Forms of Aspartic Acid in Human Urine

It was found by amino acid analysis before and after acid hydrolysis of human urine that most glutamic and aspartic acid was in bound form, while glycine, glutamic and aspartic acids accounted for about 70% of bound amino acids. Fractions rich in peptides containing aspartic acid were obtained by chromatography on various columns, and 7 peptides containing aspartic acid were isolated from these fractions. It may be inferred from these results and from the literatures that there are numerous oligopeptides containing aspartic acid in human urine.     

Complete amino acid analysis of peptides and proteins after hydrolysis by a mixture of Sepharose-bound peptidases

Incubation with a mixture of Sepharose-bound peptidases was shown to result in the quantitative release of amino acids from certain peptides and S-aminoethylated proteins. Subtraction of the low background values of amino acids generated by the enzymes enables amino acid ratios of corticotrophin-(1-24)-tetracosapeptide to be determined with a standard deviation on repeat digestions of 3-5%. Good values were obtained for amino acids that are completely or partially destroyed on acid hydrolysis, i.e. tryptophan, tyrosine, serine, asparagine and glutamine. Experiments with peptides containing d-amino acids showed that the enzyme mixture is stereospecific and could therefore be used to detect the presence of d-residues in peptides. The enzyme mixture completely hydrolyses peptide fragments obtained after Edman degradation and should therefore be useful for determining sequences of peptides containing acid-labile amino acid residues. The activities of the bound enzymes were unaltered over a period of 7 months and they provide a simple, reproducible procedure for the quantitative determination of amino acids in peptides and proteins containing l-amino acids.     

An assay for acidic peptide substrates of protein kinases

The assay of acidic peptides as substrates for protein kinases has not been as easy to perform as testing basic peptides or polypeptides. We have developed a simple, rapid, and cost-effective procedure that allows the design and testing of potential peptide substrates without the constraints imposed by the phosphocellulose filter paper method (the need to incorporate positively charged residues into the peptide sequence). The technique combines the chelation of 32Pi by acid molybdate with PEI-cellulose chromatography. In this way the migration of 32P-labeled Pi, ATP, and protein are impeded while phosphopeptide is eluted in 1.5 ml from a 0.25-ml disposable column. In order to validate the assay we used two angiotensin II analogues as peptide substrates for the protein tyrosine kinase pp60c-src. The assay results using the new procedure were compared to those of the phosphocellulose filter paper technique. We also demonstrated the use of this method to test linear and cyclic peptides that could not be assayed with the phosphocellulose paper technique. This assay will aid those who are attempting to determine the substrate specificity of protein kinases.     

Escherichia coli peptide binding protein OppA has a preference for positively charged peptides

The Escherichia coli peptide binding protein OppA is an essential component of the oligopeptide transporter Opp. Based on studies on its orthologue from Salmonella typhimurium, it has been proposed that OppA binds peptides between two and five amino acids long, with no apparent sequence selectivity. Here, we studied peptide binding to E. coli OppA directly and show that the protein has an unexpected preference for basic peptides. OppA was expressed in the periplasm, where it bound to available peptides. The protein was purified in complex with tightly bound peptides. The crystal structure (up to 2.0 Å) of OppA liganded with the peptides indicated that the protein has a preference for peptides containing a lysine. Mass spectrometry analysis of the bound peptides showed that peptides between two and five amino acids long bind to the protein and indeed hinted at a preference for positively charged peptides. The preference of OppA for peptides with basic residues, in particular lysines, was corroborated by binding studies with peptides of defined sequence using isothermal titration calorimetry and intrinsic protein fluorescence titration. The protein bound tripeptides and tetrapeptides containing positively charged residues with high affinity, whereas related peptides without lysines/arginines were bound with low affinity. A structure of OppA in an open conformation in the absence of ligands was also determined to 2.0 Å, revealing that the initial binding site displays a negative surface charge, consistent with the observed preference for positively charged peptides. Taken together, E. coli OppA appears to have a preference for basic peptides.     

Identification of the major sites of enzymic glycosylation of myelin basic protein

In the presence of porcine submaxillary N-acetylgalactosaminyltransferase and uridine diphospho-N-acetyl-D-galactosamine, approx. 1.2-1.5 mol of N-acetylgalactosamine were transfered per mol of myelin basic protein. Tritium-labelled N-acetylgalactosamine-labelled basic protein was digested with trypsin and the peptides were separated by HPLC and the radioactivity measured. Most of the radioactivity was associated with three peptide peaks (I, II and III) containing 17, 69 and 6% of the total radioactivity, respectively. The remaining radioactivity was distributed amongst several peptides, each containing less than 2.5% of the total radioactivity. Glycosylation of the basic proteins isolated from human, bovine and guinea pig myelins showed that they were all equally good acceptors. In spite of differences in the peptide profiles of the basic proteins from different species, the distribution of radioactivity between the three peptide peaks was similar for all the species studied. The transfer of N-acetylgalactosamine to peptide II was much faster than to peptides I and III. The apparent Km values of the three peptides were within a narrow range of 0.52-0.63 mM, whereas the Vmax values were considerably different. The glycosylated peptide peaks (I, II and III) were separated by electrophoresis, the radioactivity measured, and amino acid compositions determined after hydrolysis. The major radioactive peptides of the human basic protein were identified with tryptic peptides containing the following sequences: (formula; see text)     

Specific binding of mitochondrial protein precursors to liposomes containing cardiolipin

In vitro synthesized precursors of several mitochondrial proteins, including P-450(SCC), adrenodoxin, and malate dehydrogenase, bound to liposomes prepared from mitochondrial phospholipids, but not to those from microsomal phospholipids. When liposomes were prepared from various pure phospholipids, adrenodoxin precursor was bound only to the liposomes that contained cardiolipin. The liposomes containing other phospholipids did not show the binding affinity for the precursor. The binding was observed only with the precursor peptides of adrenodoxin and malate dehydrogenase, and their mature forms were not bound to the liposomes. The binding of the precursors was dependent on the concentration of cardiolipin in the liposomes. Liposomes containing various cardiolipin derivatives with modified polar head groups showed very different binding affinity for adrenodoxin precursor, suggesting the importance of the structure of the polar head of the cardiolipin molecule. Two or three positively charged amino acid residues in the extension peptide of P-450(SCC) precursor were replaced by neutral amino acid residues by site-directed mutagenesis. The mutated P-450(SCC) precursors did not bind to the liposomes containing cardiolipin. The results indicated that mitochondrial protein precursors have specific affinity for cardiolipin, and the affinity was due to the interaction between the extension peptides of the precursors and the polar head of the cardiolipin molecule.     

Location and dynamics of basic peptides at the membrane interface: electron paramagnetic resonance spectroscopy of tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid-labeled peptides

The attractive interaction between basic protein domains and membranes containing acidic lipids is critical to the membrane attachment of many proteins involved in cell signaling. In this study, a series of charged model peptides containing lysine, phenylalanine, and the spin-labeled amino acid tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) were synthesized, and electron paramagnetic resonance (EPR) spectroscopy was used to determine their position on the membrane interface and free energy of binding. When membrane-bound, peptides containing only lysine and TOAC assume an equilibrium position within the aqueous double layer at a distance of approximately 5 A from the membrane interface, a result that is consistent with recent computational work. Substitution of two or more lysine residues by phenylalanine dramatically slows the backbone diffusion of these peptides and shifts their equilibrium position by 13-15 A so that the backbone lies several angstroms below the level of the lipid phosphate. These results are consistent with the hypothesis that the position and free energy of basic peptides when bound to membranes are determined by a long-range Coulombic attraction, the hydrophobic effect, and a short-range desolvation force. The differences in binding free energy within this set of charged peptides is not well accounted for by the simple addition of free energies based upon accepted side chain partition free energies, a result that appears to be in part due to differences in membrane localization of these peptides.     

A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin

Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, alpha v beta 5, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an alpha v beta 5 ligand, vitronectin, also bound alpha v beta 5 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The alpha v beta 5 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; alpha v beta 5 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a beta 1 integrin did bind to these peptides. The interaction of alpha v beta 5 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD- containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences.     

Fractionation and structure of several hydroxyproline-containing urinary peptides, with special reference to some 3-hydroxyproline-containing peptides.

After a preliminary separation of the hydroxyproline-containing peptides on Biogel P 2, the largest peptides are fractionated on phosphocellulose and the smallest ones on QAE-Sephadex. The fractions obtained from QAE-Sephadex are subfractionated on a column of Dowex 50-M-82. The total number of hydroxyproline-containing peptides from human urine is not less than 78. Sixteen di, tri and pentapeptides have been purified, their N-terminal amino acids and amino acid compositions determined and a structure is proposed. 3 of these peptides contain 3-hydroxyproline and one of these 3 peptides probably originates from basement membrane collagen.     

The sequences of the coenzyme-binding peptide in the cytoplasmic and the mitochondrial aspartate aminotransferases from sheep liver.

The sequences of the coenzyme-binding peptide of both cytoplasmic and mitochondrial aspartate aminotransferases from sheep liver were determined. The holoenzymes were treated with NaBH4 and digested with chymotrypsin; peptides containing bound pyridoxal phosphate were then isolated. One phosphopyridoxyl peptide was obtained from sheep liver cytoplasmic aspartate aminotransferase. Its sequence was Ser-Ne-(phosphopyridoxyl)-Lys-Asn-Phe. This sequence is identical with that reported for the homologous peptide from pig heart cytoplasmic aspartate aminotransferase. Two phosphopyridoxyl peptides with different RF values were isolated from the sheep liver mitochondrial isoenzyme. They had the same N-terminal amino acid and similar amino acid composition. The mitochondrial phosphopyridoxyl peptide of highest yield and purity had the sequence Ala-Ne-(phosphopyridoxyl)-Lys-Asx-Met-Gly-Leu-Tyr. The sequence of the first four amino acids is identical with that already reported for the phosphopyridoxyl tetrapeptide from the pig heart mitochondrial isoenzyme. The heptapeptide found for the sheep liver mitochondrial isoenzyme closely resembles the corresponding sequence taken from the primary structure of the pig heart cytoplasmic aspartate aminotransferase.     

Isolation of immunizing cyanogen bromide-peptides of foot-and-mouth disease virus.

The isolated structural protein with the N-terminal amino acid threonine of foot-and-mouth disease virus, type O₁ strain Kaufbeuren was treated with CNBr and the cleavage peptides were separated by gel filtration on Sephadex G-100 followed by ion exchange chromatography on phosphocellulose. Two peptides with molecular weights of about 5.200 daltons still capable of inducing antibodies in guinea pigs were purified. The antibodies were found to neutralize the homologous foot-and-mouth disease virus as detected by the neutralization test in suckling mice. The findings strongly suggest that the primary structure of small CNBr cleavage peptides carries antigenic determinants similar to those on the native virus protein with the N-terminale amino acid threonine.     

Cyanogen bromide digestion of the avian myeloblastosis virus pp19 protein: isolation of an amino-terminal peptide that binds to viral RNA.

The avian myeloblastosis virus pp19 protein was separated from the other virus proteins by a rapid and simple purification procedure which yields milligram amounts of homogeneous protein. This protein was then fragmented by digestion with cyanogen bromide. When the mixture of the cyanogen bromide peptides was passed through a 60S avian myeloblastosis virus RNA-cellulose column, only one peptide bound with high affinity to the resin. The peptide migrated on a sodium dodecyl sulfate-polyacrylamide gel with an approximate molecular weight of 2,900 and will be referred to as the p3B peptide. This peptide was also isolated directly by chromatography of the cyanogen bromide-digested pp19 protein on a reverse-phase high-pressure liquid chromatography column. It was again the only cyanogen bromide peptide of the pp19 protein that bound to the RNA affinity resin. The p3B peptide is a basic peptide, as was seen by its rapid migration on acid-urea-polyacrylamide gels and its amino acid composition. A partial amino acid sequence analysis of the p3B peptide indicated that it was derived from the amino terminus of the intact protein. Although the p3B peptide bound to 60S RNA, it did not demonstrate the selective binding of native pp19 to regions of the RNA containing secondary structure.     

Isolation of a Drosophila gene coding for a protein containing a novel phosphatidylserine-binding motif

To elucidate the molecular basis of the binding of proteins to the membrane phospholipid phosphatidylserine (PS), we characterized PS-binding peptides isolated from a phage display library. Amino acid sequences deduced from the nucleotide sequences of over 60 phage clones isolated revealed that there was no common primary structure among these peptides, but all peptides were rich in basic amino acid residues. In particular, 15 clones encoded peptides that contained contiguous arginine residues. Characterization of two such peptides in more detail showed that they bound to PS, and to a much lower extent to other phospholipids, including phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine. Unlike other Ca2+-dependent PS-binding proteins, these peptides did not require Ca2+ for binding to PS, and the addition of Ca2+ did not alter the phospholipid specificity. Substitution of one of the two RR sequences in one peptide by alanine had no effect, but that of both sequences completely abolished the activity. Furthermore, we identified a Drosophila gene coding for a presumed nuclear protein that shares an amino acid sequence, including a RR residue, with one of the two PS-binding peptides. This protein bound to PS partly depending on the presence of the RR residue. These results allowed us to conclude that an amino acid sequence including contiguous arginine residues is a novel motif that defines Ca2+-independent PS-binding activity.     

Arsenite binding to synthetic peptides: the effect of increasing length between two cysteines

We utilized radioactive 73As-labeled arsenite and vacuum filtration methodology to determine the binding affinity of arsenite to eight synthetic peptides ranging from 13 to 24 amino acids long and containing one or two cysteines separated by 0-17 intervening amino acids. Six of the eight peptides were highly similar in amino acid sequence and were based on cysteine containing regions of the hormone-binding site of the human estrogen receptor-alpha (e.g., the sequence of peptide 28 is LEGAWCGKGVEGTEHLYSMKCKNV). The peptides with 0-14 intervening amino acids between two cysteines bound arsenite with Kd values of 2.7-20.1 uM and with Bmax values from 36 to 103 nmol/mg protein (from 0.083 to 0.19 nmol/nmol of protein). Thus, increasing the number of intervening amino acids from 0 to 14 made very little difference in the observed Kd values for arsenite, a surprising finding. Therefore, these peptides are flexible in solution and effectively contain a dithiol high affinity binding site for arsenite. Peptide 17 with two C separated by 19 amino acids bound arsenite with a Kd of 123 uM and a Bmax of 41.8 nmol/mg. The monothiol peptide 19 bound arsenite with a Kd of 124 uM and a Bmax of 26 nmol/mg protein. All experimental binding curves fit well to a one site binding model.     

Characterization of a novel collagen chain in human placenta and its relation to AB collagen.

A novel collagen chain, termed alpha C, has been isolated from human placenta by limited pepsin digestion. The collagen containing the alpha C chain copurifies with placental AB collagen during selective salt precipitation but is virtually absent from fetal birth membranes, which contain relatively larger amounts of AB. Both native AB and alpha C-containing collagens are resistant to human skin collagenase under conditions that support cleavage of type I by greater than 90%. The alpha C chain was separated from alpha B by phosphocellulose chromatography and subsequently from alpha P by chromatography on CM-cellulose. Its amino acid composition is distinct from alpha A and alha B although all three chains posses compositional features in common; the carbohydrate content of the alpha C chain was intermediate between those of alpha A and alpha B. Analysis by NaDodSO4-polyacrylamide gel electrophoresis of peptides produced by CNBr cleavage and by limited digestion with the enzyme mast cell protease indicated different and unique products for the alpha A, alpha B, and alpha C chains. The data support the existence of another collagen chain which is related to the alpha A and alpha B chains but which is structurally unique. The proteins containing these chains may in turn comprise a subfamily of collagen isotypes which represents a divergence from and/or specialization of the type IV basement membrane collagens.     

Isolation of phosphorylated peptides and proteins on ion exchange papers.

A simple technique for the isolation of ³²P-labeled peptides has been adapted from the ion exchange method of Witt and Roskoski (Anal. Biochem.66, 253, 1975). Protein kinase reaction mixtures are acidified and pipetted onto phosphocellulose papers. A variety of peptide and protein substrates are shown to bind to the ion exchange papers under conditions in which contaminating [γ-³²P]ATP is removed by washing in acetic acid. The capacity of phosphocellulose papers for the phosphopeptide, Leu-Arg-Arg-Ala-Ser(P)-Leu-Gly, exceeds 375 nmol per paper (2 × 2 cm). The method is limited to relatively basic peptides but is applicable to most proteins. Large numbers of samples can be processed simultaneously with no compromise in sensitivity or reliability of results.     

Amino acid sequence of lysozyme from baboon milk

Reduced alkylated baboon milk lysozyme was subjected to digestion with trypsin. The resulting peptides were purified by a combination of Dowex 1 (X2) and chromatography and electrophoresis on paper. The amino acid sequence of these peptides was determined in detail chiefly by the Edman procedure. Alignment of the tryptic peptides into a single chain containing 130 amino acids was established chiefly by homology with human milk lysozyme; 14 replacements were noted between the two enzymes. Baboon milk lysozyme was devoid of methionine and contained six basic amino acids (arginine residues) less than human milk lysozyme.     

Preparation of Peptides Containing Any Desired Amino Acid: Methionyl Peptides of Bovine Rhodopsin

A general method is described which allows the identification and preparation of peptides containing any amino acid of interest. The method has been applied to isolation of the methionyl peptides from a peptic digest of oxidized bovine rhodopsin. The peptide digestion mixture is first partially separated by ion exchange column chromatography. Location of peptides containing the desired amino acid is performed by amino acid analysis of acid hydrolyzed column fractions by high voltage paper electrophoresis. Peptides are further purified and prepared by peptide mapping, elution, and amino acid analysis using inexpensive high capacity techniques. Peptide sequencing is performed by a manual dansyl-Edman method well adapted for rapidly processing large numbers of samples. The methods are particularly well suited for detection and preparation of peptides containing amino acids for which there is no specific detection method.     

Aggregation of RecA-derived peptides on single-stranded oligonucleotides triggered by schiff base-mediated crosslinking

We here show that single-stranded oligonucleotides containing 5-formyl-2'-deoxyuridine (fdU) can crosslink the peptides derived from the DNA binding site of RecA protein through a Schiff base formation. The ability of crosslinking of fdU-containing oligonucleotides was investigated using a series of peptides whose amino acid residues spanning the center of the RecA-derived peptide were sequentially replaced with lysine. Circular dichroism (CD) spectroscopy, gel mobility shift assay and sedimentation experiment demonstrated that crosslinking reaction proceeded efficiently only when the peptides bound to the oligonucleotides.     

Identification of the major sites of enzymic glycosylation of myelin basic protein

In the presence of porcine submaxillary N-acetylgalactosaminyltransferase and uridine diphospho-N-acetyl-D-galactosamine, approx. 1.2–1.5 mol of N-acetylgalactosamine were transfered per mol of myelin basic protein. Tritium-labelled N-acetylgalactosamine-labelled basic protein was digested with trypsin and the peptides were separated by HPLC and the radioactivity measured. Most of the radioactivity was associated with three peptide peaks (I, II and III) containing 17, 69 and 6% of the total radioactivity, respectively. The remaining radioactivity was distributed amongst several peptides, each containing less than 2.5% of the total radioactivity. Glycosylation of the basic proteins isolated from human, bovine and guine pig myelins showed that they were all equally good acceptors. In spite of differences in the peptide profiles of the basic proteins from different species, the distribution of radioactivity between the three peptide peaks was similar for all the species studies. The transfer of N-acetylgalactosamine to peptide II was much faster than to peptides I and III. The apparent K_m values of the three peptides were within a narrow range of 0.52–0.63 mM, whereas the V_max values were considerably different. The glycosylated peptide peaks (I, II and III) were separated by electrophoresis, the radioactivity measured, and amino acid compositions determined after hydrolysis. The major radioactive peptides of the human basic protein were identified with tryptic peptides containing the following sequences:

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