Primary structure of and studies on Acanthamoeba actophorin.

We determined the amino acid sequence of the actin monomer binding/actin filament severing protein actophorin from Acanthamoeba castellanii by automated Edman degradation of peptide fragments and by sequencing of full-length cDNA. Actophorin consists of 138 amino acids (calculated molecular weight of 15,543) and shares a high degree of sequence similarity to other low molecular weight actin monomer sequestering proteins, especially vertebrate cofilin, vertebrate actin depolymerizing factor/destrin, and echinoderm depactin. Actophorin is smaller and does not contain a nuclear localization sequence like the related vertebrate proteins. Southern blot analysis indicates that actophorin is a single-copy gene; however, Northern blots show two distinct mRNA species of 1 and 0.9 kb in size. Homogeneous recombinant actophorin purified from Escherichia coli is indistinguishable from the native protein in its physical properties and in biochemical assays of its interaction with actin, but is less reactive with three monoclonal antibodies raised against the native protein. The NH2 terminus of native actophrin is blocked, while the initiating methionine residue is removed from recombinant actophorin. This difference has no measurable effect on activity. By fluorescent antibody staining of Acanthamoeba, actophorin colocalizes with actin filaments in the cortical cytoplasm, especially at the leading edge of the cell. Additionally, actophorin binds phosphatidylinositol 4',5'-bisphosphate. The recombinant actophorin forms X-ray diffraction quality crystals of superior quality in poly(ethylene glycol)/2-propanol and, like the native crystal form, belongs to space group P2(1)2(1)2(1).

Acanthamoeba actin and profilin can be cross-linked between glutamic acid 364 of actin and lysine 115 of profilin.

Acanthamoeba profilin was cross-linked to actin via a zero-length isopeptide bond using carbodiimide. The covalently linked 1:1 complex was purified and treated with cyanogen bromide. This cleaves actin into small cyanogen bromide (CNBr) peptides and leaves the profilin intact owing to its lack of methionine. Profilin with one covalently attached actin CNBr peptide was purified by gel filtration followed by gel electrophoresis and electroblotting on polybase-coated glass-fiber membranes. Since the NH2 terminus of profilin is blocked, Edman degradation gave only the sequence of the conjugated actin CNBr fragment beginning with Trp-356. The profilin-actin CNBr peptide conjugate was digested further with trypsin and the cross-linked peptide identified by comparison with the tryptic peptide pattern obtained from carbodiimide-treated profilin. Amino-acid sequence analysis of the cross-linked tryptic peptides produced two residues at each cycle. Their order corresponds to actin starting at Trp-356 and profilin starting at Ala-94. From the absence of the phenylthiohydantoin-amino acid residues in specific cycles, we conclude that actin Glu-364 is linked to Lys-115 in profilin. Experiments with the isoforms of profilin I and profilin II gave identical results. The cross-linked region in profilin is homologous with sequences in the larger actin filament capping proteins fragmin and gelsolin.

Purification and characterization of a new mammalian serum protein with the ability to inhibit actin polymerization and promote depolymerization of actin filaments.

A protein with capacity to bind G-actin and the ability to inhibit polymerization and promote depolymerization of actin filaments has been isolated from the serum of rabbit. The protein, SAIP (for serum actin inhibitory protein), has been purified by affinity chromatography of serum over actin-Sepharose followed by protein fractionation with ammonium sulfate and chromatography over DEAE-cellulose. Five milligrams of purified SAIP is obtained from 100 mL of serum. Rabbit SAIP is resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two closely related polypeptides of 60000 and 56000 daltons, respectively (ratio 5.1:1). Each of these polypeptides consists of two isoelectric variants. SAIP binds to monomeric actin with a stoichiometry of 1:1 and a Kd of 0.12 microM. The SAIP-actin complex binds to DNase I. Actin polymerization is completely inhibited by incubation of actin with an equal concentration of SAIP. At equimolar concentrations to F-actin, SAIP induces complete depolymerization of the actin filaments. SAIP is also present in calf serum.

A comparative study of the in vitro polymerization of tubulin in the presence of the microtubule-associated proteins MAP2 and tau.

At concentrations of microtubule-associated proteins MAP2 and tau inducing maximal microtubule formation, MAP2 promotes higher rates and higher levels of microtubule nucleation that tau. Microtubules polymerized to steady state by tau show a 2-fold higher rate of tubulin loss compared to microtubules polymerized by MAP2. Microtubules polymerized in the presence of both MAP2 and tau contain lower levels of MAP2 (0.06 mol/mol of tubulin) and tau (0.07 mol/mol of tubulin) than microtubules polymerized in the exclusive presence of MAP2 (0.2 mol/mol of tubulin) or tau (0.25 mol/mol of tubulin). Addition of tau to microtubules polymerized by MAP2 results in incorporation of tau into microtubules (0.047 mol/mol of tubulin) and loss of MAP2 (0.1 mol/mol of tubulin). Similarly, incubation of microtubules polymerized by tau in the presence of MAP2 results in microtubule incorporation of MAP2 (0.1 mol/mol of tubulin) and loss of tau (0.08 mol/mol of tubulin). Microtubules and their ribbon precursor contain comparable levels of MAP2. Cross-sectional views of microtubules show no difference in the number of protofilaments (13 to 15) forming the MAP2 and tau microtubules. Fingerprints of cysteine-labeled tryptic peptides of two tau polypeptides (tau 4 and tau 6) are very similar and differ totally from similar fingerprints of MAP2.

The primary structure of the coat protein of the broad-host-range RNA bacteriophage PRR1.

The complete amino acid sequence of the coat protein of RNA bacteriophage PRR1 is presented. After thermolysin digestion, 26 peptides were isolated, covering the complete coat protein chain. Their alignment was established in part using automated Edman degradation on the intact protein, in part with overlapping peptides obtained by enzymic hydrolysis with trypsin, pepsin, subtilisin and Staphylococcus aureus protease, and by chemical cleavage with cyanogen bromide and N-bromosuccinimide. To obtain the final overlaps, a highly hydrophobic, insoluble tryptic peptide was sequenced for seven steps by the currently used manual dansyl-Edman degradation procedure, which was slightly modified for application on insoluble peptides. PRR1 coat protein contains 131 amino acids, corresponding to a molecular weight of 14534. It is highly hydrophobic, and the residues with ionizable side chains are distributed unevenly: acidic residues are absent in the middle third of the sequence, whereas a clustering of basic residues occurs between positions 44 and 62. PRR1 coat protein was compared with the coat proteins of RNA coliphages MS2 and Q beta, and the minimum mutation distance was calculated for both comparisons. It is highly probable that PRR1. Q beta and MS2 share a common ancestor. The basic region present in the three coat proteins is recognized as an essential structural feature of RNA phage coat proteins.

Sequence of the A-protein of coliphage MS2. I. Isolation of A-protein, determination of the NH2- and COOH-terminal sequences, isolation and amino acid sequence of the tryptic peptides.

The A-protein of coliphage MS2 was purified to a state of sufficient homogeneity to study its primary structure. The NH2-terminal sequence was determined for the first 8 residues. Comparison with the reported sequence of R17 protein (Weiner, A. M., Platt, T., and Weber, K. (1972) J. Biol. Chem. 247, 3242-3251) shows a difference at position 6 where alanine in R17 is replaced by threonine in MS2. The COOH-terminal sequence was shown to be -Arg-Leu-Ser-Arg, confirming the existence of UAG as the termination codon of the maturation protein (Comtreras, R., Ysebaert, M., Min Jou, W., and Fiers, W. (19731 Nature New Biol. 241, 99-101; Vandekerckhove, J., Nolf, F., and Van Montagu, M. C. (1973) Nature New Biol. 241, 102; Remaut E., and Fiers, W. (1972) J. Mol. Biol. 71, 243-261). Peptides obtained by enzymatic hydrolysis with trypsin were fractionated by a combination of gel filtration and paper electrophoresis and chromatography. Thirty-eight peptides were analyzed for amino acid composition and sequence. They provide information for 312 of the 393 residues of the A-protein polypeptide chain.

Sequence of the A-protein of coliphage MS2. II. Isolation and sequence determination of chymotryptic peptides.

A total of 94 different peptides have been isolated from chymotryptic digests of underivatized (35 mg) and of reduced, carboxymethylated and citraconylated (48 mg) A-protein. Their amino acid sequences provide information for 375 residues of the total of 393 amino acids of the A-protein polypeptide chain. Abnormal specificity of chymotrypsin in the digestion of citraconylated A-protein was observed.