Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors

We have studied the kinetics of the gelation process that occurs upon warming cold extracts of Acanthamoeba using a low-shear falling ball assay. We find that the reaction has at least two steps, requires 0.5 mM ATP and 1.5 mM MgCl2, and is inhibited by micromolar Ca++. The optimum pH is 7.0 and temperature, 25 degrees-30 degrees C. The rate of the reaction is increased by cold preincubation with both MgCl2 and ATP. Nonhydrolyzable analogues of ATP will not substitute for ATP either in this "potentiation reaction" or in the gelation process. Either of two purified or any one of four partially purified Acanthamoeba proteins will cross-link purified actin to form a gel, but none can account for the dependence of the reaction in the crude extract on Mg-ATP or its regulation by Ca++. This suggests that the extract contains, in addition to actin-cross-linking proteins, factors dependent on Mg-ATP and Ca++ that regulate the gelation process.     

Purification and characterization of actinogelin, a calcium-sensitive actin-accessory protein, from rat liver

Although cell-free extracts prepared from several types of free-living cells, including Ehrlich tumor cells, macrophages and sea-urchin eggs, readily form gels under low Ca2+ conditions, no such ability to induce actin-related gel has been detected in tissue-cell extracts. Ca2+ -insensitive gelation activity was discovered, however, in several tissue-cell extracts, including liver and brain, provided that the extracts were supplemented with skeletal muscle actin. Based on sodium dodecylsulfate/polyacrylamide gel electrophoretic analysis of the gel, these extracts seem to contain both a Ca2+ -insensitive gelation factor and Ca2+ -sensitive one, actinogelin. A procedure for purification of actinogelin from rat liver was developed, and the properties of actinogelin thus purified were compared with those of Ehrlich tumor cell actinogelin. No appreciable difference was found in these two proteins, and Ca2+ sensitivity (50% inhibition of gelation at 1 microM) was very similar. Some of the molecular characteristics are described, and the importance of the presence of actinogelin in tissue cells is discussed.     

The role of actin in the temperature-dependent gelation and contraction of extracts of Acanthamoeba

The temperature-dependent assembly and the interaction of Acanthamoeba contractile proteins have been studied in a crude extract. A cold extract of soluble proteins from Acanthamoeba castellanii is prepared by homogenizing the cells in a sucrose-ATP-ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid buffer and centrifuging at 136,000 g for 1 h. When this supernate of soluble proteins is warmed to room temperature, it forms a solid gel. Upon standing at room temperature, the gel slowly contracts and squeezes out soluble components. The rates of gelation and contraction are both highly temperature dependent, with activation energies of about 20 kcal per mol. Gel formation is dependent upon the presence of ATP and Mg++. Low concentrations of Ca++ accelerate the contractile phase of this phenomenon. The major protein component of the gel is actin. It is associated with myosin, cofactor, a high molecular weight protein tentatively identfied as actin-binding protein, and several other unidentified proteins. Actin has been purified from these gels and was found to be capable of forming a solid gel when polymerized in the presence of ATP, MgCl3, and KCL. The rate of purified actin polymerication is very temperature dependent and is accelerated by the addition of fragments of muscle actin filaments. These data suggest that Acanthamoeba contractile proteins have a dual role in the cell; they may generate the forces for cellular movements and also act as cytoskeletal elements by controlling the consistency of the cytoplasm.     

Regulation of contraction and thick filament assembly-disassembly in glycerinated vertebrate smooth muscle cells

Isolated smooth muscle cells and cell fragments prepared by glycerination and subsequent homogenization will contract to one-third their normal length, provided Ca++ and ATP are present. Ca++- independent contraction was obtained by preincubation in Ca++ and ATP gamma S, or by addition of trypsin-treated myosin light chain kinase (MLCK) that no longer requires Ca++ for activation. In the absence of Ca++, myosin was rapidly lost from the cells upon addition of ATP. Glycerol-urea-PAGE gels showed that none of this myosin is phosphorylated. The extent of myosin loss was ATP- and pH-dependent and occurred under conditions similar to those previously reported for the in vitro disassembly of gizzard myosin filaments. Ca++-dependent contraction was restored to extracted cells by addition of gizzard myosin under rigor conditions (i.e., no ATP), followed by addition of MLCK, calmodulin, Ca++, and ATP. Function could also be restored by adding all these proteins in relaxing conditions (i.e., in EGTA and ATP) and then initiating contraction by Ca++ addition. Incubation with skeletal myosin will restore contraction, but this was not Ca++- dependent unless the cells were first incubated in troponin and tropomyosin. These results strengthen the idea that contraction in glycerinated cells and presumably also in intact cells is primarily thick filament regulated via MLCK, that the myosin filaments are unstable in relaxing conditions, and that the spatial information required for cell length change is present in the thin filament- intermediate filament organization.     

Reactivation of cell-free models of Physarum plasmodia after myosin reconstitution

Thin-spread glycerol-extracted Physarum plasmodia were treated with N-ethylmaleimide (NEM) to block myosin-ATPase and contractility. After supplementing the models with purified plasmodial myosin, they could be reactivated and contracted upon addition of ATP. Fluorescently labeled actomyosin fibers ruptured during contraction, resulting in beaded or rod-like contraction centers. Glycerol-extracted plasmodia lose their negative Ca++-dependence during extraction. Reconstitution of NEM-treated models with plasmodial myosin partly restored this Ca++-sensitivity. Thus, either myosin or a factor associated with it seems to be involved in the Ca++-dependent regulation of cytoplasmic actomyosin contraction in Physarum. NEM-blocked models reconstituted with skeletal muscle myosin were not reactivated by ATP. The same plasmodia subsequently incubated with plasmodial myosin were able to contract.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

The role of actin in temperature-dependent gel-sol transformation of extracts of Ehrlich ascites tumor cells

Ehrlich ascites tumor cell extracts form a gel when warmed to 25 degrees C at pH 7.0 in sucrose solution, and the gel rapidly becomes a sol when cooled to 0 degrees C. This gel-sol transformation was studied quantitatively by determining the volume or the total protein of pellets of gel obtained by low-speed centrifugation. The gelation depended on nucleotide triphosphates, Mg2+, KCl, and a reducing agent. Gelation was inhibited reversibly by 0.5 microM free Ca2+, and 25--50 ng/ml of either cytochalasin B or D, but it was not affected by 10 mM colchicine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the gel was composed of six major proteins with mol wt greater than 300,000, 270,000, 89,000, 51,000, 48,000, and 42,000 daltons. The last component was identified as cell actin because it had the same molecular weight as muscle actin and bound with muscle myosin and tropomyosin. The role of actin in gelation was studied by use of actin-inhibitors. Gelation was inhibited by a chemically modified subfragment-1 of myosin, which binds with F-actin even in the presence of ATP, and by bovine pancreatic DNase I, which tightly binds with G-actin. Muscle G-actin neutralized the inhibitory effect of DNase I when added at an equimolar ratio to the latter, and it also restored gelation after its inhibition by DNase I. These findings suggest that gelation depends on actin. However, the extracts showed temperature-dependent, cytochalasin-sensitive, and Ca2+-regulated gelation as did the original extracts when the cell actin in the extracts was replaced by muscle actin, suggesting that components other than cell actin might be responsible for these characteristics of the gelation.     

Purification and characterization of myosin from bovine retina.

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca2+ and a low activity in the presence of Mg2+. The Mg2+-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal muscle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.     

Resolution of the phosphorylated and dephosphorylated cAMP-binding proteins of bovine cardiac muscle by affinity labeling and two-dimensional electrophoresis.

The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate (8-azido-cyclic [32P]AMP) was used to analyze both the cAMP-binding component of the purified cAMP-dependent protein kinase, and the cAMP-binding proteins present in crude tissue extracts of bovine cardiac muscle. 8-Azido-cyclic [32P]AMP reacted specifically and in stoichiometric amounts with the cAMP-binding proteins of bovine cardiac muscle. Upon phosphorylation, the purified cAMP-binding protein from bovine cardiac muscle changed its electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels from an apparent molecular weight of 54,000 to an apparent molecular weight of 56,000. In tissue extracts of bovine cardiac muscle, most of the 8-azido-cyclic [32P]AMP was incorporated into a protein band with an apparent molecular weight of 56,000 which shifted to 54,000 upon treatment with a phosphoprotein phosphatase. Thus a substantial amount of the cAMP-binding protein appeared to be in the phosphorylated form. Autoradiograms following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both the pure and impure cAMP-binding proteins labeled with 8-azido-cyclic [32P]AMP revealed another binding component with a molecular weight of 52,000 which incorporated 32P from [gamma-32P]ATP without changing its electrophoretic mobility. Limited proteolysis of the 56,000- and 52,000-dalton proteins labeled with 32P from either [gamma-32P]ATP.Mg2+ or 8-azido-cyclic [32P]AMP showed patterns indicating homology. On the other hand, peptide maps of the major 8-azido-cyclic [32P]AMP-labeled proteins from tissue extracts of bovine cardiac muscle (Mr = 56,000) and rabbit skeletal muscle (Mr = 48,000) displayed completely different patterns as expected for the cAMP-binding components of types II and I protein kinases. Both phospho- and dephospho-cAMP-binding components from the purified bovine cardiac muscle protein kinase were also resolved by isoelectric focusing on polyacrylamide slab gels containing 8 M urea. The phosphorylated forms labeled with 32P from either [gamma-32P]ATP or 8-azido-cyclic [32P]AMP migrated as a doublet with a pI of 5.35. The 8-azido-cyclic [32P]AMP-labeled dephosphorylated form also migrated as a doublet with a pI of 5.40. The phosphorylated and dephosphorylated cAMP-binding proteins migrated with molecular weights of 56,000 and 54,000, respectively, following a second dimension electrophoresis in sodium dodecyl sulfate. The lower molecular weight cAMP-binding component (Mr = 52,000) was also apparent in these gels. Similar experiments with the cAMP-binding proteins present in tissue extracts of bovine cardiac muscle indicate that they are predominantly in the phosphorylated form.     

Calcium-sensitive, actin-related gelation of cell extracts of thermosensitive Chinese hamster lung cells capable of anchorage-independent growth.

The extent of actin-related gelation of extracts of thermosensitive Chinese hamster lung (CHL) cells capable of anchorage-independent growth was studied quantitatively by monitoring the total protein in the gel obtained by low-speed centrifugation. The gelation depended on the presence of ATP, KCl, MgCl2, and a reducing agent. Micromolar concentrations of Ca2+ and low doses of cytochalasin B inhibited the actin-related gelation of these extracts. The gelation was more sensitive to inhibition by Ca2+ and cytochalasin B when the extracts were prepared from cells cultured at the permissive rather than the nonpermissive temperature. When various ts mutants were examined, the half-maximal inhibitory dose (HMID) of Ca2+ and cytochalasin B for gelation of extracts of cells cultured at the nonpermissive temperature was between 1.25 and 2.19 times higher than that for extracts of the same cells cultured at the permissive temperature. The values of the HMID for Ca2+ and cytochalasin B changed shortly after the shift in temperature of cell cultures from the nonpermissive to permissive temperature. When cell extracts were incubated at the permissive temperature in vitro for only 15 minutes, these changes in values of HMID were also observed. Analysis of polypeptides of cell extracts and gel pellets on polyacrylamide gel electrophoresis suggested that the decrease in amount of a high-molecular-weight actin-binding protein (250 kDa) may play an essential role in the increased sensitivity to inhibition by Ca2+ and cytochalasin B of actin-related gelation in extracts of these ts mutants.     

Potentiation of postjunctional cholinergic sensitivity of rat diaphragm muscle by high-energy-phosphate adenine nucleotides.

The cholinergic sensitivity of rat diaphragm muscle, me-sured as the magnitude of depolarization responses to repetitive, iontophoretic pulses of acetylcholine (ACh) onto neuromuscular endplates, is increased by addition of ATP to the perfusion medium. Depolarization responses begin to increase within the first min after addition of 10 mM ATP and plateau at 60% above control levels (mean value) after 4 to 6 min. Neither the magnitude nor the time course of the potentiations corresponds to changes in resting potential or membrane resistance. Other nucleotides are equally or less effective at the same concentration: ATP=ADP greater than UTP greater than AMP=GTP (=no added nucleotide control) The duration of the individual ACh responses does not increase during continuous exposure to the active nucleotides for up to 15 min except when the muscle is pretreated with eserine. Mild enzymatic predigestion of the muscle with collagenase and then protease, increasing the availability of the postjunctional membrane to bath-applied drugs, decreases the variability and increases the magnitude of the potentiation to a given dose of ATP. The dose-response curve for ATP is then more than half-maximal at 1 mM and the ranking of the other nucleotides relative to ATP is the same as without predigestion. There is an optimum Ca++ concentration for the potentiation between zero and 2 mM: potentiation is enhanced in Ca++ -free medium, partially blocked in twice-normal Ca++ medium, and totally blocked in Ca++ -free medium 10 min after a 5 min exposure to 2.5 mM EGTA. The similar Ca++ dependence of ACh receptor activation in the absence of added nucleotide suggests that ATP directly facilitates receptor activation by ACh. This facilitory action could be one of the physiological roles for the ATP released from stimulated phrenic nerve.     

Detection of actin-binding proteins in human platelets by 125I-actin overlay of polyacrylamide gels

Actin-binding proteins have been identified in human platelets with a gel-overlay technique that uses 125I-G-actin. Platelet proteins were separated on SDS polyacrylamide gels using the buffer system of Laemmli (1970, Nature [Lond.] 227:680-685). The proteins were fixed in the gels with methanol-acetic acid, the SDS was washed out, and the proteins were renatured. The gels were incubated with 125I-G-actin from rabbit skeletal muscle that was radiolabeled with 125I according to the method of Bolton and Hunter (1973, Biochem. J. 133:529-538) and has been shown to retain biological activity. After nonspecifically bound radioactivity was washed out, gels were dried and processed for autoradiography. The 125I-G-actin binds to several proteins in human platelets, platelet extracts, and the particulate fraction. Control experiments demonstrate that the 125I-G-actin can be displaced by use of increasing amounts of unlabeled actin, that the binding is stable to 0.6 M NaCl, and that preheating the 125I-G-actin to 90 degrees C for 3 min eliminates all binding. Prominent 125I-G-actin-binding activities were present at Mr 90,000 and 40,000. The binding to the 90,000 Mr protein appears to be at least partially Ca++ sensitive, whereas the binding to the 40,000 Mr protein does not. 125I-G-actin bound to proteins in the SDS gels can be fixed in situ and compared directly with the stained gel. This technique should prove generally useful in identification and purification of some actin-binding proteins from cells and tissues.     

Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction

Treatment of chick embryo fibroblasts with 0.5% Triton® X-100 extracts most of the cell protein, leaving an organized part of the cell structure attached to the tissue culture dish. This “Triton cytoskeleton” consists largely of intermediate-sized filaments and bundles of microfilaments. SDS polyacrylamide gel electrophoresis reveals that this cytoskeleton is made up of three main proteins. One protein component is 42,000 daltons and co-migrates with muscle actin. The other two components are 52,000 and 230,000 daltons and remain quantitatively associated with the cytoskeleton during the detergent extraction. The possible identity of these three protein components and their organization into a supramolecular structure is discussed.     

Effects of the thyroid status on the sarcoplasmic reticulum in slow skeletal muscle of the rat

The effects of the thyroid status on the Ca++-transporting capabilities of rat slow skeletal muscle (m.soleus) were studied. The oxalate supported Ca++-uptake activity and Ca++-loading capacity of muscle homogenates from hyperthyroid rats showed an approximate 4.2 and 2.5 fold increase, respectively, as compared to values found in the hypothyroid group. Muscle homogenates of euthyroid rats gave intermediate values. The specific activity of oxalate supported Ca++ uptake, but not the Ca++-loading capacity, of membrane preparations enriched with respect to sarcoplasmic reticulum (SR) increased in proportion to the thyroid status. This was paralleled by a 3.5 fold increase in the amount of active Ca++ pumps in the SR preparations in the transition from hypothyroidism to hyperthyroidism as determined by measurement of Ca++-dependent 32P incorporation. These observations are not explained by differences in degree of purification of the examined SR preparations. Protein profiles of the membrane preparations obtained by gel electrophoresis indicated a thyroid-hormone dependent increase in Ca++-pump content relative to other SR proteins. The results suggest that thyroid hormone stimulates the proliferation of the SR and possibly also increases the Ca++-pump density in the SR membrane.     

Induction of either contractile or structural actin-based gels in sea urchin egg cytoplasmic extract

The gel formed by warming the 100,000 g supernate of isotonic extracts of sea urchin eggs to 40 degrees C is made up of actin and two additional proteins of mol wt of 58,000 and 220,000. Actin and 58,000 form a characteristic structural unit which has now been identified in the microvilli of the urchin egg and in the filopods of urchin coelomocytes. However, egg extract gels did not contract as those from other cell types do, and the aim of these experiments was to determine the reason for this lack of contraction. Although the extracts are dialyzed to a low ionic strength, myosin is present in soluble form and makes up approximately 1% of the protein of the extract. It becomes insoluble in the presence of high ATP concentrations at 0 degrees C, and the precipitate formed under these conditions consists almost entirely of myosin. This procedure provides a simple method of isolating relatively pure myosin without affecting other extract components and functions. Contraction will follow gelation in these extracts if the temperature and time of incubation used to induce actin polymerization are reduced to minimize myosin inactivation. At the optimal ATP and KCl concentration for contraction, the contracted material has an additional 250,000 component and contains very little 58,000. The conditions found to provide maximum gel yields favor the formation of the actin-58,000-220,000 structural gel, while reduced temperature and increase in KCl concentration results in a contractile gel whose composition is similar to those reported from amoeboid cell types. Both the structural protein cores found in the egg microvilli and a gel contraction related to the amoeboid motion which is seen in later urchin embryonic development can thus be induced in vitro in the same extract.     

Purification and some properties of skeletal muscle sarcolemma Ca2+-ATPase]

Ca2+-ATPase of skeletal muscle sarcolemma has been isolated and purified. It is prepared from salt extract of sarcolemma by ammonium sulfate fractionation and further purified by gel chromatography on Sepharose 4B. The purity of preparations was evaluated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. It has been shown that Ca2+-ATPase possesses the same mobility as skeletal muscle myosin under gel chromatography on Sepharose 4B and the same mobility as myosin heavy chains in sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Membrane protein binds to rabbit skeletal muscle actin, and this complex dissociates by ATP. Interaction with actin does not change Ca2+- or Mg2+-stimulated ATPase activity. Enzyme has only one pH optimum at 7,0-7,6. Membrane protein is highly specified to calcium--ATPase activity in the presence of Mn2+ is 10% and in the presence of Sr2+, Mg2+ or Co2+ are 3-5% of the activity in the presence of Ca2+. Other nucleoside triphosphate (UTP and ITP) are hydrolyzed at lower rates than is ATP.     

Antioxidant-induced release of calcium in biological membranes

The effect of synthetic (BHT, 2,2,5,7,8-pentamethyl-6-hydroxychroman) and natural (alpha-tocopherol) antioxidants on Ca++-transporting systems was compared in platelets, brain synaptosomes, and skeletal muscle sarcoplasmic reticulum. It was shown that synthetic antioxidants, in contrast to alpha-tocopherol, induced Ca++-release manifested in platelet aggregation, stimulation of 5-hydroxytryptamine release by synaptosomes, synaptosome depolarization and inhibition of Ca++-transport and Ca++-ATPase activity in the sarcoplasmic reticulum. The disturbances of Ca++-homeostasis induced by synthetic antioxidants are considered as molecular mechanisms of complications encountered upon their application.     

Electron-cytochemical study of the localization and properties of ATPases in the isolated nuclei of rabbit skeletal muscle under normal conditions and in experimental muscular dystrophy]

The properties and localization of ATPase system in nuclei of skeletal muscle of normal rabbit and of those with experimental muscle dystrophy were studied by electron cytochemistry. The product of cytochemical reaction of ATP hydrolysis, which is a marker of ATPase activity localization in nuclear ultrastructures, was detected on the nuclear membrane, in chromatin and in the nucleolus, ATPase activity in the nuclei was detected in the presence of both, Mg2+ and Ca2+. Addition to the incubation medium, originally containing Mg2+, Na+ and K+, resulted in an increased formation of the product reaction in all the nuclear ultrastructures in both in the norm and under experimental muscle dystrophy. However, specific inhibitor of Mg2+, Na+, K+-ATPase--ouabain--suggests the absence in the nuclei of skeletal muscles of rabbit of transport ATPase working in the "Na-pump" system. The results of experiments with a specific complex of Ca2+--EGTA allow to suppose that Mg2+, Ca2+-ATPase of skeletal muscle nuclei of normal rabbits is localized in the nucleoplasm, whereas Mg2+-ATPase is found on the nuclear membrane. Using EGTA we failed to detected the localization of Mg2+, Ca2+-ATPase in nuclear ultrastructures upon experimental muscular dystrophy.     

Characterization of the ATP-phosphohydrolase activity of bovine spermatozoa flagellar extracts.

The ATP-phosphohydrolase activity of extracts prepared from bovine spermatozoa flagella (BSFE), was characterized with respect to enzyme, substrate, activator ion and salt concentration, temperature dependence and time stability. BSFE required the presence of a divalent cation for activity: Mg++ or Ca++ could function as activator; Mn++, Zn++ and Cd++ could not. EDTA, but not EGTA, was inhibitory to enzymatic activity. Ca++ inhibited the Mg++ stimulated activity. ATP was dephosphorylated more rapidly than GTP greater than CTP greater than ITP, and ADP was dephosphorylated at 40% of the rate of ATP. The magnesium activated ATPase was stimulated by potassium and inhibited by sodium ions. Activation of BSFE ATP-phosphohydrolase was maximal in the presence of Mg++ and ATP in equimolar concentrations and K+ (0.05-0.3 M) at 30 degrees C. Although the enzymatic activity of the extract was found to decrease rapidly with time, it could be maintained for up to three days by the addition of 2-beta-mercaptoethanol to the bovine spermatozoa flagellar extracts.