase activity is obtained in the absence of added Ca2+ with Mg2+ at pH 6 or below and with Mn2+ or Co2+ at pH 8. These assay solutions are contaminated by 1 to 3 muM Ca2+, which may be sufficient to account for the observed activity.     

Binding of antibodies and other proteins to nitrocellulose in acidic, basic, and chaotropic buffers.

This report compares the binding of proteins to nitrocellulose membranes in acidic buffers (pH 2 and 3) with binding in neutral buffer (pH 7), basic buffers (pH 12 and 13), 8 M urea (pH 2, 3, and 7), and 6 M guanidine hydrochloride (pH unadjusted). Initially, similar amounts of antibodies and other proteins bound to the nitrocellulose membrane in all of these buffers and solvents. However, the susceptibility of individual proteins to displacement (stripping) from the membrane by the milk blocking agent depended on both the pH and the type of buffer or solvent used to bind the proteins to the membrane. Most proteins that were bound to nitrocellulose in acidic buffers were relatively resistant to milk stripping compared to proteins bound in pH 7 buffer. After correction for the amount of antibody remaining on the membrane after the milk block, it was found that acid-bound antibodies were unchanged in biological activity when compared with the same antibodies bound at neutral pH. These results suggest that acid binding of proteins could increase the sensitivity of nitrocellulose membrane assays using a milk block.     

Deoxyribonuclease II activity in relation to cell cycle in synchronized HeLa S3 cells

Studying the activity of DNase II in relation to cell cycle in synchronized HeLa S3 cells show a two to seven fold increase in DNase II activity at those times when DNA synthesis is taking place. The peaks of DNase II activity coincide with the peaks of DNA synthesis. The increased DNase II activity could be prevented by puromycin, suggesting that the enzyme activity increased at the S phase was caused by synthesis of new molecules rather than the activation of existing molecules. Acid phosphatase (as a marker for lysosomal enzymes) does not show an induction similar to that observed for DNase II in relation to cell cycle.     

Deoxyribonuclease II purified from the isolated lysosomes of porcine spleen and from porcine liver homogenates. Comparison with deoxyribonuclease II purified from porcine spleen homogenates

Porcine spleen DNase II (EC 3.1.22.1), one of the best-characterized DNases II, is subcellularly located in lysosomes because the enzyme is co-sedimented with two of the lysosomal marker enzymes, cathepsin D and acid phosphatase. The physicochemical properties, including the subunit structure, sensitivity to iodoacetate inactivation, native molecular weight and chromatographic behavior, of the DNase II purified from the isolated lysosomes of porcine spleen are indistinguishable from those of the same enzyme purified from the whole porcine spleen homogenate. DNase II can also be extracted from porcine liver with 0.05 M H2SO4 or 0.1 M NaCl and purified from either extract by a series of column chromatographies. The purified liver DNase II from either extract has the same subunit structure (alpha-chain, Mr 35,000 and beta-chain, Mr 10,000) as the purified DNase II of porcine spleen. The two liver extracts as well as the extracts of spleen and gastric mucosa contain DNase II with very similar properties on Sephadex G-100 gel filtration, on acid polyacrylamide gel electrophoresis under non-denaturing conditions, and on isoelectric focusing. The data strongly suggest that, for the same species of animal, the DNase II activities in various tissues are associated with protein molecules of identical structure.     

The interaction of phosphorylated oligosaccharides and lysosomal enzymes with bovine liver cation-dependent mannose 6-phosphate receptor

We have analyzed the interaction of phosphorylated oligosaccharides and lysosomal enzymes with immobilized bovine liver cation-dependent mannose-6-P receptor. Oligosaccharides with phosphomonoesters were the only species that interacted with the receptor, and molecules with two phosphomonoesters showed the best binding. Lysosomal enzymes with several oligosaccharides containing only one phosphomonoester had a higher affinity for the receptor than did the isolated oligosaccharides, indicating the possible importance of multivalent interactions between weakly binding ligands and the receptor. The binding of a mixture of phosphorylated lysosomal enzymes to the cation-dependent Man-6-P receptor was markedly influenced by pH. At pH 6.3, almost all of the lysosomal enzymes bound to the receptor; whereas at pH 7.0-7.5, approximately one-third of the material passed through the column, one-third interacted weakly, and one-third bound tightly. The distribution of individual lysosomal enzyme activities was similar to that of the total material. The species of phosphorylated oligosaccharides present on the lysosomal enzymes which interacted poorly with the receptor were similar to those found on the tightly bound material and included species of oligosaccharides with two phosphomonoester groups. Isolated oligosaccharides of this type bound to the receptor over the entire pH range tested. These findings indicate that at neutral pH the phosphorylated oligosaccharides on some lysosomal enzyme molecules are oriented in a manner which makes them inaccessible to the binding site of the cation-dependent Man-6-P receptor. Since the same enzymes bind to the cation-independent Man-6-P receptor at neutral pH, at least a portion of the phosphomannosyl residues must be exposed. We conclude that small variations in the pH of the Golgi compartment where lysosomal enzymes bind to the receptors could potentially modulate the extent of binding to the two receptors.     

Growth hormone modulates the degradative capacity of muscle nucleases but not of cathepsin D in post-weaning mice

We determined whether recombinant human growth hormone (rhGH) administration might modulate the enzyme degradative capacity of the muscle lysosomal system and influence muscle growth. Muscle cathepsin D, acid RNase and DNase II activities are determined in the gastrocnemius muscle of rhGH-treated post-weaning female BALB/c mice. Linear regressions were used to analyze the relationships of each enzyme with their respective substrate. GH induced a depletion-recovery response of muscle growth through a mechanism which is similar to catch-up growth. In these conditions, cathepsin D activity decreased with age in all animals (GH: 40%; saline: 79%), showing a substantial developmental decline that could reflect changes in the rate of protein breakdown. However, the degradative capacity of cathepsin D was paradoxically unmodified in rhGH-mice compared with saline mice (according to the enzyme vs. substrate linear regression slope), in spite of the increase in enzyme activity elicited by GH. This suggests that the muscle protein breakdown is not increased by GH-treatment in post-weaning mice. The enhancement of muscle protein deposition as indicated by the augmented muscle cell size (protein:DNA ratio) of rhGH-mice (increased 178% from 25 to 50 days) vs. saline, can be attributed to a higher muscle K(RNA). In contrast, acid RNase and DNase II activities directly participate in muscle RNA and DNA degradation. Both nucleases were inhibited by GH treatment (a decrease of 48% and 63%, respectively, vs. saline at 50 days). The decrease in RNase activity suggests an inverse relation between the rate of protein synthesis (high) and acid RNase activity (low), leading to spare muscle RNA for synthesizing protein during catch-up growth. Also, low DNase II activity could contribute to inhibiting of muscle DNA degradation, facilitating muscle growth. Thus, GH seems to act as a direct modulator of the degradative capacity of skeletal muscle nucleases but not of cathepsin D, influencing DNA and RNA degradation during the depletion-recovery response to GH of gastrocnemius muscle in female post-weaning mice.     

Fusion of enveloped viruses with cells and liposomes. Activity and inactivation.

The fusion of viruses with cells and liposomes is reviewed with focus on the analysis of the final extents and kinetics of fusion. Influenza virus and Sendai virus exhibit 100% of fusion capacity with cells at pH 5 and pH 7.5, respectively. On the other hand, there may be in certain cases, a limit on the number of virions that can fuse with a single cell, that is significantly below the limit on binding. It still remains to be resolved whether this limit reflects a limited number of possible fusion sites, or a saturation limit on the amount of viral glycoproteins that can be incorporated in the cellular membrane, like the case of virus fusion with pure phospholipid vesicles, in which the fusion products were shown to consist of a single virus and several liposomes. Both viruses demonstrate incomplete fusion activity towards liposomes of a variety of compositions. In the case of Sendai virus, fusion inactive virions bind essentially irreversibly to liposomes. Yet, preliminary results revealed that such bound, unfused virions can be released by sucrose gradient centrifugation. The separated unfused virions subsequently fuse when incubated with a "fresh" batch of liposomes. We conclude, therefore, that the fraction of initially bound unfused virions does not consist of dective particles, but rather of particles bound to liposomes via "inactive" sites. Details of the low pH inactivation of fusion capacity of influenza virus towards cells and liposomes are presented. This inactivation is caused by protonation and exposure of the hydrophobic segment of HA2, and affects primarily the fusion rate constants. Some degree of inactivation also occurs when virions are bound to cellular membranes.     

Deoxyribonuclease II from the Icelandic scallop (Chlamys islandica): isolation and partial characterization

A deoxyribonuclease (DNase) was isolated from viscera of the cold-adapted marine bivalve Icelandic scallop. The 42 kDa DNase was shown to be a single polypeptide which catalyses DNA hydrolysis in the absence of divalent cations. The isolated enzyme showed maximal activity at pH 6 and no activity above pH 7.2 against native DNA. The scallop DNase was slightly more susceptible to heat denaturation than porcine DNase II and makes double-strand breaks in circular DNA substrate as the porcine enzyme. The N-terminal sequence of the scallop DNase was shown to be closely similar to DNase II (EC 3.1.22.1) proteins from other organisms. The scallop DNase is in addition to plancitoxin I from A. planci, the only DNase II enzyme isolated from marine invertebrates.     

Characterization of human alpha fetoprotein charge microheterogeneity during fetal development.

Aliquotes of human amniotic fluid (AF), fetal serum (FS), and cord blood (CB) were obtained as by-products of routine clinical diagnostic procedures at term or in the second trimester of pregnancy. When samples of CB were applied to a pH 5.5-4 chromatofocusing gradient, three isoforms of AFP could be resolved; a pl 4.57 form (isoform IA, 52% AFP), a pl 4.27 form (isoform IB, 43% AFP), and one species that was bound to the column but could be eluted with 1.0 M NaCl (isoform II, pl less than 4.00, 5% AFP). Term AF displayed a profile similar to that observed in term CB. When samples of 15-20-week gestation AF were chromatofocused, the immunoreactive AFP recovered was distributed between isoform IA and IB (60%) and isoform II (40%). FS and AF obtained from same pregnancy (23-26 weeks) displayed an identical chromatofocusing profile. Aliquotes of AF subjected to conA revealed 83% reactive variants compared with greater than 95% reactive variants for CB. FS displayed a conA profile identical to CB. When individual CB charge isoforms were isolated and subjected to conA analysis, greater than 97% of the AFP bound to conA. In contrast, when AFP isoform IA and IB were isolated from midgestation AF, approximately 22% of the AFP did not bind to the lectin while 100% of isolated AFP isoform II eluted as the reactive variant. These data suggest that human AFP exists as at least three charge and two lectin variants and that the charge profile may change during fetal development.     

Changes in allosteric properties of phosphofructokinase bound to erythrocyte membranes.

Human and rabbit erythrocyte membranes prepared by hypotonic hemolysis contained 5 to 15% of the phosphofructokinase in the erythrocytes. The membrane-bound phosphofructokinase can be eluted by a saline wash. Human erythrocyte and rabbit muscle phosphofructokinase bind to the saline-washed membranes. This binding is specific for the inner surface of the membrane. The amount of phosphofructokinase bound is dependent on pH; at pH 7, 6 times more enzyme is bound than at pH 7.5. Unlike free phosphofructokinase, the membrane-bound phosphofructokinase is not inhibited by ATP or 2,3-diphosphoglycerate, and its fructose-6-P saturation curve is nonsigmoidal.     

Implications of CAD and DNase II in ischemic neuronal necrosis specific for the primate hippocampus

The exact molecular mechanism of ischemic neuronal death still remains unclear from rodents to primates. A number of studies using lower species animals have suggested implication of apoptosis cascade, while using monkeys the authors recently claimed necrosis cascade by calpain-induced leakage of lysosomal cathepsins (calpain-cathepsin hypothesis). This paper is to study implications of apoptotic versus necrotic cascades for the development of hippocampal CA1 neuronal death in the primate brain undergoing complete global ischemia. Here, we focused on two terminal cell death effectors; caspase-activated DNase (CAD) and lysosomal enzyme DNase II, in the monkey CA1 sector undergoing 18 min ischemia. The expressions of their mRNA and proteins, and the subcellular localizations as well as ultrastructure and specific DNA gel electrophoresis were examined. Expression of CAD was much less in the normal brain, compared with the lymph node or heart tissues. On day 1 after ischemia, however, CAD mRNA and protein were significantly increased in the CA1 sector, and then CAD protein immunohistochemically showed a translocation from the perikarya into the nucleus. Activated DNase II protein was significantly increased on days 2 and 3 after ischemia, and also showed a similar translocation indicating lysosomal leakage. Although the post-ischemic CA1 neurons showed positive terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining on days 3-5, they showed eosinophilic coagulation necrosis on light microscopy, and frank membrane disruption and mild chromatin condensation on electron microscopy. Furthermore, DNA smear pattern typical for necrosis was observed instead of DNA laddering. These data altogether suggest that the post-ischemic CA1 neuronal death of the monkey occurs not by apoptosis but by necrosis with participations of lysosomal enzymes DNase II and cathepsins as well as CAD. The interactions between apoptotic (caspase-3 and CAD) and necrotic (calpain, cathepsin and DNase II) cascades should be studied further.     

Use of Surface Plasmon Resonance and Biolayer Interferometry for the Study of Protein–Protein Interactions on the Example of an Enzyme of a Glycosyl Hydrolase Subtype (EC 3.2.1) and Specific Antibodies to It

Two previously obtained, full-size, fully human antibodies that reversibly bind the active form of an enzyme belonging to the subtype EC 3.2.1, which is used for substitutive enzymatic therapy in lysosomal storage diseases, have been characterized by surface plasmon resonance and biolayer interferometry methods. It was shown under normal physiological conditions that the designed antibodies specifically bound with the antigen (KD ~ 10^–8 M) and rapidly dissociated at neutral pH in 60% ethylene glycol while leaving the enzymatic activity unchanged. Dan ue to their properties, the developed antibodies can be used in industry as affinity ligand in the isolation of therapeutic substances of the enzyme.     

Transformation of Bacillus subtilis by DNA bound on montmorillonite and effect of DNase on the transforming ability of bound DNA.

The equilibrium adsorption and binding of DNA from Bacillus subtilis on the clay mineral montmorillonite, the ability of bound DNA to transform competent cells, and the resistance of bound DNA to degradation by DNase I are reported. Maximum adsorption of DNA on the clay occurred after 90 min of contact and was followed by a plateau. Adsorption was pH dependent and was greatest at pH 1.0 (19.9 micrograms of DNA mg of clay-1) and least at pH 9.0 (10.7 micrograms of DNA mg of clay-1). The transformation frequency increased as the pH at which the clay-DNA complexes were prepared increased, and there was no transformation by clay-DNA complexes prepared at pH 1. After extensive washing with deionized distilled water (pH 5.5) or DNA buffer (pH 7.5), 21 and 28%, respectively, of the DNA remained bound. Bound DNA was capable of transforming competent cells (as was the desorbed DNA), indicating that adsorption, desorption, and binding did not alter the transforming ability of the DNA. Maximum transformation by bound DNA occurred at 37 degrees C (the other temperatures evaluated were 0, 25, and 45 degrees C). DNA bound on montmorillonite was protected against degradation by DNase, supporting the concept that "cryptic genes" may persist in the environment when bound on particulates. The concentration of DNase required to inhibit transformation by bound DNA was higher than that required to inhibit transformation by comparable amounts of free DNA, and considerably more bound than free DNase was required to inhibit transformation by the same amount of free DNA. Similarly, when DNA and DNase were bound on the same or separate samples of montmorillonite, the bound DNA was protected from the activity of DNase.     

N-acetyl-beta-d-glucosaminidase in marmoset kidney, serum and urine.

N-Acetyl-beta-D-glucosaminidase activities were determined in homogenates of marmoset kidney, in serum and in urine by using the 4-methylumbelliferyl substrate. The enzyme activity was separated into several components by DEAE-cellulose ion-exchange chromatography, starch-gel electrophoresis and isoelectric focusing. The kidney contained two major forms of the enzyme, A and B, which had similar pH optima and Km values. The A-form bound to DEAE-cellulose at pH 6.8, migrated towards the anode on starch-gel electrophoresis and had a pI of 5.0. The B-form did not bind to DEAE-cellulose at pH 6.8, remained near the origin on starch-gel electrophoresis and had a pI of 7.64. The isoenzymes also differed in heat stability, the B-form being the more stable. Serum contained B-form activity and, in addition, two intermediate forms (I1 and I2) were loosely bound to DEAE-cellulose. The serum A-form activity was less firmly bound to DEAE-cellulose than was the tissue A-form and was designated As. Serum from a pregnant marmoset contained a form which may be analogous to the human P-isoenzyme. Urine contained only a small amount of B-form activity, the majority being present in the A-form. The kidney A- and B-forms both had mol.wts. of 96000--100000 and the activity was predominantly lysosomal. Partial purification of the kidney A isoenzyme was undertaken. Immunoprecipitation studies indicated a relationship between marmoset kidney A-form and human liver A-form activity.     

Binding of triple helix forming oligonucleotides to sites in gene promoters

A class of triplex-forming oligodeoxyribonucleotides (TFOs) is described that can bind to naturally occurring sites in duplex DNA at physiological pH in the presence of magnesium. The data are consistent with a structure in which the TFO binds in the major groove of double-stranded DNA to form a three-stranded complex that is superficially similar to previously described triplexes. The distinguishing features of this class of triplex are that TFO binding apparently involves the formation of hydrogen-bonded G.GC and T.AT triplets and the TFO is bound antiparallel with respect to the more purine-rich strand of the underlying duplex. Triplex formation is described for targets in the promoter regions of three different genes: the human c-myc and epidermal growth factor receptor genes and the mouse insulin receptor gene. All three sites are relatively GC rich and have a high percentage of purine residues on one strand. DNase I footprinting shows that individual TFOs bind selectively to their target sites at pH 7.4-7.8 in the presence of millimolar concentrations of magnesium. Electrophoretic analysis of triplex formation indicates that specific TFOs bind to their target sites with apparent dissociation constants in the 10(-7)-10(-9) M range. Strand orientation of the bound TFOs was confirmed by attaching eosin or an iron-chelating group to one end of the TFO and monitoring the pattern of damage to the bound duplex DNA. Possible hydrogen-bonding patterns and triplex structures are discussed.     

Transformation of Bacillus subtilis by DNA bound on montmorillonite and effect of DNase on the transforming ability of bound DNA.

The equilibrium adsorption and binding of DNA from Bacillus subtilis on the clay mineral montmorillonite, the ability of bound DNA to transform competent cells, and the resistance of bound DNA to degradation by DNase I are reported. Maximum adsorption of DNA on the clay occurred after 90 min of contact and was followed by a plateau. Adsorption was pH dependent and was greatest at pH 1.0 (19.9 micrograms of DNA mg of clay-1) and least at pH 9.0 (10.7 micrograms of DNA mg of clay-1). The transformation frequency increased as the pH at which the clay-DNA complexes were prepared increased, and there was no transformation by clay-DNA complexes prepared at pH 1. After extensive washing with deionized distilled water (pH 5.5) or DNA buffer (pH 7.5), 21 and 28%, respectively, of the DNA remained bound. Bound DNA was capable of transforming competent cells (as was the desorbed DNA), indicating that adsorption, desorption, and binding did not alter the transforming ability of the DNA. Maximum transformation by bound DNA occurred at 37 degrees C (the other temperatures evaluated were 0, 25, and 45 degrees C). DNA bound on montmorillonite was protected against degradation by DNase, supporting the concept that "cryptic genes" may persist in the environment when bound on particulates. The concentration of DNase required to inhibit transformation by bound DNA was higher than that required to inhibit transformation by comparable amounts of free DNA, and considerably more bound than free DNase was required to inhibit transformation by the same amount of free DNA. Similarly, when DNA and DNase were bound on the same or separate samples of montmorillonite, the bound DNA was protected from the activity of DNase.     

The effects of elevated pH and high salt concentrations on tubulin

The effects of incubating phosphocellulose-purified bovine tubulin at 4 degrees C in nucleotide-free buffers at alkaline pH or at high concentrations of NaCl, KCl, (NH4)2SO4, or NH4Cl have been studied. At pH greater than or equal to 7.5 or at NaCl concentrations greater than or equal to 0.7 M, tubulin releases bound nucleotides irreversibly and loses, with apparent first-order kinetics, the ability to assemble into microtubules. In 0.1 M 1,4-piperazinediethanesulfonic acid buffer, pH 6.9, in the presence of 1.3 M NH4Cl, tubulin undergoes more rapid loss of capacity to assemble than it does in NaCl and KCl, but 1.3 M (NH4)2SO4 causes no detectable change in tubulin after 1-h incubation. Incubation at high pH or at high neutral salt concentrations also causes an apparently irreversible change in the ultraviolet difference spectrum and in the sedimentation velocity profile of tubulin. At elevated salt concentrations a decrease of approximately 10% in the molar ellipticity within the wavelength range 220-260 nm is observed. The changes that occur during 1-h exposure to pH 8.0 can be completely prevented by including 1 mM guanosine 5'-triphosphate (GTP) or 4 M glycerol in the buffer, but those which occur at pH 9.0 cannot be prevented by these additions. In 1 M NaCl when the ratio of bound guanine nucleotide to tubulin reaches approximately 1.0, tubulin loses the abilities to assemble into microtubules and to bind colchicine. The rate of loss of nucleotide in 2 M NaCl is decreased in the presence of 1 mM GTP, and tubulin is protected almost completely from 1 M NaCl-induced loss of GTP (and retains the ability to exchange [3H]GTP as well) in the presence of bound colchicine. Investigators who anticipate exposing tubulin to buffers of elevated pH or high concentrations of chaotropic salts should be extremely cautious in interpreting the resulting data unless they can demonstrate that irreversible alteration of the protein has not occurred.     

The binding requirements of monkey brain lysosomal enzymes to their immobilised receptor protein

The lysosomal enzyme binding protein (receptor protein) isolated from monkey brain was immobilised on Sepharose 4B and used to study the binding of brain lysosomal enzymes. The immobilised protein could bind \-D-glucosaminidase, α-D-mannosidase, α-L-fucosidase and2-D-glucuronidase. The bound enzymes could be eluted either at an acid pH of 4.5 or by mannose 6-phosphate but not by a number of other sugars tested. Binding could be abolished by prior treatment of the lysosomal enzymes with sodium periodate. Alkaline phosphatase treatment of the enzymes did not prevent the binding of the lysosomal enzymes to the column but decreased their affinity, as seen by a shift in their elution profile, when a gradient elution with mannose 6-phosphate was employed. These results suggested that an ‘uncovered’ phosphate on the carbohydrate moiety of the enzymes was not essential for binding but can enhance the binding affinity.     

Association of deoxyribonuclease I with the pointed ends of actin filaments in human red blood cell membrane skeletons

We have characterized the interaction of bovine pancreatic deoxyribonuclease I (DNase I) with the filamentous (F-)actin of red cell membrane skeletons stabilized with phalloidin. The hydrolysis of [3H]DNA was used to assay DNase I. We found that DNase I bound to a homogenous class of approximately equal to 2.4 X 10(4) sites/skeleton with an association rate constant of approximately 1 X 10(6) M-1 S-1 and a KD of 1.9 X 10(-9) M at 20 degrees C. Phalloidin lowered the dissociation constant by approximately 1 order of magnitude. The DNase I which sedimented with the skeletons was catalytically inactive but could be reactivated by dissociation from the actin. Actin and DNA bound to DNase I in a mutually exclusive fashion without formation of a ternary complex. Phalloidin-treated red cell F-actin resembled rabbit muscle G-actin in all respects tested. Since the DNase I binding capacity of the skeletons corresponded to the number of actin protofilaments previously estimated by other methods, it seemed likely that the enzyme binding site was confined to one end of the filament. We confirmed this premise by showing that elongating the red cell filaments with rabbit muscle actin monomers did not appreciably add to their capacity to bind or inhibit DNase I. Saturation of skeletons with cytochalasin D or gelsolin, avid ligands for the barbed end of actin filaments, did not reduce their binding of DNase I. Furthermore, neither cytochalasin D nor DNase I alone blocked all of the sites for addition of monomeric pyrene-labeled rabbit muscle G-actin to phalloidin-treated skeletons; however, a combination of the two agents did so. In the presence of phalloidin, the polymerization of 300 nM pyrenyl actin on nuclei constructed from 5 nM gelsolin and 25 nM rabbit muscle G-actin was completely inhibited by 35 nM DNase I but not by 35 nM cytochalasin D. We conclude that DNase I associates uniquely with and caps the pointed (slow-growing or negative) end of F-actin. These results imply that the amino-terminal, DNase I-binding domain of the actin protomer is oriented toward the pointed end and is buried along the length of the actin filament.     

Conformational control of ovoperoxidase catalysis in the sea urchin fertilization membrane

The ovoperoxidase-catalyzed oxidation of iodide has been investigated as a function of pH for the homogeneous enzyme and for ovoperoxidase incorporated into several forms of the egg fertilization membrane. The pH dependent hysteresis previously observed in purified ovoperoxidase (Deits, T. L., Shapiro, B. M. (1985) J. Biol. Chem. 260, 7882-7888) is entirely absent in ovoperoxidase incorporated into the mature fertilization membrane, where the enzyme is bound noncovalently in vivo. The pH activity profile of ovoperoxidase incorporated into the mature fertilization membrane closely resembles the profile observed only transiently in purified ovoperoxidase subjected to a rapid downward pH shift. These observations can be accounted for by our previously presented mechanism for ovoperoxidase hysteresis (ibid.). We hypothesize that ovoperoxidase, upon incorporation into the fertilization membrane, is restricted to a limited subset of the conformational states available to the purified enzyme. This matrix-dependent conformational restriction is a novel control mechanism that serves to enhance the catalytic activity of ovoperoxidase upon its assembly into the fertilization membrane and thereby modulates ovoperoxidase catalysis in the vicinity of the developing egg.