The interaction of hemin with skeletal muscle actin

The ability of actin to interact with hemin was studied. It was found that the Soret absorption band of hemin changes in the presence of actin and that hemin is capable of quenching the fluorescence intensity of actin. These findings were indicative of hemin binding to actin. The binding constant for the high affinity site was calculated to be 5.3 X 10(6) M-1. The amounts of native G- and F-actin were estimated by their DNAase I inhibition activity. It was observed that the binding of hemin to G-actin is followed by a slow decrease in the ability of actin to inhibit DNAase I activity and to polymerize upon addition of salts. Binding of hemin to F-actin resulted in a gradual depolymerization of the filaments, to an inactivated form, as expressed by a reduction in the ability of hemin-bound F-actin to inhibit DNAase I activity in the absence as well as in the presence of guanidine-HCl. Electron microscopy studies further corroborated these findings by demonstrating that: (1) hemin-bound G-actin failed to show formation of polymers when salts were added; (2) a marked reduction in the amount of actin polymers was observed in the specimens examined 24 h after mixing with hemin. It is suggested that the elevated amounts of free hemin formed under pathological conditions, might be toxic to cells by interfering with actin polymerization cycles.     

The interaction of bovine pancreatic deoxyribonuclease I and skeletal muscle actin

The rate of exchange of actin-bound nucleotide is decreased by a factor of about 20 when actin is complexed with DNAase I without affecting the binding constant of calcium for actin. Binding constants of DNAase I to monomeric and filamentous actin were determined to be 5 X 10(8) M-1 and 1.2 X 10(4) M-1 respectively. The depolymerisation of F-actin by DNAase I appears to be due to a shift in the G-F equilibrium of actin by DNAase I. Inhibition of the DNA-degrading activity of DNAase I by G-actin is of the partially competitive type.     

Actin polymerization induced by pulsed electric stimulation of bone cells in vitro

Electric field pulses, capacitively applied to tissue cultures of embryonic bone cells, were shown to induce changes in the state of cellular actin. Three actin states could be defined by DNAase I inhibition. A rapidly (20-30 s) inhibiting fraction, attributed to monomeric G-actin, amounts to 55% of total actin in nonstimulated cells. An additional fraction of 8% required approx. 20 min to reach full inhibition and was tentatively defined as polymeric 'F'-actin. The remaining 37% could be detected only after treatment of the cells with 0.75 M guanidine hydrochloride, which dissociates actin from all its protein interactions. This fraction, N-actin (network actin) is believed to represent F-actin integrated into some supramolecular structure, where it is not accessible to DNAase I. Upon short electric stimulation the distribution changed to 40% G-actin, 12% F-actin and 48% N-actin. 3-Isobutyl-1-methylxanthine (IBMX; an inhibitor of cAMP phosphodiesterase), depletion of extracellular calcium, and calmodulin inhibitors abolished this field effect.     

Selective assay of monomeric and filamentous actin in cell extracts,using inhibition of deoxyribonuclease I

A simple and selective assay for monomeric and filamentous actin is presented, based on the inhibition of DNAase I by actin. In mixtures of monomeric and filamentous actin, only the monomeric form is measured as DNAase inhibitor. The total amount of actin in a sample can be determined after depolymerization of F actin with guanidine hydrochloride. The assay is rapid enough to detect changes in the polymerization state of actin in vitro over time intervals as short as 3 min. Data characterizing unpolymerized and filamentous actin pools in extracts of human platelets, lymphocytes and HeLa cells are presented.     

Nonenzymatic glycosylation of and oxidative damage to actin in vitro and in vivo

A study was made the influence exerted by non-enzymatic glycosylation (glycation) and oxidative destruction on structural and functional parameters of actin (free NH2-groups, advanced glycation end product and bityrosine cross-linking content, DNase inhibition by G-actin and myosin Mg(2+)-ATPase activation by F-actin). The functional properties of actin were shown to change under high molecular weight product formation and oxidative destruction: the extent of DNAase I inhibition decreases (from 70 to 40%) and the extent of myosin Mg(2+)-ATPase decreases (by 40%). Carnosine prevents actin oligomer formation and oxidative destruction which favours preservation of the protein functional properties.     

Conformational state of thin myofilament proteins in normal and chronically failing heart

Circular dichroism (CD) spectra of myocardial G-actin significantly differ from those of F-actin, and the spectra of G- and F-actins differ from those of myocardial tropomyosin, native tropomyosin and alpha-actinin. In heart failure in man and experimental animals, characterized by a significantly decreased ability of the contractile protein system to generate force, considerable changes in the tertiary structure of Straub G-actin are observed. During polymerization a monomer of this actin is included in F-actin as a promoter without corresponding conformational changes of a part of G-actin globule; G-actin from the failing myocardium loses its conformational mobility. According to CD data the secondary protein structure is not altered. CD spectra analysis with regard to the regions of aromatic amino acid residue localization in active sites of actin suggests that the sites of actin-myosin and actin-actin interactions do not assume the conformation necessary for normal functioning of thin filaments.     

Unfolding energetics of G-alpha-actin: a discrete intermediate can be re-folded to the native state by CCT

Nascent actin requires interactions with the highly conserved and essential eukaryotic chaperonin-containing TCP-1 (CCT) for its correct folding to the native state in vivo. Biochemical and structural analysis of the interaction between actin and CCT has been studied extensively but the underlying energetics and kinetics of the CCT-dependent actin folding process are not understood. We investigated the unfolding and folding pathways of actin, using stopped flow fluorescence and biochemical techniques. By using very low concentrations of actin, taking account of temperature and ATP concentration dependences we were able to determine accurately the activation energy of unfolding to a stable intermediate, I(3). Use of the fluorescent calcium chelator Quin-2 and consideration of the ATP concentration dependence on the unfolding rate has allowed the intrinsic kinetics to be linked to the accepted reaction scheme for actin denaturation. A free energy of -28.7(+/-0.2) kJ mol(-1) was determined for the loss of ATP from Ca-free G-actin, in good agreement with previous studies. Understanding the K(eq) value for this step then allowed the temperature dependence of the unfolding reaction of co-factor-free actin to be evaluated, yielding an activation energy for the unfolding of G-actin of 81.3(+/-3.3) kJ mol(-1). By chemical coupling of the extrinsic probe, Alexa Fluor 488 to cysteine 374 of native alpha-actin, we were able to follow the binding and folding of I(3) by CCT, observing for the first time, in vitro re-folding of EDTA-denatured G-actin. The high value of the activation energy between native actin and a non-native folding intermediate (I(3)) is characteristic of a partially folded, molten globule state expected to contain partial secondary structure.     

Actin—membrane interactions: Association of G-actin with the red cell membrane

Chemically tritiated actin from rabbit skeletal muscle was used to investigate the association of G-actin with the red cell membrane. The tritiated actin was shown to be identical to unmodified actin in its ability to polymerize and to activate heavy meromyosin ATPase. Using sealed and unsealed red cell ghosts we have shown that G-actin binds to the cytoplasmic but not the extracellular membrane surface of ghosts. Inside-out vesicles which have been stripped of endogenous actin and spectrin by low-ionic-strength incubation bind little G-actin. However, when a crude spectrin extract containing primarily spectrin, actin, and band 4.1 is added back to stripped vesicles, subsequent binding of G-actin can be increased up to 40-fold. Further, this crude spectrin extract can compete for and abolish G-actin binding to unsealed ghosts. Actin binding to ghosts increases linearly with added G-actin and requires the presence of magnesium. In addition, actin binding is inhibited by cytochalasin B and DNAase I. Negative staining reveals an abundance of actin filaments formed when G-actin is added to reconstituted inside-out vesicles but none when it is added to unreconstituted vesicles. These observations indicate that added G-actin binds to the red cell membrane via filament formation nucleated by some membrane component at the cytoplasmic surface.     

Effect of DNAase I on muscle tropomyosin polymerization

DNAase I, an endonuclease which interacts with G-actin, also affects tropomyosin polymerization. With chicken pectoralis or bovine cardiac ventricle tropomyosin, DNAase I both prevents tropomyosin from polymerizing and disrupts already formed tropomysin filaments. DNAase I and filament tropomyosin can also form a precipitable complex. In the electron microscope, the complex is observed as irregularly margined stellate-shaped structures with a maximum size of 9 micron. Isolated DNAase I-tropomyosin stellate complex consists of a 2:1 molar ratio of DNAase I and tropomyosin, suggesting that each tropomyosin subunit can bind DNAase I.     

Ecdysterone induction of actin synthesis and polymerization in a Drosophila melanogaster cultured cell line

Actin pools have been evaluated in Drosophila melanogaster Kc 0% cells, through an actin assay based on differential inhibition of DNase I by globular (G) and filamentous (F) actin. Total actin represents about 4 % of total proteins and 54 % is G-actin. In ecdysterone treated cells (0.1 μM), the total actin content increases up to 9 % of total proteins after 3 days of treatment. Ecdysterone induces increase of G-actin as well as F-actin. Increase of both actins, detectable after only 24 hrs of treatment, is roughly parallel during the first two days of treatment. For longer hormonal treatment, actin polymerization is more important than accumulation of G-actin. Indirect immunofluorescence microscopy with antibodies to exogeneous DNase I suggests that actin is widely distributed in the whole cytoplasm before and after ecdysterone treatment. These results suggest that ecdysterone induces actin synthesis and polymerization in Drosophila melanogaster cells.     

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.     

Snake venom cardiotoxin induces G-actin polymerization

Snake venom cardiotoxin showed the ability to induce polymerization of G-actin from rabbit skeletal muscle in a low ionic strength buffer composed of 0.2 mM CaCl2/0.2 mM ATP/0.5 mM mercaptoethanol/2.0 mM Tris-HCl, pH 8.0. The activity was enhanced greatly when 0.4 mM MgCl2 was present in the buffer and could be inhibited if G-actin was preincubated with deoxyribonuclease I. Furthermore, the DNAase could also partially depolymerize actin polymer previously formed by the interaction of G-actin with the toxin.     

Investigation of the actin-deoxyribonuclease I interaction using a pyrene-conjugated actin derivative

The interaction of deoxyribonuclease I with muscle actin was studied with the aid of a pyrenyl derivative of the actin [Kouyama, T., & Mihashi, K. (1981) Eur. J. Biochem. 114, 33-38] that increases its quantum yield by an order of magnitude on polymerization. It is shown that this derivative copolymerizes with unlabeled G-actin in a random manner and will also bind to deoxyribonuclease with inhibition of enzymic activity. The derivative affords a highly sensitive means of following nucleated polymerization. Preincubation of F-actin with deoxyribonuclease at a concentration of 5% or less of that of total subunits causes inhibition of polymerization of additional G-actin onto the filaments. In red cell membranes that contain stabilized short filaments of actin such that the concentration of filament ends is large relative to monomers, complete inhibition of nucleated polymerization of G-actin is achieved by preincubation with deoxyribonuclease. The results indicate that binding of DNase occurs at the "plus" ends of the actin filaments. Competition with cytochalasin E, which is known to have a high affinity for the plus or preferentially growing ends of F-actin, can be observed. Whereas the activity of deoxyribonuclease in the 1:1 complex with G-actin is inhibited, the enzyme attached to the ends of filaments appears to be fully active. This causes a reduction in the inhibition of enzymic activity with increasing F-actin concentration, presumably by reason of a change in the partition of the enzyme between monomers and filament ends. The degree of inhibition increases with time, however, as the actin depolymerizes. Implications for measurements of actin monomer concentrations by the deoxyribonuclease assay procedure are considered.     

Rabbit skeletal muscle F-actin can be stable at low ionic strength, provided trace amounts of Ca2+ are absent.

Addition of low concentrations (0.2--2.0 mM) of EGTA to rabbit skeletal muscle G-actin in the presence of ATP caused increase in viscosity. The effect is probably due to chelation of Ca2+. EGTA-polymerized actin was sedimented in the ultracentrifuge as a pellet which could be depolymerized in the presence of Ca2+ and then repolymerized. Electron microscopy indicated that formation of filamentous actin which appears to be somewhat more flexible than F-actin obtained by polymerization with KCl. The EGTA-polymerized actin was dissociated by DNAase I faster than KCl-polymerized actin. F-Actin can thus be stable also in very low ionic strength media if Ca2+ is removed whereas for G-actin to be the only form of the protein in such media, micromolar concentrations of Ca2+ must be present.     

Association kinetics and binding constants of nucleoside triphosphates with G-actin.

The dissociation of the complex between 1:N6-ethenoadenosine, 5'-triphosphate (xiATP) and G-actin was initiated by dilution to concentrations between 1 micronM and 5 nM and monitored by the fluorescence change of xiATP. The results were quantitatively explained by a two-step mechanism: a reversible dissociation of the actin-nucleotide complex followed by a fast irreversible inactivation of nucleotide-free G-actin. Under normal conditions (0.8 mM CaCl2, pH 8.2,21 degrees C), the rate-limiting step was the dissociation of the nucleotide-G-actin complex. The half-time of the dissociation of xiATP from G-actin was 290 s as compared to only 13 s for the following denaturation step of nucleotide-free actin. 1 mM EDTA highly accelerated the dissociation step and, regardless of its concentration, the complex dissociated quantitatively within 1 min. Addition of Ca2+ within 20 s after EDTA addition induced a re-association of xiATP with nucleotide-free but still native G-actin. This reversal was kinetically resolved by means of a multimixing stopped-flow apparatus. The association rate constant was 6 X 10(6) M-1s-1. From the association and dissociation rate constant, a value of 2.5 X (10(9) M-1 was calculated for the binding constant of xiATP to G-actin. The binding constant of ATP (1.4 X 10(10) M-1) was derived from the relative binding constant of xiATP and ATP as determined by fluorescence titration of xiATP-G-actin with ATP. These binding constants are 10(3)-10(4) times higher than values reported earlier on the basis of more indirect data.     

Effects of the actin-binding protein DNAase I on cytoplasmic streaming and ultrastructure of Amoeba proteus

Microinjection of DNAase I, which is known to form a specific complex with G-actin, induces characteristic changes in cytoplasmic streaming, locomotion and morphology of the contractile apparatus of A. proteus. Light microscopical studies show pronounced streaming originating from the uroid and/or the retracting pseudopods, which ceases 10--15 min after injection of DNAase I, at a time when ultrasctructural studies show that the actin filament system is very much reduced. These results suggest that a controlled reversible equilibrium between soluble and polymerized forms of actin is a necessary requirement for amoeboid movement. The topographic distribution of contractile filaments beneath the plasma membrane visualized by correlated light- and electron microscopy of DNAase I-injected cells establishes the importance of the membrane-bound filamentous layer for three major aspects of streaming: (1) Streaming originates by local contractions of a cell membrane-associated filament layer at the uroid and/or retracting pseudopods, creating a pressure flow. (2) This flow continues beneath the membrane, which is stabilized by filaments in the lateral regions between the posterior end, with a high hydrostatic pressure, and the anterior end, with a low hydrostatic pressure. (3) Pseudopods or extending areas are created by a local destabilization of the cell periphery caused by the separation of the filamentous layer from the plasma membrane.     

The interaction of 5'-nucleotidase purified from chicken gizzard and actin, and the reversible loss of the inhibitory capacity of actin on deoxyribonuclease I

Evidence is presented for a direct interaction of the intrinsic membrane protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) purified from avian smooth muscle (chicken gizzard) and the cytoskeletal component actin. Two different modes of interaction can be discerned: firstly, an immediate inhibitory effect of preferentially filamentous actin (F-actin) on the enzymic (i.e., AMPase) activity of 5'-nucleotidase and a direct binding of this enzyme to immobilized F-actin. Since these effects are suppressed by the addition of myosin subfragment 1, binding of 5'-nucleotidase appears to occur along the F-actin filament axis. Secondly, a time- and 5'-nucleotidase concentration-dependent transformation of also preferentially F-actin into a form unable to inhibit the enzymic activity of deoxyribonuclease I (DNAase I). This desensitization of actin versus DNAase I is not due to a denaturation process and was found to be reversible after addition of ATP. Furthermore, it does not seem to effect the ability of actin to bind to DNAase I. The transformation is accompanied by the hydrolysis of actin-bound nucleotide into adenosine, which remains bound to actin. Therefore, the desensitization of actin versus DNAase I appears to be due to a nucleotide-dependent conformational change of actin. An unidentified contamination of the 5'-nucleotidase preparations to a varying degree with ADPase and ATPase activities appears to be responsible for the desensitization process, although a synergistic role of these activities and 5'-nucleotidase cannot be excluded.     

The quantitation of G- and F-actin in cultured cells

An improved method to quantitate the amounts of filamentous (F-actin) and monomeric (globular) actin (G-actin) in cultured cells was developed. Cells are lysed into a myosin-containing buffer and F-actin is removed by centrifugation. The pelleted F-actin is then depolymerized to G-actin in a 1 mM ATP-containing buffer for 1 h before measuring the levels of G-actin using the DNase I inhibition assay. Partitioning of G-actin in the supernatant (greater than 95%) and recovery of actin in both fractions (greater than 85%) were measured by adding [3H]actin to cultured cells. Actin in the separated fractions is stable for at least 72 h at 0 degree C. Asynchronous monolayer cultures of Chinese hamster ovary (CHO) cells contain 2.5 +/- 0.2% of the total protein as actin with 72.4 +/- 5.7% as F-actin. About 10% of this F-actin is not associated with the readily sedimented Triton-cytoskeleton. CHO cells grown in suspension contain 55.8% of the actin as F-actin; following plating about 90 min is required for these cells to flatten and for the F-actin level to reach the monolayer value of about 70%.     

A DNase I binding/immunoprecipitation assay for actin

An actin assay which employs the competition between labeled and unlabeled rabbit skeletal muscle actin for DNase I has been developed. Iodination of actin by the method of Bolton and Hunter results in the incorporation of approximately 0.5 mol of 125-iodine/incorporation of approximately 0.5 mol of 125-iodine/mol of actin. This 125I-actin retained the ability to bind to DNase I and inhibit enzymatic activity. The 125I-actin-DNase complex can be precipitated by the addition of a monospecific rabbit antibody to DNase I. The efficiency of this immunoprecipitation step is improved by the use of a second sheep anti-rabbit gamma-globulin. Using this immunoprecipitation assay, there is a linear displacement of the DNase I-bound 125I-actin by rabbit skeletal muscle actin standards or by the actin present in tissue and cell extracts. Using 17.5 ng of DNase I and approximately 500 pg of 125I-actin, 50% inhibition of binding was obtained with 23 ng of unlabeled actin. Reducing the amount of DNase I to 2 ng results in 50% inhibition of binding with 4 ng of unlabeled actin and an increase in the estimated sensitivity of the assay from 1.7 to 0.24 ng. The slopes of the displacement curves generated with both vertebrate and invertebrate non-muscle actins are parallel to rabbit skeletal muscle actin. This observation indicates approximately equal actin-DNase I binding affinities and suggests a high degree of conservation of the actin-DNase I binding site. The assay is useful for measuring the pools of F- and G-actin in a wide range of cells.     

State of actin in gastric parietal cells

Remodeling of theapical membrane-cytoskeleton has been suggested to occur when gastricparietal cells are stimulated to secrete HCl. The present experimentsassayed the relative amounts of F-actin and G-actin in gastric glandsand parietal cells, as well as the changes in the state of actin onstimulation. Glands and cells were treated with a Nonidet P-40extraction buffer for separation into detergent-soluble (supernatant)and detergent-insoluble (pellet) pools. Two actin assays were used toquantitate actin: the deoxyribonuclease I binding assay to measureG-actin and F-actin content in the two pools and a simple Western blotassay to quantitate the relative amounts of actin in the pools.Functional secretory responsiveness was assayed by aminopyrineaccumulation. About 5% of the total parietal cell protein is actin,with about 90% of the actin present as F-actin. Stimulation of acidsecretion resulted in no measurable change in the relative amounts ofG-actin and cytoskeletal F-actin. Treatment of gastric glands withcytochalasin D inhibited acid secretion and resulted in a decrease inF-actin and an increase in G-actin. No inhibition of parietal cellsecretion was observed when phalloidin was used to stabilize actinfilaments. These data are consistent with the hypothesis thatmicrofilamentous actin is essential for membrane recruitment underlyingparietal cell secretion. Although the experiments do not eliminate theimportance of rapid exchange between G- and F-actin for the secretoryprocess, the parietal cell maintains actin in a highly polymerizedstate, and no measurable changes in the steady-state ratio of G-actin to F-actin are associated with stimulation to secrete acid.