By the use of electrophoretic and spectrophotometric methods two types of endopeptidases were demonstrated in crude extracts of Escherichia coli: one was active on N‐acetyl‐dl‐phenyl‐alanine‐2‐naphtyl ester and the other on N‐benzoyl‐l‐arginine‐p‐nitroanilide. The two activities were separated by gel filtration on Sephadex G‐100. After gel electrophoresis of crude extracts, three bands of activity toward acetyl‐phenylalanine‐naphthyl ester were visualized. The enzyme corresponding to the band with the strongest esterolytic activity was also responsible for casein‐hydrolytic activity in gel between pH 6 and 8; it was isolated and characterized. Starting with 450 g of wet cells, about 3 mg of a purified enzyme were obtained. This represented a recovery of 16% and almost 1000‐fold purification. The isolated enzyme, designated as protease I, was homogeneous by electrophoresis and sucrose‐gradient centrifugation. Its molecular weight was estimated by two different methods to be about 43000. The enzyme hydrolysed N‐acetyl‐dl‐phenylalanine‐2‐naphthyl ester, a chymotrypsin substrate, but was inactive upon several synthetic substrates for enzymes with carboxypeptidase‐A and trypsin‐like specificity. The esterolytic activity was inhibited by DFP, whereas it was resistant to phenyl methyl‐sulfonylfluoride even after prolonged treatment. Metal chelating agents, sulfhydryl reagents and several metal ions were without effect. The proterolytic activity of the purified enzyme was confirmed by its ability to breakdown native E. coli polynucleotide phosphorylase.
We have previously identified a macrophage 70-kDa, actin-bundling protein as a constituent of actin-based cytoplasmic gel and showed that its association with or dissociation from cytoplasmic gels was remarkably affected by submicromolar calcium. In this study, we purified the 70-kDa protein from soluble cytosolic extracts and carried out a more detailed characterization. The amino acid sequences of four peptidic fragments, obtained from the purified protein by enzymatic or chemical cleavage, were completely or nearly identical to those of L-plastin, a protein initially identified in transformed cells from solid tumors (Goldstein & Leavitt, 1985). By Western blot analysis of normal cells and tissues using specific anti-70-kDa protein antibodies, the 70-kDa molecule was detected only in hematopoietic cells. The 70-kDa protein bound to actin with apparent Kd values of 1.8 and 5.5 microM in the absence and presence of 20 microM free calcium, respectively. Cross-linking activity measured by falling-ball viscosimetry was optimal at free calcium lower than 0.15 microM but was progressively inhibited at higher calcium concentrations, within the physiological range. Half-maximal inhibition occurred at 1.6 microM free calcium. No severing of actin filaments by the 70-kDa protein was observed in any of these assays or previously (Pacaud & Harricane, 1987). Major conformational changes of the protein, as measured by the fluorescence emission intensity of tyrosine residues, occurred at free calcium concentration ranging between 0.15 and 1.5 microM. Magnesium did not mimic the calcium effect. The results suggest that the 70-kDa protein possesses both high-affinity sites and selectivity for calcium.(ABSTRACT TRUNCATED AT 250 WORDS)
Proteins which bind to actin filaments in macrophages were investigated by developing a procedure for the isolation of cytoplasmic gels. The gels were found to consist of five major constituents: actin, filamin and the 105-kDa, 70-kDa and 55-kDa components. Prolonged exposure of this macromolecular complex to high-ionicstrength buffer solubilized almost all the proteins, leaving behind the 55-kDa component along with a large amount of actin. Gel filtration of the solubilized extract led to the isolation of five constituents comprising actin, filamin, the 105-kDa and 70-kDa polypeptides, plus a molecular species which eluted at the position of a 280-kDa globular protein. The biochemical and immunological properties of the 105-kDa component were analogous to those of a-actinin. Although several attempts were made to correlate the three other constituents (280-kDa, 70-kDa and 55-kDa) with known cytoskeletal proteins, their identity remains to be established. a-Actinin, and the 280-kDa and 70-kDa species all exhibited the ability to co-sediment with F-actin and to pack actin filaments into bundles. The bundling activity of the 70-kDa protein was significantly decreased in the presence of micromolar concentrations of calcium, while the activity of the 280-kDa protein was not. Such a Ca'+-sensitive protein could be very important in controlling the local cytoplasmic viscosity.It is now well established that most animal cells contain ordered arrays of three major fiber systems : microfilaments, microtubules and intermediate filaments. Because of their highly dynamic properties, microfilaments (actin filaments) are increasingly the subject of ultrastructural and biochemical investigations at the cellular level and in cytosolic extracts.Under appropriate conditions, cytosolic preparations from many cells can undergo a rapid transition from a soluble to a gel-like phase. This rise in viscosity is induced by a rapid polymerization of monomeric actin. Substantial variations in cytoplasmic consistency had also been observed during cell locomotion. It had been subsequently found that conditions which affect the viscoelastic properties of the cytoplasm, such as transient changes in free Ca", also influence actin-myosin interactions (for reviews see [l -31). These findings led Hellewell and Taylor [4] to propose that transitions from the sol to gel state may be directly involved in the generation of cytoplasmic movements.More recently, the use of techniques involving microinjection of fluorescently labeled proteins into living cells [S] has allowed a direct correlation to be established between the reorganization of actin filament bundles and motile activities in fibroblasts [6, 71. Therefore, the rapid assembly and disassembly of microfilaments is probably required for cell motility. The biochemical and structural bases for these dynamic cellular events are, however, very poorly understood. They are probably related to the properties of the actin molecule itself and also to its interactions with actin-associated proteins. Ma...
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