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...

Protease I, a periplasmic endopeptidase from Escherichia coli has been further purified by a modified procedure. While the purified protein consists of a single polypeptide chain of about 21 000 daltons, its molecular weight in dilute salt solution was estimated to be near 43000, suggesting that the enzyme has a marked tendency to dimerize. It has only one disulphide bond and is very sensitive to urea.In agreement with previous evidence of a chymotrypsin-like specificity, hydrolytic assays of various p-nitrophenyl esters of N-substituted amino acids showed that phenylalanine and tyrosine derivatives are the best substrates for the enzyme. The K, (app) for N-benzoyloxycarbonyl-L-tyrosinep-nitrophenyl ester at pH 7.5 in 100 mM sodium phosphate buffer at 25 "C was found to be 0.2 mM. In contrast to chymotrypsin, protease I is unable to hydrolyse N-acetyl-L-phenylalanine ethyl ester and its tyrosine analogue. Moreover, the enzyme appears devoid of amidase activity and exhibits a low activity upon polypeptides. At 37 "C, it cleaves the carboxymethylated B-chain of bovine insulin at four points : Phe25-Tyr26, Phe24-Phe25, Leu"-Tyrt6 and Ser9-His". From a detailed study of peptides bonds hydrolyzed, it was concluded that protease I has a stringent requirement for both residues forming the scissile bond, and appears to possess an extended hydrophobic binding site.

A method has been devised to study the influence of Ca2+ on the in vitro formation of actin gel networks. Under appropriate conditions low-Ca' + cytosolic extracts ( < 1 nM) from macrophages rapidly formed a macromolecular complex composed of actin, filamin, a-actinin and two new proteins of 70 kDa and 55 kDa. [Pacaud, M. (1986) Eur. J . Biochem. 156, Increasing concentrations of free Ca2+ to 1 -2 pM resulted in complete inhibition of the association of 70-kDa protein, a protein which associates actin filaments into parallel arrays. Concentrations of Ca2+ greater than 3 pM caused incorporation of two additional proteins, gelsolin and a 18-kDa polypeptide, with no change in either the actin or a-actinin content of the cytoskeletal structures. Use of a polyacrylamide gel overlay technique with 12sI-calmodulin revealed that a high-M, calmodulin-binding protein analogous to spectrin was also associated with these structures when micromolar Ca2+ was present. Similar assays with 45CaC12 indicated that the 70-kDa protein binds Ca2+ with high affinity. It is thus suggested that Ca2+ might regulate the dynamic assembly of microfilaments through several target proteins, gelsolin, the 70-kDa protein and calmodulin.Calcium ions play a key role in the initiation and regulation of a variety cell functions (reviewed in 11, 21). Several lines of evidence suggest that local variations in intracellular concentrations of free Ca2 + in macrophage and neutrophils may be involved in regulating the organisational state of actin filaments and such stimulus-elicited responses as shape changes, chemotaxis or phagocytosis [3 -61. Cell movements are related to the structural and dynamic properties of cytoplasm and a number of investigations have revealed that Ca2+ also acts as a regulator of the gel/sol transformations of cytoplasm or cytosolic extracts (reviewed in [l]). Controlled interconversions between monomeric, polymeric or higher organised states of actin are thought to be responsible for the viscoelastic properties of cytoplasms ; while interactions between myosin and organised arrays of actin filaments (microfilaments) are likely to be required for the generation of motile force (reviewed in 17, 81).Although the biochemical basis for the role that Ca2+ plays in the dynamics of microfilaments is poorly understood, this process is assumed to be mediated by Ca2+-induced alterations in the association of actin with actin-related proteins. It is conceivable that the functional state of some actinbinding proteins could be modulated by Ca2 + either directly or indirectly. Indirect regulation is known to involve calmodulin, a ubiquitous Ca2 +-receptor protein that regulates a variety of Ca2+-dependent functions (reviewed in [9, 101). For instance, the Ca2 +-dependent interaction of calmodulin with myosin light-chain kinase activates this enzyme, which in turn stimulates the actin-activatable ATPase of myosin 17, Correspondence to M. Pacaud, Biochimie CNRS -INSERM, B.P. 5051, F-34033 Montpellier Cedex, France 111. On the other hand, two...

Hydrolytic activities of isolated membrane fractions of Escherichia coli against chromogenic substrates, p-nitrophenyl ester and ,-naphthyl ester derivatives of N-substituted amino acids, were investigated by spectrophotometric and electrophoretic methods. Although detergents were absolutely necessary for the solubilization of enzymes, the amount of solubilized activities was increased by adding salt, such as NaCl or KCl. Two esterases were identified and separated by PAGE and by chromatography of the solubilized proteins in the presence of detergent. One hydrolyzed the alanine derivatives preferentially, whereas the other was mainly active on phenylalanine derivatives. Only the first was inactivated by diisopropyl fluorophosphate, a serine hydrolase inhibitor. Whereas the chymotrypsin-like enzyme was equally distributed between the inner and the outer membrane, the alanine activity was only detected in the inner membrane. They were both resistant to extraction with high salt concentrations, indicating their integral association with membranes. A study of the accessibility of these enzymes to their substrate in membrane vesicles with known polarity suggests that both alanine and phenylalanine activities are localized near the external surface of the cytoplasmic (inner) membrane. However, the phenylalanine activity (chymotrypsin-like enzyme) appears to be deeply buried inside the outer membrane. Because of its insensitivity to diisopropyl fluorophosphate, this last esterase seems to be distinct from the previously isolated periplasmic endopeptidase, protease I, which is also a chymotrypsin-like enzyme. Several lines of evidence indicate that membrane-bound proteolytic enzymes may play an important role in bacterial and eucaryotic cell physiology. The occurrence of limited proteolysis in bacteria has been reported in processes such as the transfer of secreted proteins (reviewed in references 5, 7, 26), membrane biogenesis (10, 12), and penetration of toxins into sensitive cells (22). However, the mechanism and physiological significance of these specific cleavages are still obscure. It is, therefore, of interest to undertake a detailed study of the enzymes involved in these reactions. All endopeptidases so far purified and characterized in Escherichia coli are soluble proteins. One of them, protease I (19, 20), is localized in the periplasmic space, whereas the other two, protease II (17, 18) and protease III (3) are found in the cytoplasm. Very little is known, however, about endopeptidases existing in membranes. Attempts to characterize particulate enzymes have been hampered by the lack of suitable solubilization procedures, instability of the solu

Under appropriate conditions macrophage cytosolic extracts can form a three-dimensional gel network of cross-linked actin filaments. These cytoplasmic gels are mainly composed of actin, filamin, alpha-actinin, and two new proteins of about 70,000 and 55,000 Mr (70 and 55 K). The behaviour of 70 K protein was found to be remarkably affected by Ca2+. Ca2+ treatment of isolated cytoplasmic gels led to the selective solubilization of the 70 K protein along with a 17 K polypeptide. Half-maximal recovery in the supernatant fraction was obtained from about 0.15 microM free Ca2+. The cytoplasmic gel constituents solubilized in high ionic strength buffer were able to re-assemble into an insoluble actin network when returned to near physiological ionic conditions. However, the inclusion of micromolar Ca2+ prevented the re-association of 70 K protein with actin in these complexes. As compared to the 70 K protein, alpha-actinin was fully resistant to any variations in Ca2+ concentrations. On the other hand, purified 70 K protein displayed the property of assembling actin filaments into bundles at low Ca2+ concentrations (less than 0.15 microM). However, the bundling activity decreased progressively at higher Ca2+, as detected by co-sedimentation and electron microscopy of the 70 K protein-actin mixtures. Half-maximal inhibition was observed at about 0.3 microM free Ca2+. Re-assembly of actin filaments into bundles occurred after chelation of Ca2+ by EGTA, indicating that the inhibitory effect of Ca2+ was reversible. Severing of actin filaments by 70 K protein was not observed in any of the solution conditions used. The Ca2+-dependent inhibition of the ability of 70 K protein to interact with actin networks resulted in a marked distortion of the overall organization of actin filaments, as revealed by thin-section electron microscopy of cytoplasmic gels formed in the presence and absence of Ca2+. Large zones of oriented bundles of filaments were replaced by a looser mesh. When the actin gel constituents were re-assembled in the presence of Ca2+ and exogenous gelsolin, it was also observed that the filament bundles (essentially generated by alpha-actinin) collapsed into dense aggregates. Furthermore, gelsolin did not significantly affect the ability of actin to re-combine with other proteins. The data presented here and previously led us to suspect that the Ca2+ control of the functional state of 70 K protein might be one of the prime factors in the triggering of rapid assembly and disassembly of microfilaments within macrophages.

N-Acetyl-D-arginine linked to an agarose matrix has been used to purify protease I1 from Escherichia coli by affinity chromatography. The specific adsorption of protease I1 to this absorbent was achieved in 220 mM potassium phosphate buffer pH 7.6, and the enzyme was eluted with L-arginine.Enzyme preparations from cells harvested at late log phase have been resolved into two molecular species which differ in specific activity, kinetic constants and carbohydrate content. Both species appeared homogeneous by electrophoresis in conventional buffers and also in the presence of sodium dodecyl sulfate. Only one enzyme species was obtained by the same procedure using bacteria harvested at the middle of exponential growth.We have previously demonstrated the presence of an enzyme with trypsin-like activity in crude extracts of Escherichia coli [l]. This enzyme, designated as protease 11, has been recently purified by a conventional method and some of its properties were investigated [2]. It hydrolyzes the synthetic substrates of trypsin and is inhibited by common inhibitors such as diisopropylphosphofluoridate, tosyl lysine chloromethyl ketone and amidines. Nevertheless it is unaffected by peptidic trypsin inhibitors. Protease I1 is also competitively inhibited by two enantiomeric forms of N-substituted derivatives of arginine and by free L-arginine. Taking advantage of the particularity that a number of neutral proteolytic enzymes are capable of binding but not hydrolyzing, significantly, the enantiomeric substrate analog [3,4], we have developed a method of purification of protease I1 by affinity chromatography on N-acetyl-D-arginine coupled to an agarose matrix. The procedure has enabled us to prepare the homogeneous enzyme in relatively simple steps with a final recovery greater than previously obtained by the conventional method : 30 -37 instead of 15 -25 %. Moreover two enzyme forms has been isolated from cell extracts from bacteria harvested at late log phase.