Molecular organization of the sex steroid-binding protein (SBP) of human plasma

Several years ago this laboratory presented evidence that SBP is a dimer composed of two subunits having similar molecular weights. The question of whether or not these subunits are identical and therefore products of a single gene remained unanswered. We now report that the two polypeptide chains are identical and that SBP is a homodimer. The experimental approach was to reduce and [14C]alkylate cystine residues in human SBP, digest the product with trypsin or cyanogen bromide and determine the number of unique amino acid sequences around each [14C]carboxymethylcysteine residue. Only four unique sequences were found when all the radioactive peptides were analyzed. Since there are eight half-cystine residues per dimer, the results support a homodimeric structure.     

Disulphide bridges of the heavy chain of human immunoglobulin G2

Amino acid sequences around the disulphide bridges of the heavy chain of an immunoglobulin of the gamma2 subclass have been studied. The protein was digested with pepsin and the digest fractionated by Sephadex. Screening of the eluate by one-dimensional electrophoresis of oxidized and unoxidized samples was used as an assay and pools of fractions were prepared. Identification by diagonal electrophoresis of several inter- and intra-chain disulphide bridges was done on the pooled fractions. The inter-heavy-chain bridged peptide included four cystine residues. Comparison with proteins of other human subclasses indicated that the intrachain bridges identified are the bridges of the invariable section of gamma2 heavy chains. The amino acid sequence of one cysteic acid peptide that may have been derived from the variable part of the molecule was determined. Partial reduction followed by carboxymethylation with radioactive iodoacetate of two proteins of the gamma2 class showed a number of labelled peptides that could be identified as being related to the inter-chain bonded cystine residues.     

The structure of the mutant dihydrofolate reductase from Streptococcus faecium. Partial sequence and order of the limited tryptic and cyanogen bromide peptides.

The major form of dihydrofolate reductase from a methotrexate-resistant mutant (strain A) of Streptococcus faecium var. Durans has been purified on a large scale. Amino acid analysis of this form of the enzyme (isoenzyme 2) reveals an absence of cystine or cysteine, and sedimentation studies indicate a molecular weight of 20,800. The NH2-terminal sequence was determined by Edman degradation of the intact protein and the COOH terminus by selective tritiation and by carboxypeptidase treatment. After the action of trypsin on the citraconylated protein, seven of the expected nine peptides were purified from the digest, and after cyanogen bromide treatment of the unmodified protein, all seven of the anticipated peptides were isolated. The amino acid composition of all of these peptides has been established as well as their complete or partial sequences. From the results it was possible to order these peptides within the sequence and to establish the sequence of the NH2-terminal 60 residues and the COOH-terminal 11 residues.     

Degradation and debittering of a tryptic digest from beta-casein by aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2.

The mode of action of purified aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2 on a complex peptide mixture of a tryptic digest from bovine beta-casein was analyzed. The oligopeptides produced in the tryptic digest before and after aminopeptidase N treatment were identified by analysis of the N- and C-terminal amino acid sequences and amino acid compositions of the isolated peptides and by on-line liquid chromatography-mass spectrometry. Incubation of purified peptides with aminopeptidase N resulted in complete hydrolysis of many peptides, while others were only partially hydrolyzed or not hydrolyzed. The tryptic digest of beta-casein exhibits a strong bitter taste, which corresponds to the strong hydrophobicity of several peptides in the tryptic digest of beta-casein. The degradation of the "bitter" tryptic digest by aminopeptidase N resulted in a decrease of hydrophobic peptides and a drastic decrease of bitterness of the reaction mixture.     

A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin.

We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.     

Immunoglobulin κ-chains: Comparative sequences in selected stretches of Bence-Jones proteins

Three type K Bence-Jones proteins have been fully reduced and carboxymethylated with high-specific-activity iodo[(14)C]acetate. A tryptic digest and a chymotryptic digest of each protein were fractionated on a Sephadex column and the radioactive peptides were purified by paper electrophoresis. All the proteins studied had five unique carboxymethylated cysteine sequences. Three of these were identical with the exception of a single substitution, and two had some variations. The common peptides could be placed in the C-terminal half of the molecule. The variations around the other two half-cysteine residues provided information about the nature of the variability of the primary sequence of immunoglobulin kappa-chains. The results are consistent with the hypothesis that the chains derive from a common ancestor by somatic mutation of a small number of genes or by gene doubling and selection in the course of evolution. The isolation of the N-terminal peptide in methionine-containing Bence-Jones proteins is also described.     

Studies on the sub-units of triose phosphate isomerase

The sub-unit structure of rabbit muscle triose phosphate isomerase was studied by determination of the number of unique cysteine peptides. Alkylation of the thiol groups with radioactive iodoacetate in the presence of guanidine hydrochloride gave the S-carboxy[¹⁴C]methyl derivative of the protein. This was digested with trypsin, and the radioactive peptides were fractionated by ion-exchange chromatography; four main radioactive peaks were obtained, one of which contained two radioactive peptides. Peptide `maps' of the tryptic digest showed five main spots. The relationship between the members of both sets of five peptides was established. The radioactive peptides were characterized, and the results indicated the presence of five unique cysteine residues in the protein. Since there are approximately ten thiol groups/molecule, there are two closely related or identical sub-units. Studies of the terminal residues bear out this suggestion; only one kind of N-terminal residue (alanine) and one kind of C-terminal residue (glutamine) were detected. These results are in accord with the evidence from crystallography.     

Identification of the serine residue phosphorylated by protein kinase C in vertebrate nonmuscle myosin heavy chains

Two-dimensional mapping of the tryptic phosphopeptides generated following in vitro protein kinase C phosphorylation of the myosin heavy chain isolated from human platelets and chicken intestinal epithelial cells shows a single radioactive peptide. These peptides were found to comigrate, suggesting that they were identical, and amino acid sequence analysis of the human platelet tryptic peptide yielded the sequence -Glu-Val-Ser-Ser(PO4)-Leu-Lys-. Inspection of the amino acid sequence for the chicken intestinal epithelial cell myosin heavy chain (196 kDa) derived from cDNA cloning showed that this peptide was identical with a tryptic peptide present near the carboxyl terminal of the predicted alpha-helix of the myosin rod. Although other vertebrate nonmuscle myosin heavy chains retain neighboring amino acid sequences as well as the serine residue phosphorylated by protein kinase C, this residue is notably absent in all vertebrate smooth muscle myosin heavy chains (both 204 and 200 kDa) sequenced to date.     

Serine-324 of myosin's heavy chain is photoaffinity-labeled by 3'(2')-O-(4-benzoylbenzoyl)adenosine triphosphate

A portion of the active site of rabbit skeletal myosin near the ribose ring of ATP can be labeled by the photoaffinity analogue 3'(2')-O-(4-benzoylbenzoyl)adenosine triphosphate (Bz2ATP). The specificity of the photolabeling was assured by first trapping [14C]Bz2ATP at the active site by use of thiol cross-linking agents [Mahmood, R., Cremo, C., Nakamaye, K., & Yount, R. (1987) J. Biol. Chem. 262, 14479-14486]. Five radioactive peptides were isolated by high-performance liquid chromatography after extensive trypsin and subtilisin digestion of photolabeled myosin subfragment 1. Four of these peptides were sequenced by Edman techniques, and all originated from a region with the sequence Gly-Glu-Ile-Thr-Val-Pro-Ser-Ile-Asp-Asp-Gln, which corresponds to rabbit myosin heavy chain residues 318-328. The fifth labeled peptide had an amino acid composition appropriate for residues 312-328. Amino acid composition, radiochemical analysis, and sequence data indicate that Ser-324 is the major amino acid residue photolabeled by Bz2ATP. Spectrophotometric evidence indicates that the benzophenone carbonyl group has inserted into a C-H bond from either the alpha- or beta-carbon of serine. These results place Ser-324 at a distance of 6-7 A from the 3'(2') ribose oxygens of ATP bound at the active site of myosin.     

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.     

The amino acid sequence of ribonuclease U1, a guanine-specific ribonuclease from the fungus Ustilago sphaerogena

The complete amino acid sequence of ribonuclease U1 (RNase U1), a guanine-specific ribonuclease from a fungus, Ustilago sphaerogena, was determined by conventional protein sequencing, using peptide fragments obtained by several enzymatic cleavages of the performic acid-oxidized protein. The oxidized protein was first cleaved by trypsin and the resulting peptides were purified and their amino acid sequences were determined. These tryptic peptides were aligned with the aid of overlapping peptides isolated from a chymotryptic digest of the oxidized protein. The amino acid sequence thus deduced was further confirmed by isolation and analysis of peptides obtained by digestion of the oxidized protein with lysyl endopeptidase. The location of the disulfide bonds was deduced by isolation and analysis of cystine-containing peptides from a chymotryptic digest of heat-denatured RNase U1. These results showed that the protein is composed of a single polypeptide chain of 105 amino acid residues cross-linked by two disulfide bonds, having a molecular weight of 11,235, and that the NH2-terminus is blocked by a pyroglutamate residue. It has an overall homology with other guanine-specific or related ribonucleases, and shows 48% identity with RNase T1 and 38% identity with RNase U2.     

Specific modification of the condensation domain of fatty acid synthase and the determination of the primary structure of the modified active site peptides

Fatty acid synthase from the uropygial gland of goose was inactivated by iodoacetamide with a second-order rate constant of 1.3 M-1 S-1 at pH 6.0 and 25 degrees C. Of the seven component activities of the synthase, only the condensation activity was significantly inhibited by iodoacetamide modification. Since preincubation of the enzyme with acetyl-CoA, but not with malonyl-CoA, protected the enzyme from inactivation by iodoacetamide, it is suggested that iodoacetamide probably modified the primer-binding thiol group at the condensation active site. Determination of the stoichiometry of modification was done using [1-14C]iodoacetamide that was purified by high-performance liquid chromatography. Graphical analysis of the data showed that binding of 1.2 carboxamidomethyl groups per subunit of fatty acid synthase would result in complete inhibition of the enzyme activity, suggesting that there is one condensation domain per subunit of fatty acid synthase. Analysis of the tryptic peptide map of the enzyme that was modified with [1-14C]iodoacetamide in the presence and absence of acetyl-CoA revealed that acetyl-CoA prevented the labeling of a major radioactive peptide and a minor radioactive peptide. These two peptides were purified by high-performance liquid chromatography. Amino acid analysis of these two peptides revealed that the major radioactive peptide contained S-carboxymethylcysteine while the minor radioactive peptide did not. However, the latter peptide contained beta-alanine, suggesting that this peptide was from the acyl carrier protein segment of fatty acid synthase and that the iodoacetamide treatment resulted in modification of the pantetheine thiol, although to a lower extent than the primer-binding thiol. The sequence of the primer-binding active site peptide from the condensation domain was H2N-Gly-Pro-Ser-Leu-Ser-Ile-Asp- Thr-Ala-Cys(carboxamidomethyl)-X-Ser-Ser-Leu-Met-Ala-Leu-Glu-Asn-A la-Tyr-Lys- COOH, the first reported sequence of the condensation active site from a vertebrate fatty acid synthase. The acyl carrier protein segment showed extensive sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the phosphopantetheine attachment, and the sequence was H2N-Asp-Val-Ser-Ser-Leu- Asn-Ala-Asp-Ser-Thr-Leu-Ala-Asp-Leu-Gly-Leu-Asp-Ser(4'-phosphopanteth ein e) -Leu-Met-Gly-Val-Glu-Val-Arg-COOH.     

The purification and amino acid sequence of toxin CM-13b from Naja haje annulifera (Egyptian cobra) venom.

Toxin CM-13b was purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose. The toxin comprises 65 amino acid residues and is cross-linked by five disulphide bridges. The complete amino acid sequence of toxin CM-13b was elucidated. The reduced and S-carboxymethylated toxin was digested with trypsin and chymotrypsin and the peptides purified by DEAE-cellulose chromatography and chromatography or electrophoresis on paper. The amino acid sequences of the intact toxin and its constituent peptides were determined by the Edman-Begg procedure, either through the use of the automatic sequenator or by manual manipulation. The chymotryptic digest provided the necessary overlapping peptides for aligning the tryptic peptides. The primary structure of toxin CM-13b shows a high degree of homology with that of protein S4C11 from Naja melanoleuca venom[1], but their toxicities are very different.     

Inactivation of the F1-ATPase from the thermophilic bacterium PS3 by 5'-p-fluorosulfonylbenzoylinosine at 65 degrees C is accompanied by modification of beta-tyrosine-364

A major radioactive peptide, T1, was resolved by high-performance liquid chromatography from a tryptic digest prepared from the F1-ATPase from the thermophilic bacterium PS3 which had been inactivated with p-fluorosulfonylbenzoyl[3H]inosine. Two radioactive peptides, T1P1 and T1P2, were isolated from a peptic digest of T1 by high-performance liquid chromatography. The sequences of T1P1 and T1P2 were shown to be E-E-H-X-Q-V-A-R and E-E-H-X-Q, respectively, where X corresponds to derivatized Tyr-364 of the beta subunit.     

Identification of the thiol groups in human ceruloplasmin

Human ceruloplasmin was attached to activated thiol-Sepharose via its thiol groups and was then digested with pepsin. After appropriate washings the thiol peptides were eluted by reduction and were carboxymethylated and purified by column chromatography and electrophoresis. Amino acid sequencing showed that the peptides were derived from five different areas in the molecule and together accounted for 92 residues, six of which were cysteines. Since one of the peptides contained two cysteines it seemed evident that, prior to the reductive elution of the peptides, one of these had been paired in a disulfide bridge with one of the four remaining thiol peptides present in the mixture. The disulfide was isolated and identified by digesting the immobilized protein with pepsin followed by trypsin. The second (tryptic) digestion released the disulfide peptide. Three of the true thiol peptides obtained occur in regions of sequence that have already been reported and which account for 564 of the approximately 1050 residues present in the protein. Three of them also show about 40% identity with each other, whereas no relatedness is observed with the fourth. The three related peptides are, moreover, clearly homologous to the copper-binding areas in the small blue plant and bacterial proteins plastocyanin and azurin. Homologous regions are also evident when the peptides are compared to the two sequences reported for the blue oxidase, fungal laccase, one of which contains a disulfide bridge.     

Characterization of bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase from aspen.

Bispecific O-methyltransferase (OMT, EC 2.1.1.68) which catalyses the meta-specific methylation of caffeic acid and 5-hydroxyferulic acid was purified to homogeneity from the developing secondary xylem of aspen (Populus tremuloides). The enzyme was purified by conventional techniques and affinity chromatography on S-adenosyl-L-homocysteine agarose using substrate elution. The enzyme has a M(r) of 40,000 as determined by SDS-PAGE. Amino acid sequences of three peptides produced from a proteolytic digest of bispecific OMT were obtained by automated Edman degradation. Polyclonal antibodies raised against the purified OMT reacted strongly to OMT in both purified and unpurified fractions in western blots. Addition of an equal concentration of anti-OMT IgG to crude extract protein inhibited OMT activity by more than 70%.     

Amino acid sequence of beta-galactosidase. VI. Limited tryptic digestion of the citraconylated protein and sequences of tryptic peptides.

Hydrolysis with trypsin of citraconyl-carboxy-methyl-beta-galactosidase was carried out under limiting conditions. No Asp-Arg-X sequences were cleaved and many large peptides were produced. Butanol extraction from dilute acid proved very useful for separating the more hydrophobic fragments. Peptides were purified and sequenced. From this digest and two earlier preparations, all 80 theoretically possible tryptic fragments have been isolated and their structures determined.     

Localization of the two free thiol groups in the porcine pancreatic alpha-amylase I sequence

Porcine pancreatic alpha-amylase I, a single 496 residue long polypeptide chain, contains 5 disulfide bridges and 2 free -SH groups. The conditions for specific blocking of native amylase either with radioactive N-ethyl maleimide or with labeled iodoacetic acid were determined. Under these conditions 2 moles of blocking reagent are incorporated per mole of amylase. [14C]-S-succinimido amylase was cleaved by CNBr and the resulting peptides were purified. Only one of them the CNBr 2 + 3 peptide (178 residues) was found labeled. Ts1 a 33-residue peptide containing the whole radioactivity was purified from the tryptic digest of this large fragment. After reduction and carboxymethylation Ts1A, (22 residues) was obtained which contains 2 moles of succinyl-Cys and one mole of CM-Cys per mole of peptide. Chymotryptic digestion of Ts1A yielded 2 equally labeled peptides: C1 (16 residues) and C2 (6 residues). Automated sequencing of both peptides and counting of the PTH-amino acids shows that the free cysteines are only 15 residues apart in the sequence.     

Structural studies of α-crystallin

1. alpha-Crystallin has been isolated from the cortex of ox lens by isoelectric precipitation followed by chromatography on DEAE-cellulose. The amino acid composition is in agreement with that reported for alpha-crystallin prepared by a different method. There is one thiol group/20000g. of protein (20000 is the order of magnitude of the sub-unit molecular weight), and disulphide bonds are absent. 2. The thiol group has been alkylated with radioactive iodoacetate in the presence of urea. 3. Partial acid hydrolysis of the alkylated protein gives, according to the conditions, mainly three radioactive peptides or nearly exclusively one radioactive dipeptide. The dipeptide is N-seryl-(S-carboxymethyl)cysteine, Ser-CMCys. The two other peptides are probably the tripeptides related to Ser-CMCys. 4. The simplest interpretation of these results is that the sequence around the cysteine residue is a common structural feature of the sub-units of alpha-crystallin.     

The amino acid sequences of the tryptic, chymotryptic and peptic peptides from the L-2 light chain of rabbit skeletal muscle myosin.

The amino acid sequences of the tryptic peptides from the aminoethylated L-2 light chain of rabbit skeletal muscle myosin were determined by various enzymatic hydrolyses, partial hydrolysis with dilute acetic acid and Edman degradation. The amino acid sequences of the chymotryptic and peptic peptides from the carboxymethylated L-2 light chain were partially analysed in the same manner as the tryptic peptides. The primary structure of the L-2 light chain of rabbit skeletal muscle myosin was deduced from the above results.