A monoclonal antibody against human vitamin-D-binding protein for the analysis of genetic variation in the group-specific component system (Gc)

Summary We have developed a murine hybridoma cell line that is stable in secreting a monoclonal antibody (hDBP-1) directed against the group-specific component (Gc) molecule. The hDBP-1 is monospecific for Gc and does not crossreact with human albumin, which has 23% of its amino acid residues identical with vitamin-D-binding protein (DBP). The subclass of the antibody is IgG1 for the heavy chain, the light chain being of the kappa type. Isoelectric focusing discloses four major bands for the hDBP-1 with isoelectric points between pH 6.5 and 7.8. Binding to the antigen at different pH values was determined: there is high affinity in the physiological range and no binding at pH 3.5 and lower. In the presence of high salt concentrations, binding was reduced to about 50% at 1.5 M NaCl. The hDBP-1 recognizes the common human Gc types and the Gc of all apes and old world monkeys. No reaction was observed with the Gc of other mammals such as horses, cattle, rats, rabbits, sheep, goats and pigs. By testing hDBP-1 against 77 of the more than 120 known rare human Gc variants, it could be shown that this monoclonal antibody cannot recognize seven of these rare variants and can only poorly recognize nine. The binding site of hDBP-1 to Gc is not related to the binding site of Gc with G-actin: it recognizes Gc, the binary complex between Gc and G-actin, as well as the ternary complex between Gc, G-actin and DNase I. Competition assays with vitamin D₃ and Gc in enzyme-linked immunosorbent assay indicate that the epitope of hDBP-1 on the Gc molecule may be related to the vitamin-D₃-binding site.     

Protection of actin against proteolysis by complex formation with deoxyribonuclease I

G-actin bound to deoxyribonuclease I (DNase I) is resistant to digestion by trypsin and chymotrypsin. In the absence of DNase I, G-actin is cleaved by these proteases to yield a 33 500 molecular weight core protein which is not degraded further. The major sites of proteolytic action in the amino acid sequence of actin have been identified as being adjacent to residues arginine-62 and lysine-68 for trypsin and leucine-57 for chymotrypsin. These residues are rendered inaccessible to proteases in the buffer by complex formation with DNase I. Digestion of G-actin with pronase from Streptomyces griseus yields fragmentation patterns that are similar to those observed with trypsin and chymotrypsin. This is likely to be because the specificities of the major constituents of pronase resemble those of trypsin and chymotrypsin. Again, complex formation with DNase I protects the otherwise vulnerable bonds in actin against proteolysis. Incubation with subtilisin Carlsberg leads to complete digestion of G-actin. No subtilisin-resistant core protein accumulates during the incubation. Protection of G-actin when complexed to DNase I is less than complete in this case but still is significant. This is interpreted in terms of the broad specificity of subtilisin and the observed fragmentation pattern of free G-actin when treated with subtilisin.     

Group-specific component (vitamin D binding protein) prevents the interaction between G-actin and profilin

Profilin purified from human platelets formed a 1:1 molar ratio complex with rabbit skeletal muscle G-actin but was displaced by purified serum Gc (vitamin D binding protein) in a dose-dependent fashion as assessed by chromatography and ultrafiltration. This suggested that Gc and profilin competed for the same binding area on G-actin, with Gc-G-actin complexes being more stable than profilin-G-actin complexes in vitro. The binding domain for Gc on G-actin was localized to a 16,000-Da C-terminal fragment of G-actin generated by Staphylococcus aureus V8 protease, as judged by comigration on two-dimensional electrophoresis and also by overlaying electrophoresis gels with 125I-Gc. Previous studies have reported that residues 374 and 375 of G-actin are essential for binding of profilin. In this study, experiments involving tryptic removal of Cys-374 labeled with the fluorescent probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)-ethylenediamine showed that these C-terminal amino acids were not necessary for interaction with Gc.     

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.     

Susceptibility of mammalian deoxyribonucleases I (DNases I) to proteolysis by proteases and its relationships to tissue distribution: biochemical and molecular analysis of equine DNase I

Equine (Equus caballus) deoxyribonuclease I (DNase I) was purified from the parotid gland, and its 1295-bp cDNA was cloned. The mature equine DNase I protein consisted of 260 amino acid residues. The enzymatic properties and structural aspects of the equine enzyme were closely similar to those of other mammalian DNases I. Mammalian DNases I are classified into three types--pancreatic, parotid and pancreatic-parotid-based on their tissue distribution; as equine DNase I showed the highest activity in the parotid gland, it was confirmed to be of the parotid-type. Comparison of the susceptibility of mammalian DNases I to proteolysis by proteases demonstrated a marked correlation between tissue distribution and sensitivity/resistance to proteolysis; pancreatic-type DNase I shared properties of resistance to proteolysis by trypsin and chymotrypsin, whereas parotid-type DNase I did not. In contrast, pancreatic-parotid-type DNase I exhibited resistance to proteolysis by pepsin, whereas the other enzyme types did not. However, site-directed mutagenesis analysis revealed that only a single amino acid substitution could not account for acquisition of proteolysis resistance in the mammalian DNase I family during the course of molecular evolution. These properties are compatible with adaptation of mammalian DNases I for maintaining their activity in vivo.     

Myosin subfragment 1 and structural elements of G-actin: effects of S-1(A2) on sequences 39-52 and 61-69 in subdomain 2 of G-actin.

The effect of myosin on the structure of two sequences on G-actin, a loop between residues 39 and 52 and a segment between residues 61 and 69 from the NH2-terminus, was probed by limited proteolytic digestions of G-actin in the presence of the myosin subfragment 1 isozyme S-1(A2). Under the experimental conditions of this work, no polymerization of actin was induced by S-1(A2) [Chen & Reisler (1991) Biochemistry 30, 4546-4552]. S-1(A2) did not change the rates of subtilisin and chymotryptic digestion of G-actin at loop 39-52. In contrast to this, the second protease-sensitive region on G-actin, segment 61-69, was protected strongly by S-1(A2) from tryptic cleavage. The minor if any involvement of loop 39-52 in S-1 binding was confirmed by determining the binding constants of S-1(A2) for pyrene-labeled G-actin (1.2 x 10(6) M-1), subtilisin-cleaved pyrenyl G-actin (0.3 x 10(6) M-1), and DNase I-pyrenyl G-actin complexes (0.3 x 10(6) M-1). Consistent with this, the activity of DNase I, which binds to actin loop 39-52 [Kabsch et al. (1990) Nature 347, 37-44], was inhibited almost equally well by actin in the presence and absence of S-1(A2). These results confirm the observation that DNase I and S-1(A2) bind to distinct sites on actin [Bettache et al. (1990) Biochemistry 29, 9085-9091] and demonstrate myosin-induced changes in segment 61-69 of G-actin.     

Crystallization and preliminary crystallographic data of chicken gizzard G-actin . DNase I complex and Physarum G-actin . DNase I complex.

Smooth muscle G-actin from chicken gizzard and Physarum plasmodium G-actin both interact with DNase I and form 1 : 1 complexes. These complexes were crystallized by using polyethylene glycol 6000 as a precipitant. Both crystals belong to the same orthorhombic space group P2(1)2(1)2(1). The cell dimensions of chicken gizzard G-actin.DNase I complex are a=42.00 +/- 0.07 A, b=225.3 +/- 0.4 A, and c=77.4 +/- 0.1 A, while those of Physarum G-actin.DNase I complex are a=42 A, b=221 A, and c=77 A.     

Role of actin in the responses of adrenal cells to ACTH and cyclic AMP: inhibition by DNase I

Erythrocyte ghosts were loaded with pancreatic DNase I and fused with Y- 1 adrenal tumor cells to test the possibility that this enzyme might inhibit the steroidogenic responses of the cells to ACTH and cyclic AMP. Fusion of erythrocyte ghosts loaded with DNase I, but not those containing albumin, ovalbumin, boiled DNase I, or DNase I with excess G- actin, inhibited the increase in production of 20 alpha- dihydroprogesterone produced by ACTH and dibutyryl cyclic AMP; inhibition was concentration-dependent with 50% inhibition by 3 X 10(7) molecules of DNase I per cell. It was found that inhibition by DNase I was exerted at the step in the steroidogenic pathway at which cholesterol is transported to mitochondria where steroidogenesis begins. This was shown by measuring transport of cholesterol into the inner mitochondrial membrane, by measuring the production of pregnenolone by isolated mitochondria and by demonstrating that DNase I was without effect on the conversion of pregnenolone to 20 alpha- dihydroprogesterone (an end-product of steroid synthesis). The actin content of Y-1 cells was measured by two methods based upon inhibition of DNase I and by SDS gels following centrifugation. The cells were found to contain 2-3 X 10(7) molecules of actin per cell of which two- thirds is present as G-actin. Since DNase I is known to bind to G-actin to give a one to one complex, these and other findings suggest that at least some of the G-actin in the cells may be necessary for the steroidogenic responses to ACTH and cyclic AMP.     

Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells.

Lamellipodium protrusion is linked to actin filament disassembly in migrating fibroblasts [Cramer, 1999: Curr. Biol. 9:1095-1105]. To further study this relationship, we have identified a method to specifically and sensitively detect G-actin in distinct spatial locations in motile cells using deoxyribonuclease I (DNase I). Although DNase I can bind both G- and F-actin in vitro [Mannherz et al., 1980: Eur. J. Biochem. 95:377-385], when cells were fixed in formaldehyde and permeabilized in detergent, fluorescently-labelled DNase I specifically stained G-actin and not F-actin. 92-98% of actin molecules were stably retained in cells during fixation and permeabilization. Further, increasing or decreasing cellular G-actin concentration by treating live cells with latrunculin-A or jasplakinolide, respectively, caused a respective increase and decrease in DNase I cell-staining intensity as expected. These changes in DNase I fluorescence intensity accurately reflected increases and decreases in cellular G-actin concentration independently measured in lysates prepared from drug-treated live cells (regression coefficient = 0.98). This shows that DNase I cell-staining is very sensitive using this method. Applying this method, we found that the ratio of G-/F-actin is lower in both the lamellipodium and in a broad band immediately behind the lamellipodium in migrating compared to non-migrating fibroblasts. Thus, we predict that protrusion of the lamellipodium in migrating fibroblasts requires tight coupling to filament disassembly at least in part because G-actin is relatively limited within and behind the lamellipodium. This is the first report to directly demonstrate high sensitivity of cell-staining for any G-actin probe and this, together with the ready commercial accessibility of fluorescently-labelled DNase I, make it a simple, convenient, and sensitive tool for cell-staining of G-actin.     

Molecular, biochemical and immunological analyses of porcine pancreatic DNase I.

Deoxyribonuclease I (DNase I) was purified 26500-fold in 39% yield from porcine pancreas to electrophoretic homogeneity using three-step column chromatography. The purified enzyme was inhibited by an antibody specific to the purified enzyme but not by G-actin. A 1303 bp cDNA encoding porcine DNase I was constructed from total RNA from porcine small intestine using a rapid amplification of cDNA ends method, followed by sequencing. Mature porcine DNase I protein was found to consist of 262 amino acids. Unlike all other mammalian DNase I enzymes that are inhibited by G-actin, porcine DNase I has H65 and S114 instead of Y65 and A114, which presumably results in the lack of inhibition. Porcine DNase I was more sensitive to low pH than rat or bovine enzymes. Compared with their primary structures, the amino acid at position 110 was N in porcine enzyme, but S in rat and bovine enzymes. A porcine mutant enzyme in which N was substituted by S alone at position 110 (N110S) became resistant to low pH to a similar extent as the rat and bovine enzymes.     

Effect of Hypothyroidism on Different Forms of Actin in Rat Cerebral Neuronal Cultures Studied by an Improved DNase I Inhibition Assay

An improved DNase I inhibition assay for the filamentous actin (F-actin) and monomeric actin (G-actin) in brain cells has been developed. Unlike other methods, the cell lysis conditions and postlysis treatments, established by us, inhibited the temporal inactivation of actin in the cell lysate and maintained a stable F-actin/G-actin ratio for at least 4-5 h after lysis. The new procedure allowed separate quantitation of the noncytoskeletal F-actin in the Triton-soluble fraction (12,000 g, 10 min supernatant) that did not readily sediment with the Triton-insoluble cytoskeletal F-actin (12,000 g, 10 min pellet). We have applied this modified assay system to study the effect of hypothyroidism on different forms of actin using primary cultures of neurons derived from cerebra of neonatal normal and hypothyroid rats. Our results showed a 20% increase in the Triton-insoluble cytoskeletal F-actin in cultures from hypothyroid brain relative to normal controls. In the Triton-soluble fraction, containing the G-actin and the noncytoskeletal F-actin, cultures from hypothyroid brain showed a 15% increase in G-actin, whereas the F-actin remained unaltered. The 10% increase in total actin observed in this fraction from hypothyroid brain could be totally accounted for by the enhancement of G-actin. The mean F-actin/G-actin ratio in this fraction was about 30% higher in the cultures from normal brain compared to that of the hypothyroid system, which indicates that hypothyroidism tends to decrease the proportion of noncytoskeletal F-actin relative to G-actin.     

Structural aspects of skeletal muscle G-actin molecule as studied by proteolytic digestion: effect of nucleotide

Trypsin and chymotrypsin were used as probes of conformation of G-actin molecule. The pattern of fragments produced has been analyzed by sodium dodecyl sulfate gel electrophoresis. G-actin is known to be nonrefractory to proteolysis [Jacobson, G.R., and Rosenbusch, J.P. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2742-2746]. It is really true that G-actin is cut easily into a 33-kDa fragment by trypsin or chymotrypsin, but only when free ATP is present in the medium. After the removal of free ATP from the medium, G-actin became more refractory to proteolysis. The amounts of degradation of G-actin depended on the ATP concentration in the medium with saturating at about 0.5 mM. epsilon-ADP also had the effect and its fluorescence spectrum was changed on the addition of G-actin. After the removal of free ATP, G-actin still bound 1 mol/mol of ATP. So, the present results suggest the presence of a second ATP interaction site on G-actin and that ATP interaction at this site induces conformational changes in G-actin molecule.     

Covalent modification of G-actin by pyridoxal 5'-phosphate: polymerization properties and interaction with DNase I and myosin subfragment 1.

Pyridoxal 5'-phosphate (PLP), a lysine-specific reagent, has been used to modify G-actin. At pH 7.5, PLP reacted with 1.7-2 lysines on G-actin. Limited proteolytic digestion experiments indicated that, in agreement with previous works, essentially lysine-61 was modified in a 1:1 fashion by PLP, other lysines being much less reactive. A PLP-derivatized affinity label of ATP binding sites, AMPPLP, reacted with two additional lysines that do not appear to be located in the ATP site on G-actin. PLP-G-actin did not polymerize spontaneously up to 30 microM; however, it retained other essential native properties of G-actin. PLP-actin bound to the barbed ends of actin filaments with an equilibrium dissociation constant of 4 microM and prevented dilution-induced depolymerization like a capping protein. PLP-actin copolymerized with unmodified actin. The stability of F-actin copolymers decreased with the fraction of PLP-actin incorporated, consistent with a model within which the actin-PLP-actin interactions in the copolymer are 50-fold weaker, and PLP-actin-PLP-actin interactions are 200-fold weaker than regular actin-actin interactions. PLP-actin bound DNase I with an equilibrium association constant of 2 nM-1, i.e., 10-fold lower than that of unmodified actin. PLP modification did not affect the binding of G-actin to myosin subfragment 1. However, polymerization of PLP-actin by myosin subfragment 1 was not observed in low ionic strength buffers, whereas PLP-F-actin-S1 filaments, in which the stoichiometry PLP-actin:S1 is 1:1, were formed with an apparent critical concentration of 4.5 microM in the presence of 0.1 M KCl.     

Hydrogen/deuterium exchange mass spectrometry of actin in various biochemical contexts

Hydrogen/deuterium exchange mass spectrometry (H/D MS) of monomeric actin (G-actin), polymeric actin (F-actin), phalloidin-bound F-actin and G-actin complexed with DNase I provides new insights into the architecture of F-actin and the effects of phalloidin and DNase I binding. Although the overall pattern of deuteration change supports the gross features of the Holmes F-actin model, two important differences were observed. Most significantly, no change in deuteration was observed in the critical "hydrophobic plug" region, suggesting this feature may not be present. Polymerization also produced deuteration increases for peptide fragments containing the ATP phosphate-binding loops, suggesting G-actin transitions to a more "open" conformation upon polymerization. However, polymerization produced decreases in deuteration mainly localized to the "inner", filament-axis side as predicted by the Holmes model. Mapping the phalloidin-induced decreases in F-actin deuteration onto the Lorenz binding site produced a single common patch straddling two monomers across the 1-start helix contact, again consistent with the Holmes architecture. Finally, both DNase I and phalloidin were able to alter the deuteration of regions distal to their respective binding sites. These results highlight the great opportunities for H/D MS to exploit high-resolution structures for detailed studies of the organization and dynamics of complex molecular assemblies.     

Subtilisin-cleaved actin: polymerization and interaction with myosin subfragment 1

Homogeneous preparations of actin cleaved into two fragments, the N-terminal 9- and C-terminal 36-kDa peptides, were achieved by proteolysis of G-actin with subtilisin at 23 degrees C at a 1:1000 (w/w) ratio of enzyme to actin. The subtilisin cleavage site was identified by sequence analysis to be between Met-47 and Gly-48. Although under nondenaturing conditions the two fragments remained associated to one another, the cleavage affected macromolecular interactions of actin. The rates of cleaved actin polymerization by MgCl2, KCl, and myosin subfragment 1 (S-1) were slower and the critical concentrations for this process were higher than in intact protein. Intact and cleaved actin formed morphologically indistinguishable filaments and copolymerized in the presence of MgCl2. The affinity of actin for S-1 was decreased by about 10-fold due to subtilisin cleavage, but the S-1 ATPase activity was activated to the same Vmax value by both intact and cleaved actins. DNase I inhibition measurements revealed lower affinity of cleaved actin for DNase I than that of intact protein. These results are discussed in terms of actin's structure.     

Change in the actin-myosin subfragment 1 interaction during actin polymerization

To better characterize the conformational differences of G- and F-actin, we have compared the interaction between G- and F-actin with myosin subfragment 1 (S1) which had part of its F-actin binding site (residues 633-642) blocked by a complementary peptide or "antipeptide" (Chaussepied, P., and Morales, M. F. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471-7475). Light scattering, sedimentation, and electron microscopy measurements showed that, with the antipeptide covalently attached to the S1 heavy chain, S1 was not capable of inducing G-actin polymerization in the absence of salt. Moreover, the antipeptide-carrying S1 did not change the fluorescence polarization of 5-[2-(iodoacetyl)-aminoethyl]aminonaphthalene-1-sulfonic acid (1,5-IAEDANS)-labeled G-actin or of 1,5-IAEDANS-labeled actin dimer, compared to the control S1. This result, interpreted as a lack of interaction between G-actin and antipeptide-carrying S1, was confirmed further by the following experiments: in the presence of G-actin, antipeptide.S1 heavy chain was not protected against trypsin and papain proteolysis, and G-actin could not be cross-linked to antipeptide.S1 by 1-ethyl-3[-3-(dimethylamino)propyl]carbodiimide. In contrast, similar experiments showed that antipeptide.S1 was able to interact with nascent F-actin and with F-actin. Thus, blocking the stretch 633-642 of S1 heavy chain by the antipeptide strongly inhibits G-actin-S1 interaction but only slightly alters F-actin-S1 contact. We, therefore postulate that this stretch of skeletal S1 heavy chain is essential for G-actin-S1 interaction and that the G-F transformation generates new S1 binding site(s) on the actin molecule.     

Localization of selenium-binding protein at the tips of rapidly extending protrusions

Cell protrusive motility underlies cell fundamental biological processes such as cell growth, locomotion, and migration. Here I showed that selenium-binding protein (SBP) was exclusively located at the leading edges of rapidly growing protrusions in newly plated T98G glioma cells, and at the growing tips of the neurites in SH-SY5Y neuroblastoma cells. Double staining by anti-SBP antibody and deoxyribonuclease (DNase I) that labels monomeric G-actin or phalloidin that labels filamentous F-actin showed that the SBP-positive area was overstained by DNase I but, surprisingly, was not stained by phalloidin. When the cells were incubated with chemicals which block actin polymerization or activity of phosphatidylinositol 3-kinase, recruitment of SBP and G-actin at the cell margin was still observed, showing that their recruitment precedes actin polymerization. Taken together, I suggest that SBP may be involved in the initial sequential events in rapid cell outgrowth, such as determining direction of cell outgrowth and recruitment of actin monomer.     

Decline of contractility during ischemia-reperfusion injury: actin glutathionylation and its effect on allosteric interaction with tropomyosin

The severity and duration of ischemia-reperfusion injury is hypothesized to play an important role in the ability of the heart subsequently to recover contractility. Permeabilized trabeculae were prepared from a rat model of ischemia-reperfusion injury to examine the impact on force generation. Compared with the control perfused condition, the maximum force (F_max) per cross-sectional area and the rate of tension redevelopment of Ca²⁺-activated trabeculae fell by 71% and 44%, respectively, during ischemia despite the availability of a high concentration of ATP. The reduction in F_max with ischemia was accompanied by a decline in fiber stiffness, implying a drop in the absolute number of attached cross bridges. However, the declines during ischemia were largely recovered after reperfusion, leading to the hypothesis that intrinsic, reversible posttranslational modifications to proteins of the contractile filaments occur during ischemia-reperfusion injury. Examination of thin-filament proteins from ischemic or ischemia-reperfused hearts did not reveal proteolysis of troponin I or T. However, actin was found to be glutathionylated with ischemia. Light-scattering experiments demonstrated that glutathionylated G-actin did not polymerize as efficiently as native G-actin. Although tropomyosin accelerated the time course of native and glutathionylated G-actin polymerization, the polymerization of glutathionylated G-actin still lagged native G-actin at all concentrations of tropomyosin tested. Furthermore, cosedimentation experiments demonstrated that tropomyosin bound glutathionylated F-actin with significantly reduced cooperativity. Therefore, glutathionylated actin may be a novel contributor to the diverse set of posttranslational modifications that define the function of the contractile filaments during ischemia-reperfusion injury. force; troponin; cooperativity     

Alkaline deoxyribonuclease activity in mouse teratocarcinoma cells: variation of enzyme levels during differentiation.

Embryonal carcinoma (EC) cells contain an alkaline DNase whose specific activity is much higher than their differentiated derivatives. After partial purification on CM-Sephadex, fractions eluted at 0.15 M NaCl contain a DNase activity which is inhibited by G-actin. The possible role of this alkaline DNase activity in maintaining the unpolymerized state of actin filaments in EC cells is discussed.