Isolation of a minor species of actin from the nuclei of Acanthamoeba castellanii

Actin was extracted from isolated nuclei of Acanthamoeba castellanii and purified to homogeneity under nondenaturing conditions by diethylaminoethylcellulose and Sephadex G-100 chromatography. The pure protein has the same molecular weight as cytoplasmic Acanthamoeba actin and a very similar amino acid composition. Isoelectrofocusing shows that nuclear actin is slightly more acidic than the major cytoplasmic species, and comparative analysis of peptides from tryptic and cyanogen bromide digests shows that both actins are very similar but not chemically identical. In an assay that is specific for most actins, the inhibition of DNase I through the formation of a 1:1 G-actin-DNase I complex, the nuclear and cytoplasmic actins are equally effective. By use of a similar procedure for the purification of both actins, it is estimated that the amount of nuclear actin is about 1.5% of the amount of cytoplasmic actin, a major protein of the amoeba. It is concluded that a minor isoelectric species of actin associates selectively with the nuclei of A. castellanii.     

Isolation of Escherichia coli synthesized recombinant eukaryotic proteins that contain epsilon-N-acetyllysine.

Recombinant porcine (rpST) and bovine somatotropins (rbST) synthesized in Escherichia coli contain the amino acid, epsilon-N-acetyllysine. This amino acid was initially discovered in place of the normal lysine144 in a modified reversed-phase HPLC (RP-HPLC) species of rpST. Mass spectrometry and amino acid sequencing of a tryptic peptide isolated from this RP-HPLC purified protein were used to identify this altered residue as epsilon-N-acetyllysine. Ion-exchange chromatography was utilized to prepare low isoelectric point (pI) forms of rpST and rbST, which are enriched in epsilon-N-acetyllysine. Electrospray mass spectrometry demonstrated that the majority of the protein in these low pI fractions contained species 42 Da larger than normal. Immobilized pH gradient electrophoresis (IPG) of the ion-exchange purified low pI proteins was used to isolate several monoacetylated species of rpST and rbST. The location of the acetylated lysine in each IPG-purified protein was determined by tryptic peptide mapping and amino acid sequencing of the altered tryptic peptides. Amino acid analyses of enzymatic digests of rpST and rbST were also used to confirm the presence of epsilon-N-acetyllysine in these recombinant proteins. These data demonstrate that a significant portion of rpST and rbST produced in E. coli contain this unusual amino acid.     

Isolation and structural characterization of three isoforms of recombinant consensus alpha interferon

Recombinant DNA-derived consensus alpha interferon was expressed in Escherichia coli and purified. Isoelectric focusing of this purified protein indicated the presence of three isoelectric subforms of pI 6.1, 6.0, and 5.7. These three subforms were preparatively separated by isoelectric focusing using Immobiline polyacrylamide gel and did not exhibit apparent differences in biological activity and tertiary structure. The pI 5.7 subform could also be separated from the pI 6.1 and 6.0 subforms by reverse-phase HPLC. Automated N-terminal amino acid sequence analysis of the pI 6.1 and 6.0 subforms yielded sequences corresponding to the methionyl and des-methionyl forms of the protein, respectively. Sequence analysis of the pI 5.7 subform indicated that its N terminus is blocked. To further determine the structure of the blocking moiety in the pI 5.7 subform, a blocked N-terminal tryptic peptide was isolated from HPLC peptide mapping of the S-carboxymethylated derivative. Results obtained from mass spectroscopic and amino acid analyses of this peptide suggest that it is blocked with an acetyl group at the N-terminal cysteine residue.     

Physical and chemical characterization of a horse serum carboxylesterase

The serine carboxylesterase from horse serum was characterized by amino acid composition, peptide mapping, molecular and subunit weights, and sequencing of the amino acids around the essential serine residue at the active site. A protocol was developed for using reversed-phase high-performance liquid chromatography as the final step to obtain homogeneous preparations of horse serum carboxylesterase. Amounts sufficient for determining the amino acid composition and for peptide maps were obtained from a partially purified starting material which contained approximately 55% carboxylesterase. The amino acid composition, like the subunit weight (70,800 +/- 1400), was similar to the corresponding values reported for other serine carboxylesterases. However, the amino acid sequence of the tryptic digest fragment containing the essential nucleophilic seryl residue differed significantly from the corresponding sequences of other mammalian serine carboxylesterases.     

Isolation and characterization of fatty acid binding proteins from mammary tissue of lactating rats.

A protein fraction with fatty acid binding activity has been isolated from mammary tissue from lactating rats by a process involving DEAE-cellulose ion-exchange chromatography, heat treatment, CM-cellulose ion-exchange chromatography and finally ammonium sulphate precipitation. The purified fraction migrated as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 14400. However, when this protein fraction was electrophoresed under non-dissociating conditions, two species were observed in a 4:1 ratio. The two components were separated using h.p.l.c. Both bind fatty acids and appear to have similar amino acid compositions although exhibiting different pI values of 4.8 and 4.9. The mammary fatty acid binding proteins appear to be very similar to the fatty acid binding protein isolated from rat heart based on the electrophoretic mobilities and amino acid composition. The major mammary form (pI 4.9) has been partially sequenced and the amino acid sequences obtained can be aligned with 67 residues of the revised rat heart amino acid sequence [Heuckeroth, Birkenmeier, Levin & Gordon (1987) J. Biol. Chem. 262, 9709-9717]. Both mammary species also showed immunochemical identity to rat heart fatty acid binding protein when tested with an anti-serum raised against the heart protein. Anti-sera raised against the minor mammary form (pI 4.8) specifically precipitated this form under non-denaturing conditions but both forms after they had been denatured. Quantitative immunoassays using the anti-(heart fatty acid binding protein) serum showed that concentrations of the fatty acid binding proteins present in mammary cytosols increase during lactation and increase further after feeding a high-fat diet.     

Phosphorylation of beta-crystallin B2 (beta Bp) in the bovine lens

Three major 32P-labeled polypeptides were found in the soluble fraction of bovine lenses cultured in a medium containing [32P]orthophosphate. Two of the polypeptides corresponded to the phosphorylated A and B chains of alpha-crystallin. In this communication, the third polypeptide is now identified. This polypeptide is characterized by a molecular weight of 27,000 and a pI of 6.6, eluted exclusively in the beta Low fraction of a CL-6B gel filtration separation of lens soluble material, and could be further purified by DE52 anion exchange chromatography. The only 32P-labeled amino acid detected was phosphoserine. A single 32P-labeled peptide was observed after tryptic digestion and two-dimensional mapping. The amino acid sequence of the purified peptide is Gly-Ala-Phe-His-Pro-Ser-Ser. This sequence exactly matches the expected C-terminal tryptic fragment, residues 198-204, of the bovine beta-crystallin B2. The results of carboxypeptidase A digestion of the 32P-labeled peptide suggest that only Ser203 is phosphorylated. By using the catalytic subunit of the cAMP-dependent protein kinase, purified beta B2 was phosphorylated in vitro, generating a single 32P-labeled polypeptide with the identical pI as the phosphorylated polypeptide obtained from lens culture. On the basis of these data, the Mr 27,000 32P-labeled polypeptide is identified as the phosphorylated form of the beta-crystallin B2.     

The mammalian hypusine-containing protein, eukaryotic initiation factor 4D. Structural homology of this protein from several species

A single cellular protein of Mr approximately 18,000 and pI near 5.1, recently identified as eukaryotic translation initiation factor eIF-4D, contains the unusual amino acid hypusine [N epsilon-(4-amino--2-hydroxybutyl)lysine] formed post-translationally from lysine with a structural contribution from the polyamine spermidine. When the 3H-labeled hypusine-containing protein isolated from Chinese hamster ovary (CHO) cells that were grown with radioactive polyamine is digested with trypsin and the digest is subjected to two-dimensional separation, a single radioactive peptide is seen. A labeled peptide that occupies this same position is found in a digest of the [3H]hypusine protein from human lymphocytes and the single hypusine-containing tryptic peptide from purified rabbit reticulocyte eIF-4D also moves to this identical position. Stepwise Edman degradation of the tryptic digest of CHO cell hypusine-protein releases the radioactivity as a single peak in accordance with our earlier evidence for a single hypusine residue per molecule of eIF-4D. The similar patterns of radioactive peptides obtained from tryptic digests of radioiodinated eIF-4D from CHO cells, human lymphocytes, and rabbit reticulocytes suggest a highly conserved primary structure for this protein.     

Inhibition of actin polymerization by a synthetic dodecapeptide patterned on the sequence around the actin-binding site of cofilin

Cofilin is an F-actin side-binding and -depolymerizing protein with an apparent molecular mass of 21 kDa. By means of the end label fingerprinting method, the amino acid residue on cofilin sequence cross-linked to actin by zero length cross-linker, 1-ethyl-3-(3-dimethylamino propyl)carbodiimide, was identified as Lys112 and/or Lys114. A synthetic dodecapeptide patterned on the sequence around the actin-cross-linking site of cofilin (Trp104-Met115) inhibited the binding of cofilin to actin. Moreover, the dodecapeptide was found to be a potent inhibitor of actin polymerization. Thus, we conclude that the dodecapeptide sequence constitutes the region essential for the actin-binding and -depolymerizing activity of cofilin. A sequence similar to the dodecapeptide is found in other actin-depolymerizing proteins, destrin, actin-depolymerizing factor, and depactin. Therefore, the dodecapeptide sequence may be a consensus sequence essential for actin-binding and -depolymerizing activity in actin-depolymerizing proteins.     

The EGF receptor is an actin-binding protein

In a number of recent studies it has been shown that in vivo part of the EGF receptor (EGFR) population is associated to the actin filament system. In this paper we demonstrate that the purified EGFR can be cosedimented with purified filamentous actin (F-actin) indicating a direct association between EGFR and actin. A truncated EGFR, previously shown not to be associated to the cytoskeleton, was used as a control and this receptor did not cosediment with actin filaments. Determination of the actin-binding domain of the EGFR was done by measuring competition of either a polyclonal antibody or synthetic peptides on EGFR cosedimentation with F-actin. A synthetic peptide was made homologous to amino acid residues 984-996 (HL-33) of the EGFR which shows high homology with the actin-binding domain of Acanthamoeba profilin. A polyclonal antibody raised against HL-33 was found to prevent cosedimentation of EGFR with F-actin. This peptide HL-33 was shown to bind directly to actin in contrast with a synthetic peptide homologous to residues 1001-1013 (HL-34). During cosedimentation, HL-33 competed for actin binding of the EGFR and HL-34 did not, indicating that the EGFR contains one actin-binding site. These results demonstrate that the EGFR is an actin-binding protein which binds to actin via a domain containing amino acids residues 984-996.     

Lamprey 48-kDa lens protein represents a novel class of crystallins

SDS-PAGE revealed a major Mr 48 000 polypeptide of pI around 8 in the water-soluble fraction of lamprey lenses. It occurs as a monomeric protein, and its amino acid composition and tryptic peptides show no resemblances to alpha-, beta-, gamma- or delta-crystallin. Immunoblotting with antiserum against the 48-kDa protein revealed an immunologically related polypeptide of similar Mr in reptiles, several birds and a fish, but showed no cross-reactivity with any other water-soluble lens component. The 48-kDa protein is not detected in many birds and fishes, and in the investigated mammals and amphibians.     

Actin-binding Verprolin Is a Polarity Development Protein Required for the Morphogenesis and Function of the Yeast Actin Cytoskeleton

Yeast verprolin, encoded by VRP1, is implicated in cell growth, cytoskeletal organization, endocytosis and mitochondrial protein distribution and function. We show that verprolin is also required for bipolar bud-site selection. Previously we reported that additional actin suppresses the temperature-dependent growth defect caused by a mutation in VRP1. Here we show that additional actin suppresses all known defects caused by vrp1-1 and conclude that the defects relate to an abnormal cytoskeleton. Using the two-hybrid system, we show that verprolin binds actin. An actin-binding domain maps to the LKKAET hexapeptide located in the first 70 amino acids. A similar hexapeptide in other acting-binding proteins was previously shown to be necessary for actin-binding activity. The entire 70– amino acid motif is conserved in novel higher eukaryotic proteins that we predict to be actin-binding, and also in the actin-binding proteins, WASP and N-WASP. Verprolin-GFP in live cells has a cell cycle-dependent distribution similar to the actin cortical cytoskeleton. In fixed cells hemagglutinin-tagged Vrp1p often co-localizes with actin in cortical patches. However, disassembly of the actin cytoskeleton using Latrunculin-A does not alter verprolin's location, indicating that verprolin establishes and maintains its location independent of the actin cytoskeleton. Verprolin is a new member of the actin-binding protein family that serves as a polarity development protein, perhaps by anchoring actin. We speculate that the effects of verprolin upon the actin cytoskeleton might influence mitochondrial protein sorting/function via mRNA distribution.     

The 135 kDa actin-bundling protein fromLilium longiflorum pollen is the plant homologue of villin

Summary Actin microfilaments, which are essential for cell growth and cytoplasmic streaming in pollen tubes, are closely dependent on actin-binding proteins for their organization and regulation. We have purified the plant 135 kDa actin-bundling protein (P-135-ABP) fromLilium longiflorum pollen and determined that its amino acid composition is highly similar to members of the villin-gelsolin family of proteins. We used antibodies against P-135-ABP to probe an expression cDNA library ofL. longiflorum pollen and isolated a full-length clone (ABP135) that corresponds to a 106 kDa polypeptide. The deduced amino acid sequence ofABP135 shows homology with members of the villin-gelsolin family of proteins and contains the characteristic six repeats of this family, as well as an extended carboxy-terminal domain that includes the villin headpiece preceded by a highly variable region. Using two-dimensional polyacrylamide gel electrophoresis we detected at least 5 isoforms of P-135-ABP, with isoelectric points (pI) ranging between 5.6 to 5.9. The most abundant P-135-ABP isoform has a pI of 5.8, closely approximating the pI predicted from the deducedABP135 amino acid sequence. These data, together with the partial amino acid sequence from a proteolytic peptide of the protein, indicate that P-135-ABP is a plant villin. Immuno-detection of Lilium villin in rapidly frozen pollen tubes localized it to actin bundles. Lilium villin is also ubiquitously expressed in all tissues tested. Since villins, like gelsolins, are also Ca²⁺-dependent severing, capping, and nucleating proteins, Lilium villin may participate in F-actin fragmentation and nucleation in the apex of the pollen tube where there is steep Ca²⁺ gradient.Abbreviations BMM butyl methyl-methacrylate - PPI polyphos-phoinositides - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Subunit structure and amino acid composition of xylose isomerase from Streptomyces albus.

The subunit structure and amino acid composition of xylose isomerase from Streptomyces albus have been examined. A native molecular weight of 165,000 determined by sedimentation equilibrium was reduced to 43,000 when the protein was treated with 6 M guanidine hydrochloride. No further reduction in molecular weight was observed when potential disulfide bridges of xylose isomerase were reduced and alkylated, indicating that the protein was devoid of interchain disulfide bonds. NH2-terminal analysis using [3H]dansyl chloride showed 0.86 residues of methionine per Mr equals 41,500 unit. Analysis of the native protein with an automated protein sequenator revealed the presence of only one degradable polypeptide chain. Fractionation of the soluble tryptic peptides of S-[14C]carboxymethyl xylose isomerase by ion exchange chromatography and one-dimensional paper electrophoresis yielded 37 to 43 peptides. When the acid-insoluble tryptic peptides were dissolved and analyzed using gel filtration techniques, and additional four peptides were found. A unique radioactive tryptic peptide containing S-carboxymethylcysteine was found among the soluble peptides, confirming cysteine as the limiting amino acid residue in the amino acid composition of xylose isomerase. On the basis of its lysine and arginine content, the number of tryptic peptides is consistent with the hypothesis that the native xylose isomerase is a tetramer of four very similar or identical subunits of Mr equals 41,500, associated by noncovalent bonds.     

Identification and purification of calcium ion dependent modulators of actin polymerization from bovine thyroid

We describe the purification of Ca2+-dependent actin modulator proteins from bovine thyroid using DNase I affinity chromatography and diethylaminoethylcellulose chromatography. The 40K actin modulator has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 40 000 and an isoelectric point of 8.1. Its amino acid composition is different from previously described actin-associated proteins and thyroid actin. On the basis of the centrifugation assay and the DNase I inhibition assay, the actin complexed with the 40K protein is G-actin in its conformation rather than F-actin oligomers. Substoichiometric concentrations of the 40K protein rapidly inhibit actin polymerization in the presence of physiological concentrations of Ca2+ and Mg2+. An 80K actin modulator also has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 80 000 and an isoelectric point of 6.35-7.0. Its amino acid composition is different from those of villin, gelsolin, and leukocyte actin polymerization inhibitor. On the basis of the DNase inhibition assay and the centrifugation assay, the nonprecipitable actin associated with the 80K protein was F-actin in its conformation. The 80K protein acts very efficiently as a Ca2+-dependent nucleator for actin assembly and reduces its viscosity. In addition to the 40K and 80K actin modulators, 91K and 95K actin-associated proteins were partially purified. The 91K-95K fraction has similar activity to the 80K protein regarding precipitation of F-actin. The 125I-G-actin polyacrylamide gel overlay technique [Snabes, M. C., Boyd, A.E., & Bryan, J. (1981) J. Cell Biol. 90, 809-812] revealed that both the 91K and 95K proteins bind 125I-actin after sodium dodecyl sulfate (NaDodSO4) electrophoresis while the 80K and 40K proteins do not. Thyroid 91K protein comigrated with a human platelet 91K actin binding protein on NaDodSO4 gels and may be similar to macrophage gelsolin. The 95K protein may be similar to villin, the intestinal cytoskeletal protein.     

Platelet glycoprotein Ib beta is phosphorylated on serine 166 by cyclic AMP-dependent protein kinase

Platelet responses are inhibited by agents such as prostaglandin E1 that increase the cytoplasmic concentration of cyclic AMP. Inhibition is thought to result from phosphorylation of specific proteins. One protein that becomes phosphorylated is glycoprotein (GP) Ib beta, a component of the GP Ib.IX complex. We have suggested that phosphorylation of GP Ib beta inhibits the collagen-induced polymerization of actin. The aim of the present study was to identify the amino acid(s) in GP Ib beta that is phosphorylated. Purified GP Ib.IX complex was phosphorylated by the catalytic subunit of purified bovine cyclic AMP-dependent protein kinase in the presence of [gamma-32P]ATP. Phosphoamino acid analysis showed that in GP Ib beta, [32P]phosphate was incorporated only into serine and was in a single tryptic peptide. Amino acid sequencing showed that this peptide was from the cytoplasmic domain of GP Ib beta and encompassed residues 161-175. A single serine residue, serine 166, contained the radiolabel. To determine whether the same residue was phosphorylated in intact platelets, GP Ib beta was isolated from 32P-labeled platelets before or after their exposure to prostaglandin E1. In both cases, radiolabel was present in phosphoserine and was in a single tryptic peptide. This peptide was the same as that which was phosphorylated in the purified GP Ib.IX complex, as shown by its identical mobility on two-dimensional tryptic maps, the presence of a positively charged residue in the fourth position, and the presence of the radiolabel in the sixth position of the peptide. This study shows that when cyclic AMP concentrations rise in platelets, the cytoplasmic domain of GP Ib beta is phosphorylated on serine 166, probably by cyclic AMP-dependent protein kinase. We suggest that phosphorylation of this residue may contribute to the inhibitory actions of cyclic AMP by inhibiting collagen-induced polymerization of actin.     

Characterization and binding properties of human fetal lung fatty acid-binding proteins

When delipidated Mr>10,000 cut-off human fetal lung cytosol was separated on gel filtration and ion-exchange chromatography on Auto-FPLC system, two fatty acid-binding proteins (FABPs) of pI 6.9 and pI 5.4 were purified to homogeneity. On Western blotting analysis with the anti-human fetal lung pI 6.9 FABP, these two proteins showed immunochemical cross reactivity with each other and with purified hepatic FABPs but not with cardiac or gut FABP. These two FABPs have identical molecular mass of 15.2 kDa, which is slightly higher than that of the hepatic proteins (14.2 kDa). Carbohydrate covalently linked to FABPs, that may substantially add to the molecular mass, was not detected in the purified protein preparations. Amino acid analysis revealed that both the proteins have same amino acid composition each containing one Trp residue that is lacking in hepatic FABP. Different isoforms of lung FABP exhibited different binding ability for their natural ligands. These proteins bind palmitoyl CoA with higher affinity than oleic acid. pI 6.9 FABP can more rapidly and efficiently transfer fatty acid than can pI 5.4 FABP from unilammelar liposomes. Thus these FABPs may play a critical role in fatty acid transport during human fetal lung development.Abbreviations AO anthroyloxy - 12-AS 12-(9-anthroyloxy)stearic acid - FABP fatty acid-binding protein - NBD-PE [N-(4-nitrobenzo-2-oxa-1,3-diazole)phosphatidylethanolamine - Pal-CoA palmitoyl coenzyme A - PITC phenylisothiocyanate - PBS phosphate-buffered saline - PtdCho phosphatidylcholine - SUV small unilamellar vesicle - Tris tris(hydroxymethyl) amino methane     

The nature of post-translational formation of MM creatine kinase isoforms

Isoforms (derived from the same isoenzyme but distinguished by differences in isoelectric point) of MM creatine kinase appear in plasma after myocardial infarction. They are formed by conversion of the tissue form of creatine kinase (MM-A, pI 7.80) to progressively more acidic species (MM-B, pI 7.50) and MM-C (pI 7.20) after release into the circulation. To define the changes responsible for myocardial MM creatine kinase isoform formation in humans and dogs, purified isoforms were treated with trypsin or cyanogen bromide. The digests were fractionated by reverse-phase high pressure liquid chromatography. Comparison of proteolytic maps showed that MM-A and MM-C were each characterized by a single, unique peptide peak. Maps of MM-B creatine kinase contained both of these peaks. Sequence analysis and comparison with the complete amino acid sequence of MM creatine kinase showed that the peptide unique to MM-A corresponded to the COOH-terminal tryptic or CNBr peptide. The peptide unique to MM-C was shown to have the same amino acid composition except for lysine (the COOH-terminal amino acid). Thus, isoform formation is characterized by the successive removal of the COOH-terminal lysine residue from one M subunit at a time resulting in the conversion of MM-A to isoforms MM-B and MM-C.     

Biochemical and preliminary crystallographic characterization of the vitamin D sterol- and actin-binding by human vitamin D-binding protein

Vitamin D-binding protein (DBP), a multi-functional serum glycoprotein, has a triple-domain modular structure. Mutation of Trp145 (in Domain I) to Ser decreased 25-OH-D(3)-binding by 80%. Furthermore, recombinant Domain I (1-203) and Domain I + II (1-330) showed specific and strong binding for 25-OH-D(3), but Domain III (375-427) did not, suggesting that only Domains I and II might be required for vitamin D sterol-binding. Past studies have suggested that Domain III is independently capable of binding G-actin. We exploited this apparently independent ligand-binding property of DBP to purify DBP-actin complex from human serum and rabbit muscle actin by 25-OH-D(3) affinity chromatography. Competitive (3)H-25-OH-D(3) binding curves for native DBP and DBP-actin complex were almost identical, further suggesting that vitamin D sterol- and actin-binding activities by DBP might be largely independent of each other. Trypsin treatment of DBP produced a prominent 25 kDa band (Domain I, minus 5 amino acids in N-terminus), while actin was completely fragmented by such treatment. In contrast, tryptic digestion of purified DBP-actin complex showed two prominent bands, 52 (DBP, minus 5 amino acids in the N-terminus) and 34 kDa (actin, starting with amino acid position 69) indicating that DBP, particularly its Domains II and III were protected from trypsin cleavage upon actin-binding. Similarly, actin, except its N-terminus, was also protected from tryptic digestion when complexed with DBP. These results provided the basis for our studies to crystallize DBP-actin complex, which produced a 2.5 A crystal, primitive orthorhombic with unit cell dimensions a=80.2A, b=87.3A, and c=159.6A, P2(1)2(1)2(1) space group, V(m)=2.9. Soaking of crystals of actin-DBP in crystallization buffer containing various concentrations of 25-OH-D(3) resulted in cracking of the crystal, which was probably a reflection of a ligand-induced conformational change in the complex, disrupting crystal contacts. In conclusion, we have provided data to suggest that although binding of 25-OH-D(3) to DBP might result in discrete conformational changes in the holo-protein to influence actin-binding, these binding processes are largely independent of each other in solution.     

Primary structure of the southern bean mosaic virus coat protein: First results

Studies on the southern bean mosaic virus coat protein have established the molecular weight of this protein, its amino acid composition, the nature of its C-terminal amino acid, and the blockage of the N-terminal residue by an acetyl group. After hydrolysis of the protein by trypsin, the hydrolysate was fractionated by ion-exchange chromatography. Among the purified tryptic peptides were isolated the N- and the C-terminal peptides where sequences were determined, principally by mass spectrometry.