Amino acid sequences around the disulphide bridges and methionine residues of porcine pepsin

1. The amino acid sequences around three disulphide bridges and four methionine residues of porcine pepsin were studied by using diagonal electrophoresis methods. 2. Two of the three disulphide bridges were in small loops of five and six residues. The sequence around one of the two half-cystine residues of the third disulphide bridge had a large number of acidic residues. 3. The sequence of a tetrapeptide containing phosphoserine was also determined. 4. Four unique methionine-containing sequences were constructed. The information is sufficient for the determination of the overlaps in the cyanogen bromide fragments of pepsin. 5. The usefulness of diagonal methods in the study of protein structure, the relative positions of cystinyl and methionyl residues in porcine pepsin and the homology between pepsin and rennin are discussed.     

Amino acid sequences around the pyridoxal-5'-phosphate-binding sites of phenol hydroxylase

Phenol hydroxylase was labelled with pyridoxal 5'-phosphate. A radioactive label was introduced by using sodium boro[3H]hydride to reduce the initially formed Schiff's base. The labelled enzyme was digested with Staphylococcus V8 protease. Labelled peptides were isolated and their sequences were determined. The label could be located to three different lysyl residues. Sequence similarities with the known structures of p-hydroxybenzoate hydroxylase and glutathione reductase are discussed. The positions of the labelled sequences, relative to the bound ligands at the active site, are proposed on the basis of such sequence similarities.     

Amino acid sequences of cytochrome b5 from human, porcine, and bovine erythrocytes and comparison with liver microsomal cytochrome b5

The amino acid sequences of human, porcine, and bovine erythrocyte cytochromes b5 which are soluble and present in the cytosol have been determined. In addition, the partial sequences of microsome-bound liver cytochrome b5, namely the sequence of the N-terminal region and joint region between the heme-containing and membranous part, have been established for human and porcine sources. All the cytochromes b5 from erythrocyte and liver contained N-acetylated N-termini. Of the 97 amino acid residues of erythrocyte cytochrome b5, residues 1-96 were identical with those of the liver protein of the same species. However, residue 97 (C-terminal residue) was proline for human erythrocyte cytochrome b5 and serine for the porcine protein, while residues 97 (joint region) of human and porcine liver cytochromes b5 were threonine. These findings indicate that the two forms of cytochrome b5 are encoded by two different but closely related mRNAs.     

Terminal amino acid sequences and proteolytic cleavage sites of mouse mammary tumor virus env gene products

The mature envelope glycoproteins of mouse mammary tumor virus (gp52 and gp36) were isolated by reversed-phase high-pressure liquid chromatography. The N-terminal amino acid sequence of gp36 was determined for 28 residues. The C-terminal amino acid sequences of gp52 and gp36 were determined by carboxypeptidase digestion. The N-terminal amino acid sequence of gp52 has been reported previously (L. O. Arthur et al., J. Virol. 41:414-422, 1982). These data were aligned with the predicted amino acid sequence of the env gene product obtained by translation of the DNA sequence (S. M. S. Redmond and C. Dickson, Eur. Mol. Biol. Org. J. 2:125-131, 1983). The amino acid sequences of the mature viral proteins were in agreement with the predicted amino acid sequence of the env gene product over the regions of alignment. This alignment showed the sites of proteolytic cleavages of the env gene product leading to the mature viral envelope glycoproteins. The N-terminal amino acid sequence of gp52 starts at residue 99 of the predicted structure indicating proteolytic cleavage of a signal peptide. A dipeptide (Lys-Arg) is excised between the C-terminus of gp52 and the N-terminus of gp36. The C-terminal amino acid sequence of gp36 is identical to the sequence predicted by the codons immediately preceding the termination codon for the env gene product. The data show that there is no proteolytic processing at the C-terminal of the murine mammary tumor virus env gene product and that the env gene coding region extends into the long terminal repeat.     

Amino acid sequence analysis of the proteolytic cleavage products of the bovine immunodeficiency virus Gag precursor polypeptide.

Bovine immunodeficiency virus Gag proteins were purified from virions, and their amino acid sequences and molecular masses were determined. The matrix, capsid, and nucleocapsid (MA, CA, and NC, respectively) and three smaller proteins (p2L, p3, and p2) were found to have molecular masses of 14.6, 24.6, and 7.3 and 2.5, 2.7, and 1.9 kDa, respectively. The order of these six proteins in the Gag precursor, Pr53gag, is NH2-MA-p2L-CA-p3-NC-p2-COOH. In contrast to other retroviral MA proteins, the bovine immunodeficiency virus MA retains its N-terminal methionine and is not modified by fatty acids. In addition, the bovine immunodeficiency virus NC migrates as a 13-kDa protein in denaturing gel electrophoresis; however, its molecular mass was determined to be 7.3 kDa.     

Monoclonal antibodies against human erythrocyte band 3 protein. Localization of proteolytic cleavage sites and stilbenedisulfonate-binding lysine residues

Monoclonal antibodies against the membrane domain of human red blood cell band 3 protein have been prepared and used in topographical studies of the arrangement of the polypeptide in the membrane. One of the antibodies binds to a site near the N terminus of the membrane domain; another binds to a site near the C terminus. The latter has been used to localize a site of intracellular trypsin digestion. The cleavage site, in human band 3, corresponds to Lys-761 in mouse band 3; the site is 168 residues from the C terminus of the protein. This is the first intracellular site in the membrane domain (other than the N terminus) that has been localized in the primary structure. The antibody that binds to the N-terminal portion of the membrane domain has been used to identify a new S-cyanylation cleavage site about 7,000 daltons from the C terminus. Proteolysis/cross-linking experiments with the stilbenedisulfonate derivative H2DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) reveal that one end of the H2DIDS reacts covalently with a lysine residue that is between about 70 and 168 residues from the C terminus of band 3. In addition to placing restrictions on the location of the H2DIDS-binding lysine, these studies provide direct evidence that the C-terminal 28,000-dalton papain fragment crosses the membrane at least three times. With previous data on the remainder of the membrane domain, there is now direct evidence that the band 3 polypeptide crosses the membrane at least eight times.     

Amino acid sequences around the sites of phosphorylation in the pig heart pyruvate dehydrogenase complex.

1. When pig heart pyruvate dehydrogenase complex was phosphorylated to completion with [gamma-32P]ATP by its intrinsic kinase, three phosphorylation sites were observed. The amino acid sequences around these sites were: sequence 1, Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg; and sequence 2, Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser(P)-Tyr-Arg. 2. When phosphorylated to inactivation by repetitive additions of limiting quantities of [gamma-32P]ATP, phosphate was incorporated mainly (more than 90%) into Ser-5 of sequence 2. Phosphorylation of this site thus results in activation of pyruvate dehydrogenase. 3. If Ser-5 is phosphorylated with ATP and the enzyme then incubated with [gamma-32P]ATP, phosphorylation of the remaining sites occurred. Ser-12 of sequence 2 is phosphorylated about twice as rapidly as Ser-6 of sequence 1. 4. Incubation of pyruvate dehydrogenase with excess [gamma-32P]ATP with termination of phosphorylation at about 50% complete inactivation showed that Ser-5 of sequence 2 was phosphorylated most rapidly, but also that Ser-12 of sequence 2 was significantly (15% of total) phosphorylated. Ser-6 sequence 1 contained about 1% total P. 5. These results suggest that addition of limiting amounts of ATP produces primarily phosphorylation of Ser-5 of sequence 2 (inactivating site). This also occurs during incubation with excess ATP before complete inactivation occurs, but a greater occupancy of other sites also occurs during this treatment.     

Amino acid sequences of porcine fast and slow troponin T isoforms

In this study, 10 troponin T isoforms from adult porcine skeletal muscle messenger RNA were clarified. These were eight fast- and two slow-type isoforms. Fast-type isoforms had three and two variable exons in the N-terminal and the C-terminal region respectively. Slow-type isoforms had one variable exon in the N-terminal region.     

Amino acid sequences of substrate-binding sites in chicken liver fatty acid synthase

The amino acid sequences of three essential regions of chicken liver fatty acid synthase have been determined: that around 4'-phosphopantetheine ("carrier" site), the substrate "loading" site containing serine, and a "waiting" site for the growing fatty acid containing cysteine. The amino acid sequence of the 4'-phosphopantetheine region was determined for the acetyl-, malonyl-, hydroxybutyryl-, and butyryl-enzyme with peptides obtained by hydrolysis of the enzyme with trypsin and Staphylococcus aureus (V8) protease. The sequence region around the essential serine was obtained for the acetyl- and malonyl-enzyme. The N-terminus of the tryptic peptide was blocked. However, the same sequence is obtained for the acetyl- and malonyl-peptide after S. aureus protease digestion, suggesting that the enzyme contains a single acyl transferase rather than two separate transacylases. The sequence around the cysteine was obtained by use of a radioactive iodoacetamide label. An unusual sequence of three serines adjacent to the cysteine was found. The strong similarities between peptides from different species for all three of the regions suggest that the multifunctional polypeptides from yeast and animals have evolved from the monofunctional enzymes of lower species.     

Amino acid sequence of the alanyl peptide from cyanogen bromide cleavage of bovine plasma albumin

The amino acid sequence of the alanyl peptide from cyanogen bromide cleavage of bovine plasma albumin has been determined. This peptide has 96 residues and extends the known sequence that begins at the N terminus from 87 to 183 residues.     

Identification of proteolytic cleavage sites by quantitative proteomics

The identification of natural substrates and their cleavage sites is pivotal to defining proteolytic pathways. Here we report a novel strategy for the identification of the signature of proteolytic cleavage events based on quantitative proteomics. Lysine residues in proteins are blocked by guanidination so that free N-terminals can be labeled with amine-specific iTRAQ reagents. The quantitative nature of iTRAQ reagents allows us to distinguish N-terminals newly formed by proteolytic treatment (neoepitopes) from original N-terminals in proteins. Proteins are digested with trypsin and analyzed using MALDI-TOF/TOF mass spectrometry. Peptides labeled with iTRAQ reagents are distinguished from other peptides by exhibiting intense signature ions in tandem mass spectrometry analysis. A corresponding data acquisition strategy was developed to specifically analyze iTRAQ tagged N-terminal peptides. To validate the procedure, we examined a set of recombinant Escherichia coli proteins that have predicted caspase-3 cleavage motifs. The protein mixture was treated with active or inactive caspase-3 and subsequently labeled with two different iTRAQ reagents. Mass spectrometric analysis located 10 cleavage sites, all corresponding to caspase-3 consensus. Spiking caspase-cleaved substrate into a human cell lysate demonstrated the high sensitivity of the procedure. Moreover, we were able to identify proteolytic cleavage products associated with the induction of cell-free apoptosis. Together, these data reveal a novel application for iTRAQ technology for the detection of proteolytic substrates.     

Amino acid sequences around the cystine residues in horse growth hormone

The cystine-containing peptides of horse growth hormone were isolated and their amino acid sequences determined. Four unique half-cystine residues occur in two peptides, one containing 11 and the other, at the C-terminus of the protein, 15 amino acids. These sequences are compared with published data on growth hormones from other species.     

Control of band 3 lateral and rotational mobility by band 4.2 in intact erythrocytes: release of band 3 oligomers from low-affinity binding sites.

Band 4.2 is a human erythrocyte membrane protein of incompletely characterized structure and function. Erythrocytes deficient in band 4.2 protein were used to examine the functional role of band 4.2 in intact erythrocyte membranes. Both the lateral and the rotational mobilities of band 3 were increased in band 4.2-deficient erythrocytes compared to control cells. In contrast, the lateral mobility of neither glycophorins nor a fluorescent phospholipid analog was altered in band 4.2-deficient cells. Compared to controls, band 4.2-deficient erythrocytes manifested a decreased ratio of band 3 to spectrin, and band 4.2-deficient membrane skeletons had decreased extractability of band 3 under low-salt conditions. Normal band 4.2 was found to bind to spectrin in solution and to promote the binding of spectrin to ankyrin-stripped inside-out vesicles. We conclude that band 4.2 provides low-affinity binding sites for both band 3 oligomers and spectrin dimers on the human erythrocyte membrane. Band 4.2 may serve as an accessory linking protein between the membrane skeleton and the overlying lipid bilayer.     

Amino acid sequences C-terminal to the cross-links in bovine elastin.

All the desmosine-containing elastolytic peptides of bovine ligamentum-nuchae elastin have now been examined for amino acid sequences C-terminal to the cross-links. In addition, amino acid residues C-terminal to lysine residues in bovine tropoelastin were also examined. No tyrosine C-terminal to cross-links in bovine elastin or C-terminal to lysine in tropoelastin was detected. Apparently all the tyrosine residues C-terminal to lysine residues in pig tropoelastin are replaced with phenylalanine in bovine tropoelastin. All the data presented are consistent with the scheme proposed for the formation of desmosine and isodesmosine cross-links of elastin by Gerber & Anwar [(1975) Biochem. J. 149, 685--695].     

Identification of MUC1 proteolytic cleavage sites in vivo

Mucins are high molecular weight glycoproteins that provide a protective layer on epithelial surfaces and are involved in cell-cell interactions, signaling, and metastasis. The identification of several membrane-tethered mucins, including MUC1, MUC3, MUC4, and MUC12, has incited interest in the processing of these mucins and the mechanisms that govern their release from the cell surface. MUC1 consists of an extracellular subunit and a membrane-associated subunit. The two moieties are produced from a single precursor polypeptide by an early proteolytic cleavage event but remain associated throughout intracellular processing and transport to the cell surface. We identified the MUC1 proteolytic cleavage site and showed it to be identical in pancreas and colon cell lines and not to be influenced by the presence of heavily glycosylated tandem repeats. The MUC1 cleavage site shows homology with sequences in other cell-surface-associated proteins and may represent a common mechanism for processing of these molecules.     

Amino acid substitutions around the chromophore of the chromoprotein Rtms5 influence polypeptide cleavage

Extension of the conjugated pi-system of many all-protein chromophores with an acylimine bond is the basis for their red-shifted optical properties. The presence of this post-translational modification is evident in crystal structures of these proteins. Harsh denaturation of proteins containing an acylimine bond results in partial polypeptide cleavage. For the red fluorescent protein DsRed, the extent of cleavage is quantitative. However, this is not the case for the blue non-fluorescent chromoprotein Rtms5, even though all chromophores in tetrameric Rtms5 contain an acylimine bond. We have identified two positions around the chromophore of Rtms5 where substitutions can promote or suppress the extent of cleavage on harsh denaturation. We propose a model in which cleavage of Rtms5 is facilitated by a trans to cis isomerisation of the chromophore.     

Amino acid sequences around the cysteine residues of rabbit muscle triose phosphate isomerase

1. The nature of the subunits in rabbit muscle triose phosphate isomerase has been investigated. 2. Amino acid analyses show that there are five cysteine residues and two methionine residues/subunit. 3. The amino acid sequences around the cysteine residues have been determined; these account for about 75 residues. 4. Cleavage at the methionine residues with cyanogen bromide gave three fragments. 5. These results show that the subunits correspond to polypeptide chains, containing about 230 amino acid residues. The chains in triose phosphate isomerase seem to be shorter than those of other glycolytic enzymes.     

Amino acid sequences and phosphorylation sites of emu and rhea eggshell C-type lectin-like proteins

Avian calcified eggshell layers contain in their organic matrix one or two C-type lectin-like proteins. Previously characterized eggshell proteins of this family are chicken ovocleidin-17 (OC-17), goose ansocalcin and ostrich struthiocalcins 1 and 2 (SCA-1, SCA-2). In this report we present the amino acid sequences of two emu (Dromaius novaehollandiae) (dromaiocalcin-1 and -2; DCA-1, DCA-2) and of two rhea (Rhea americana) (rheacalcin-1 and -2; RCA-1, RCA-2) C-type lectin-like eggshell proteins, thus doubling the data set for comparison of these major specific eggshell proteins. The ratite proteins can be divided into two groups. Group 1, comprising SCA-1, DCA-1 and RCA-1, shows by 70--77% identity of sequences, the lack of phosphorylation, and a variable number (7--9) of cysteines. Group 2, consisting of SCA-2, DCA-2 and RCA-2, shows 78--85% identical sequences, 2--3 phosphorylated serines located at almost identical sites, and contains only the common set of six conserved cysteins characteristic for this family of proteins. While goose ansocalcin fits perfectly into group 1 with a sequence identity of 63--70% to the other members, no phosphorylation, and seven cysteines, chicken OC-17 was assigned to group 2 in spite of only 42--47% sequence identity (and 37--39% to group 1) because of its two phosphorylated serines and its regular set of six cysteines. At present it remains unknown why ratites, but not goose or chicken, require two different types of C-type lectin-like proteins to construct their eggshells.