Revision of the blocked N terminus of rat heart fatty acid-binding protein by liquid secondary ion mass spectrometry

The primary structure of rat heart muscle fatty acid-binding protein was investigated by liquid secondary ion mass spectrometry. The protein was digested with trypsin, chymotrypsin, and Staphylococcus aureus V8 protease and the resulting peptides were separated by reverse phase high performance liquid chromatography. The masses of the protonated molecular ions (MH+) of the tryptic, chymotryptic, and S. aureus protease peptides were determined by liquid secondary ion mass spectrometry analysis using 20-500 pmol of material. From the tryptic digest, two peptides with MH+ 1036 and 861 were initially found that did not match the published primary sequence (Sacchettini, J. C., Meininger, T. A., Lowe, J. B., Gordon, J. I., and Banaszak, L. J. (1987) J. Biol. Chem. 262, 5428-5430). The amino acid sequences of these two peptides were determined by a combination of mass spectrometry, B/E-linked scanning, and high performance tandem mass spectrometric techniques to be: (Formula: see text). These new data require that corrections be made to the previously published sequence, involving residues 1-4 and 51-52. The corrected amino sequence for rat m-FABP reveals greater homology with myelin P2, mouse adipocyte p422 protein, and intestinal fatty acid-binding protein than was previously demonstrated.     

Rat heart fatty acid-binding protein. Evidence that supports the amino acid sequence predicted from the cDNA.

The amino acid sequence of rat heart fatty acid-binding protein was re-examined by analysing the tryptic and the chymotryptic peptides, since some discrepancies have been reported between the sequences determined by protein analyses and that deduced from the cDNA analyses. Our result completely agreed with the amino acid sequence predicted from the cDNA analyses, providing evidence for the actual existence of the molecular species predicted from the cDNA.     

Rat heart fatty acid-binding protein is highly homologous to the murine adipocyte 422 protein and the P2 protein of peripheral nerve myelin

The rat heart contains an abundant cytosolic protein which binds long chain fatty acids. We have determined its primary structure by Edman degradation of peptides generated from chymotryptic, tryptic, and elastase digestions. This polypeptide (Mr = 14,992) contains 134 amino acids and has a blocked (acetylated) NH2 terminus. The sequence of rat heart fatty acid-binding protein (FABP) is remarkably similar to the murine adipocyte 422 protein and the P2 protein of peripheral nerve myelin. Computer-assisted alignment of heart FABP and 422 revealed that 82 of 132 comparable residues are identical (62%). There are 77 identities out of 131 possible matches between this protein and the human myelin P2 protein (59%). Similar comparisons demonstrate that heart FABP has significant homology to several other proteins which bind hydrophobic ligands. The rank of order of similarity to heart FABP is: 422 greater than myelin P2 greater than cellular retinoic acid-binding protein greater than cellular retinol-binding protein II greater than cellular retinol-binding protein greater than intestinal FABP greater than liver FABP. These eight sequences form a family of paralogous homologues. Heart FABP has a region of internal homology involving tandemly arrayed oligopeptides spanning residues 71-100 and 101-131. This feature is not found in the 422 and P2 sequences. The endogenous ligands bound by the 422, P2, and heart FABP sequences have not been defined. Interpretation of the biological significance of their structural similarities and differences will require information about their ligand specificities and affinities.     

Characterization of a fatty acid-binding protein from rat heart

A fatty acid-binding protein has been isolated from rat heart and purified by gel filtration chromatography on Sephadex G-75 and anion-exchange chromatography on DE52. The circular dichroic spectrum of this protein was not affected by protein concentration, suggesting that it does not aggregate into multimers. Computer analyses of the circular dichroic spectrum predicted that rat heart fatty acid-binding protein contains approximately 22% alpha-helix, 45% beta-form and 33% unordered structure. Immunological studies showed that the fatty acid-binding proteins from rat heart and rat liver are immunochemically unrelated. The amino acid composition and partial amino acid sequence of the heart protein indicated that it is structurally related to, but distinct from, other fatty acid-binding proteins from liver, intestine, and 3T3 adipocytes. Using a binding assay which measures the transfer of fatty acids between donor liposomes and protein (Brecher, P., Saouaf, R., Sugarman, J. M., Eisenberg, D., and LaRosa, K. (1984) J. Biol. Chem. 259, 13395-13401), it was shown that both rat heart and liver fatty acid-binding proteins bind 2 mol of oleic acid or palmitic acid/mol of protein. The structural and functional relationship of rat heart fatty acid-binding protein to fatty acid-binding proteins from other tissues is discussed.     

Sequence of Guinea Pig Myelin Basic Protein

This paper proposes a tentative amino acid sequence of guinea pig myelin basic protein obtained by comparison of peptide fragments of the guinea pig and bovine proteins. Analyses of the tryptic peptides confirmed the known sequence differences in the NH2-terminal half of the molecule and showed that in the COOH-terminal half of the guinea pig protein Ser131 was missing, Ala136 - His137 was deleted, Leu140 was replaced by Phe, and an extra Ala was inserted somewhere within sequence 142-151 (tryptic peptide T23 ). Sequence determination of guinea pig tryptic peptides corresponding to residues 130-134 ( T20 ), 135-138 ( T21 ), and 142-151 ( T23 ) of the bovine protein confirmed the above sequence changes and placed the extra Ala between Gly142 and His143 . The sequence of the region corresponding to bovine residues 130-143 is thus Ala-Asp-Tyr-Lys-Ser-Lys-Gly-Phe-Lys-Gly-Ala-His. No species differences were observed in the amino acid compositions of the remaining tryptic peptides obtained from the COOH-terminal half of the molecule. Based upon these results, the guinea pig basic protein contains 167 amino acid residues and has a molecular weight of 18,256.     

The primary structure of bovine cellular retinoic acid-binding protein

The complete amino acid sequence of bovine adrenal gland cellular retinoic acid-binding protein (CRABP) has been determined. The primary structure was established by analyses of cyanogen bromide fragments and peptides obtained by trypsin and Staphylococcus aureus protease digestions. The polypeptide chain of bovine CRABP comprises 136 amino acid residues. From partial sequence information, CRABP has been shown to be homologous to cellular retinol-binding protein, myelin protein P2, and the fatty acid-binding Z-protein. A comparison of the complete amino acid sequences of the members of this protein family, which also includes the rat intestinal fatty acid-binding protein, shows that CRABP is more similar to cellular retinol-binding protein and protein P2 than to the fatty acid-binding proteins. All five proteins are very similar in their NH2-terminal regions, suggesting that this part is important for a property common to the members of this protein family. This is the first report of a complete amino acid sequence of a CRABP.     

Histatins, a novel family of histidine-rich proteins in human parotid secretion. Isolation, characterization, primary structure, and fungistatic effects on Candida albicans

Histatins 1, 3, and 5 from human parotid secretion were isolated by gel filtration on Bio-Gel P-2 and reverse phase high performance liquid chromatography. The complete amino acid sequences of histatins determined by automated Edman degradation of the proteins, Staphylococcus aureus V8 protease, and tryptic peptides, are as follows: (Sequence: see text). Histatins 1, 3, and 5 contain 38, 32, and 24 amino acid residues, have molecular weights of 4929, 4063, and 3037, respectively, and contain 7 residues of histidine. Histatin 1 contains 1 mol of phosphate/mol of protein; histatins 3 and 5 lack phosphate. With the exception of Glu (residue 4) and Arg (residue 11) in histatin 1, the first 22 amino acid residues of all three histatins are identical, and the carboxyl-terminal 7 residues of histatins 1 and 3 are also identical. The sequence, -Glu-Phe-Pro-Phe-Tyr-Gly-Asp-Tyr-Gly- (residues 23-29), in histatin 1 is absent in histatin 3; and the sequence, -Gly-Tyr-Arg- (residues 23-25), in histatin 3 is absent in histatin 1. The complete sequence of histatin 5 is contained within the amino terminal 24 residues of histatin 3. The structural data suggest that histatins 1 and 3 are derived from different structural genes, whereas histatin 5 is a proteolytic product of histatin 3. All three histatins exhibit the ability to kill the pathogenic yeast, Candida albicans.     

The primary structure of a basic (pI 9.0) fatty acid-binding protein from liver of Gallus domesticus

The complete amino acid sequence of a basic (pI 9.0) fatty acid-binding protein purified from liver of Gallus domesticus was determined by automated Edman degradation of tryptic, CNBr/HFBA and Staphylococcus aureus protease peptides. The protein contains 125 amino acid residues which correspond to a molecular mass of 14094. The identification of the blocked N-terminus Ac-Ala required digestion of a SV-8 peptide with the acylamino acid-releasing enzyme prior to sequence analysis. Sequence comparison shows that chicken liver basic-FABP has a significant similarity to other proteins belonging to the superfamily of intracellular lipid molecule binding proteins. Moreover, these sequence data confirm that basic-FABP probably binds its substrate in a slightly different way when compared with other FABPs. Basic-FABP was submitted to the EMBL Data Library with an accession number of P80226     

Evidence for posttranslational modification and gene duplication of Campylobacter flagellin.

A gene encoding a flagellin protein of Campylobacter coli VC167 has been cloned and sequenced. The gene was identified in a pBR322 library by hybridization to a synthetic oligonucleotide probe corresponding to amino acids 4 to 9 of the N-terminal sequence obtained by direct chemical analysis (S. M. Logan, L. A. Harris, and T. J. Trust, J. Bacteriol. 169:5072-5077, 1987). The DNA was sequenced and shown to contain an open reading frame encoding a protein with a molecular weight of 58,945 and a length of 572 amino acids. The deduced amino acid sequence was identical to the published N-terminal amino acid sequence of VC167 flagellin and to four internal regions whose partial sequences were obtained by direct chemical analysis of two tryptic and two cyanogen bromide peptides of VC167 flagellin. The C. coli flagellin protein contains posttranslationally modified serine residues, most of which occur within a region containing two 9-amino-acid repeating peptides separated by 34 unique amino acids. Comparisons with the sequences of flagellins from other bacterial species revealed conserved residues at the amino- and carboxy-terminal regions. Hybridization data suggest the presence of a second flagellin copy located adjacent to the first on the VC167 chromosome.     

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (NADP): amino acid sequence of the subunits from isoenzyme I and structural relationship with isoenzyme II

The structural relationship between isoenzymes I and II of chloroplast glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NADP+ oxidoreductase (phosphorylating) EC 1.2.1.13) has been established at the protein level. The complete primary structure of subunits A and B of glyceraldehyde-3-phosphate dehydrogenase I from Spinacia oleracea has been determined by sequence analysis of the corresponding tryptic peptides, aligned by fragments derived from cyanogen bromide and Staphylococcus proteinase V8 digestions and by partially sequencing each intact subunit. Subunit A has an Mr of 36,225 and consists of 337 amino acid residues, whilst subunit B (Mr 39,355) consists of 368 residues. The amino acid sequence of subunit B, as determined through direct analysis of the protein, is identical to that recently deduced at cDNA level (Brinkmann et al. (1989) Plant Mol. Biol. 13, 81-94). The two subunits share a common portion of amino acid sequence which differs by 66 amino acid residues. Subunit B has an extra C-terminal sequence of 31 amino acid residues. Chloroplast glyceraldehyde-3-phosphate dehydrogenase II was partially characterized by sequencing the N-terminal portion of the intact protein and some of its tryptic peptides. The sequences of all the examined fragments fit precisely that of the corresponding regions of subunit A from glyceraldehyde-3-phosphate dehydrogenase I.     

High pressure liquid chromatography purification of UP1 and UP2, two related single-stranded nucleic acid-binding proteins from calf thymus

Two single-stranded nucleic acid-binding proteins, UP1 and UP2, that were originally reported by Herrick and Alberts (Herrick, G., and Alberts, B. (1976) J. Biol. Chem. 251, 2124-2132) have been purified to apparent homogeneity from calf thymus by high performance liquid chromatography. The amino acid sequence of UP1 (Williams, K. R., Stone, K. L., LoPresti, M. B., Merrill, B. M., and Planck, S. R. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 5666-5670) reveals that UP1 contains 195 amino acids, including one dimethylarginine residue near its COOH terminus. Further analysis of this sequence now demonstrates that UP1 contains a 91-residue internal repeat such that when residues 3-93 (the "A" region) are aligned with residues 94-194 (the "B" region), 32% of the amino acids in these two regions are identical and an additional 39% of those changes that are seen could be accomplished by single base changes. The high degree of internal homology between residues 51-61 and 143-152 and in particular the high density of aromatic and positively charged amino acids in these two regions suggest that residues 51-61 and 143-152 may constitute two independent DNA-binding sites. Solid-phase sequencing of three tryptic peptides that together account for 9% of the 39,500-dalton UP2 protein demonstrate that there is a high degree of sequence homology between UP1 and UP2. Of the 34 residues that have been sequenced in UP2, 44% are identical in both UP1 and UP2. The blocked NH2 terminus, amino acid composition, particularly with regard to its high glycine content and the presence of dimethylarginine, and molecular weight of UP2 suggest this protein is related to proteins that have previously been found associated with heterogeneous RNA. Taken together, these data indicate that both UP1 and UP2 belong to a new family of single-stranded nucleic acid-binding proteins that may be closely related to heterogeneous ribonucleoproteins.     

Primary structures of cysteine-containing peptides from the calcium ion-transporting adenosine triphosphatase of rabbit sarcoplasmic reticulum.

A preliminary investigation of the primary structure of the Ca(2+-transporting ATPase (adenosine triphosphatase) protein of rabbit skeletal-muscle sarcoplasmic reticulum is reported. The preparation of derivatives of delipidated protein in a form suitable for sequence analysis is described. Tryptic peptides containing S-carboxymethylcysteine residues were isolated from the reduced carboxymethylated protein, and their sequences were partially determined. The results are consistent with mol.wt. about 105000 for the polypeptide, and the absence of extended repeated lengths of sequence. The distribution of tryptophan and cysteine residues between large, aggregated peptides and soluble tryptic peptides shows that these residues are concentrated in different regions of the primary structure. This observation agrees with other evidence that these residues are, on the whole, widely separated in the native protein. The details of the procedures used to isolate the peptides, and the evidence for the determination of their sequences, are given Supplementary Publication SUP 50085 (30 pages), which has been deposited at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem.J. (1978) 169, 5.     

Molecular cloning of cDNA encoding human and rabbit forms of the Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum

We have cloned cDNAs encoding the rabbit and human forms of the Ca2+ release channel of sarcoplasmic reticulum. The human cDNA encodes a protein of 5032 amino acids, with a molecular weight of 563,584, which is made without an NH2-terminal signal sequence. Amino acid substitutions between rabbit and human sequences were noted in 163 positions and deletions or insertions in eight regions accounted for additional sequence differences between the two proteins. Analysis of the sequence indicates that 10 potential transmembrane sequences in the COOH-terminal fifth of the molecule and two additional, potential transmembrane sequences nearer to the center of the molecule could contribute to the formation of the Ca2+ conducting pore. The remainder of the molecule is hydrophilic and presumably constitutes the cytoplasmic domain of the protein. A 114-120 amino acid motif is repeated four times in the protein, in residues 841-954, 955-1068, 2725-2844, and 2845-2958 and a 16 amino acid part of the motif is repeated twice more in residues 1344-1359 and 1371-1386. Although the channel is modulated by Ca2+, ATP, and calmodulin, no clear high affinity Ca2(+)-binding domain of the EF hand type and no clear high affinity ATP-binding domain were detected in the primary sequence. An acidic sequence in residues 1872-1923 contains 79% glutamate or aspartate residues and this sequence is a potential low affinity Ca2(+)-binding site. Several potential calmodulin-binding sites were observed in the sequence, in the region 2800 to 3050.     

Amino acid sequence of chitinase from Streptomyces erythraeus

The amino acid sequence of chitinase from Streptomyces erythraeus was determined by the conventional method. The amino acid sequences of tryptic peptides of the reduced and S-carboxymethylated protein were determined. The tryptic peptides were aligned by overlapping the amino acid sequences of chymotryptic peptides, lysyl endopeptidase peptides and cyanogen bromide fragments. S. erythraeus chitinase consists of 290 amino acid residues with the molecular weight of 30,400 and has two disulfide bridges at Cys(45)-Cys(89) and Cys(265)-Cys(272). The enzyme has no significant homology with other chitinases, lysozymes, and other proteins.     

Identification of surface-exposed segments of apolipoprotein B-100 in the LDL particle

The isolation and amino acid sequence of eleven peptides liberated by tryptic treatment from surface-exposed regions of apolipoprotein B-100 in the native low-density lipoprotein particle are described. These peptides represent eight segments in the sequence of the B-100 protein, one of which was localised to the amino-terminal thrombolytic fragment T4 (1297 amino acids), four to the T3 fragment (2052 residues) and three to the carboxylterminal fragment T2 (1287 residues). An exposed segment was identified on each side of the T2/T3 cleavage site, in close proximity to two segments enriched in basic amino acids (residues 3147-3157 and 3359-3367 respectively). The surface exposure of this region is consistent with its contribution to the putative apo-B,E receptor binding domain. Four of the eight tryptic segments contribute to regions of proline-rich clusters. Homology between the sequence of the tryptic peptides and those predicted by cDNA cloning was complete.     

Amino acid sequence of calmodulin from wheat germ

The complete amino acid sequence of calmodulin from wheat germ was determined by isolating and sequencing the cyanogen bromide and tryptic peptides. The protein consisted of 149 amino acid residues and its amino(N)-terminus was blocked with an acetyl group. Wheat germ calmodulin lacked tryptophan and contained 1 mol each of histidine, tyrosine, cysteine, and N epsilon-trimethyllysine residues per mol of the protein. A comparison of its amino acid sequence with that of bovine brain calmodulin indicated that there were eleven amino acid subsitutions other than amide assignments, two insertions and one deletion of amino acid residues in wheat germ calmodulin.     

Tetrahymena HMG nonhistone chromosomal protein. Isolation and amino acid sequence lacking the N- and C-terminal domains of vertebrate HMG 1

The complete amino acid sequence of a high mobility group (HMG) nonhistone chromosomal protein of the ciliated protozoan Tetrahymena pyriformis (GL strain) was determined. This protein was extracted with 0.5 M HClO4 together with histone H1 (molar ratio 1:1) from the whole histone extract, then purified by gel filtration and reverse-phase HPLC. The HMG protein showed a single electrophoretic band on SDS gel electrophoresis. The amino acid sequence was determined by Edman degradation of intact protein, BrCN fragments, and their staphylococcal protease and tryptic peptides. Thus the total sequence, consisting of 99 amino acid residues and having a molecular weight of 11,626, was completely determined. Phosphorus analysis of the tryptic peptides, containing serine or threonine, showed that this HMG protein was phosphorylated at two positions, each 6-7%, and contained 0.15 mol phosphate/mol protein. This Tetrahymena HMG is rather similar to the central part of vertebrate HMG 1 in terms of the amino acid sequence and the hydropathy profile.     

Tetrahymena histone H1. Isolation and amino acid sequence lacking the central hydrophobic domain conserved in other H1 histones

The complete amino acid sequence of a single H1 histone of the protozoan Tetrahymena pyriformis was determined, following previous determinations of the sequences of histones H2B, H2A, H3, and H4. Only a single H1 species was obtained by fractionation of a 0.5 M HClO4-soluble fraction from the whole histone extract and further purification. This starting material for sequencing contained 1.1 mol/mol phosphate and showed a single electrophoretic band after dephosphorylation. The sequence determination was performed by Edman degradation of BrCN fragments, staphylococcal protease peptides, and tryptic peptides, as well as secondary peptides from one BrCN fragment and one staphylococcal protease peptide. Phosphorus analysis of the tryptic peptides, containing serine or threonine, showed that five sites of the sequence were phosphorylated to various extents (5-30%). Thus, the total sequence, consisting of 165 amino acid residues and having a molecular weight of 17,942 in the unmodified form, was completely determined. This unusually small H1 sequence differs substantially from the human spleen H1 sequence of 218 residues, having larger proportions of hydrophilic residues and smaller proportions of hydrophobic residues. Comparison of the distribution pattern of hydrophilic and hydrophobic residues, between the protozoan and human sequences, showed that the protozoan sequence lacks the central hydrophobic domain that is conserved in the known vertebrate and other H1 histones. The implications for the function of H1 are discussed from the evolutionary viewpoint.     

The cDNA and protein sequences of human lactate dehydrogenase B.

Human lactate dehydrogenase B (LDH-B) cDNA was isolated and sequenced. The LDH-B cDNA insert consists of the protein-coding sequence (999 bp), the 5' (54 bp) and 3' (203 bp) non-coding regions, and the poly(A) tail (50 bp). The predicted sequence of 333 amino acid residues was confirmed by amino acid composition and/or sequence analyses of a total of 185 (56%) residues from tryptic peptides of human LDH-B protein. The nucleotide and amino acid sequences of the human LDH-B coding region show 68% and 75% homologies respectively with those of the human LDH-A. The peptide map and amino acid composition data have been deposited as Supplementary Publication SUP 50139 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment [see Biochem. J. (1987) 241, 5].     

The primary structure of the cytotoxin alpha-sarcin

The primary structure of the cytotoxin alpha-sarcin was determined. Eighteen of the 19 tryptic peptides were purified; the other peptide has arginine only. The complete sequence of 17 of the peptides was determined; the sequence of the remaining peptide was determined in part. The sequence of the 39 NH2-terminal residues was obtained by automated Edman degradation. The carboxyl-terminal amino acids were identified after carboxypeptidase treatment. The assignment of the amino acids in the tryptic peptides was confirmed and their alignment established from the sequence of the secondary tryptic peptides obtained after cleavage of citraconylated alpha-sarcin, from the sequence of a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine peptide, from the sequence of a chymotryptic peptide, and from the sequence of a peptide obtained with Staphylococcus aureus V8 protease. alpha-Sarcin contains 150 amino acid residues; the molecular weight is 16,987. There are disulfide bridges between cysteine residues at positions 6 and 148 and between residues 76 and 132.