Quantitation of the major proteins of the human erythrocyte membrane by amino acid analysis

The proteins of the human erythrocyte membrane have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the resulting gel cut into 2-mm sections, and the amino acid content and composition of each slice measured using a sensitive method of amino acid analysis. The distribution of proteins among bands coincides closely with that estimated using staining intensity. Composition data for the major bands agree well with those reported for the purified proteins in all cases except that of band 4.5. Using quantitative amino acid analysis and resistive particle counting the total protein content of purified membranes was found to be 3.75 X 10(-13) g/cell, which is substantially less than previous estimates based on indirect methods. These data are used to calculate the number of copies of each major protein in a single erythrocyte.     

Radiation-induced reductive modifications of sulfur-containing amino acids within peptides and proteins

The complex scenario of radical stress reactions affecting peptides/proteins can be better elucidated through the design of biomimetic studies simulating the consequences of the different free radicals attacking amino acids. In this context, ionizing radiations allowed to examine the specific damages caused by H-atoms and electrons coupled with protons, thus establishing the molecular basis of reductive radical stress. This is an innovative concept that complements the well-known oxidative stress also in view of a complete understanding of the global consequences of radical species reactivities on living systems. This review summarizes the knowledge of the chemical changes present in sulfur-containing amino acids occurring in polypeptides under reductive radical conditions, in particular the transformation of Met and Cys residues into α-amino butyric acid and alanine, respectively. Reductive radical stress causing a desulfurization process, is therefore coupled with the formation of S-centered radicals, which in turn can diffuse apart and become responsible of the damage transfer from proteins to lipids. These reductive modifications assayed in different peptide/protein sequences constitute an integration of the molecular inventories that up to now take into account only oxidative transformations. They can be useful to achieve an integrated vision of the free radical reactivities in a multifunctional system and, overall, for wider applications in the redox proteomics field.     

In vitro labeling of proteins by reductive methylation: Application to proteins involved in supramolecular structures

Actin and tropomyosin, purified from both muscle and brain, and α-actinin, purified from muscle, have been labeled in vitro by reductive methylation to specific activities of greater than 10⁵ dpm/μg protein. Actin so modified bound DNase I and polymerized identically to unmodified actin. Furthermore, the spectral properties of actin did not change after labeling. The interactions of labeled tropomyosin and α-actinin with F-actin were nearly identical to those of the unmodified proteins. These modified proteins comigrated with their unmodified counterparts in both SDS-containing polyacrylamide gels and isoelectric focusing gels. The labeled actin was quantitatively extracted from SDS-containing polyacrylamide gels (yield > 98% of radioactivity applied demonstrating that all of the radioactivity was protein bound. The reductive methylation procedure worked well at pH 8.0–8.5 in either pyrophosphate buffer or Bicine buffer using formaldehyde with [³H]-sodium borohydride as the reducing agent. The procedure could also be performed at pH 7.0 in phosphate buffer using [¹⁴C]-formaldehyde with sodium cyanoborohydride as the reducing agent. Proteins so labeled are ideal for use in quantitative experiments involving protein-protein interactions.     

N-terminal amino acid analysis reveal peptide heterogeneity in major electrophoretic protein components of erythrocyte ghosts

Preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis of dansylated erythrocyte membrane proteins shows that 60% of the 1-dimethylaminophthalene-5-sulfonyl fluorescence migrates with sodium dodecylsulfate-polyacrylamide gel electrophoresis Bands 1,2 and 3. N-terminal amino acid analyses show that each of these bands contains at least five N-terminals. Bands 1 and 2 contain identical N-terminals. Most N-terminals appear constant, but sone additional N-termini vary from one donor to the next.     

Quantitative determination of N-terminal amino acids of peptides and proteins with cobalt(III) chelates.

Quantitative N-terminal peptide-bond hydrolysis with the cis-beta-hydroxyaquo(triethylenetetramine) cobal (III) ion, i.e. beta-[Co(trien)(OH)(OH2)]2+, is reported. The method has been demonstrated with 20 small peptides, a hexapeptide, bradykinin, insulin A chain (oxidized), glucagon and insulin. The procedure involves no acidic hydrolysis step and thus no destruction of labile amino acids.     

N-terminal N-myristoylation of proteins: prediction of substrate proteins from amino acid sequence

Myristoylation by the myristoyl-CoA:protein N-myristoyltransferase (NMT) is an important lipid anchor modification of eukaryotic and viral proteins. Automated prediction of N-terminal N-myristoylation from the substrate protein sequence alone is necessary for large-scale sequence annotation projects but it requires a low rate of false positive hits in addition to a sufficient sensitivity.Our previous analysis of substrate protein sequence variability, NMT sequences and 3D structures has revealed motif properties in addition to the known PROSITE motif that are utilized in a new predictor described here. The composite prediction function (with separate ad hoc parameterization (a) for queries from non-fungal eukaryotes and their viruses and (b) for sequences from fungal species) consists of terms evaluating amino acid type preferences at sequences positions close to the N terminus as well as terms penalizing deviations from the physical property pattern of amino acid side-chains encoded in multi-residue correlation within the motif sequence. The algorithm has been validated with a self-consistency and two jack-knife tests for the learning set as well as with kinetic data for model substrates. The sensitivity in recognizing documented NMT substrates is above 95 % for both taxon-specific versions. The corresponding rate of false positive prediction (for sequences with an N-terminal glycine residue) is close to 0.5 %; thus, the technique is applicable for large-scale automated sequence database annotation. The predictor is available as public WWW-server with the URL http://mendel.imp.univie.ac.at/myristate/. Additionally, we propose a version of the predictor that identifies a number of proteolytic protein processing sites at internal glycine residues and that evaluates possible N-terminal myristoylation of the protein fragments.A scan of public protein databases revealed new potential NMT targets for which the myristoyl modification may be of critical importance for biological function. Among others, the list includes kinases, phosphatases, proteasomal regulatory subunit 4, kinase interacting proteins KIP1/KIP2, protozoan flagellar proteins, homologues of mitochondrial translocase TOM40, of the neuronal calcium sensor NCS-1 and of the cytochrome c-type heme lyase CCHL. Analyses of complete eukaryote genomes indicate that about 0.5 % of all encoded proteins are apparent NMT substrates except for a higher fraction in Arabidopsis thaliana ( approximately 0.8 %).     

Specific 13C reductive methylation of glycophorin A. Possible relation of the N-terminal amino acid and the lysine residues to MN blood group specificities

Heterozygous and homozygous glycophorin A were partially and fully reductively methylated with 13C-enriched formaldehyde in the presence of sodium cyanoborohydride. Total reductive methylation modified the five lysine residues (to produce N epsilon,N-[13C]dimethyl lysine) and the N-terminal amino acid residues (N alpha,N-[13C]dimethyl serine and leucine) of glycophorins AM and AN, respectively. 13C-NMR spectra of these species indicated that the 13C-enriched methyl carbons of the five lysyl derivatives all occur at 44.1 ppm downfield from Me4Si. Titration results indicate that the pK alpha of these methylated lysines is greater than 10. The chemical shift equivalent methyl resonances of the 13C-enriched methylated N-terminal Leu derivative were found to occur at 42.8 ppm downfield from Me4Si and exhibited a normal pH titration behavior (pK alpha approximately 7.4). The methyl resonances of the N alpha,N-[13C]dimethyl Ser derivative, on the other hand, were found to exhibit chemical shift nonequivalence, indicating rotational constraints about the C alpha-N bond. The linewidths of the two methyl resonances were also found to be considerably different; this phenomenon could be eliminated by running spectra of the sample (pH approximately 5.0) at elevated temperatures (75 degrees C). This result suggested that for the N alpha,N-[13C]dimethyl Ser derivative of glycophorin AM, hindered rotation must occur about one of the N alpha-13CH3 bonds. This structural difference at the N-terminal residue of glycophorins AM and AN may be related to the MN blood group determinants displayed by these related glycoproteins.     

Recombinant protein sequences can trigger methylation of N-terminal amino acids in Escherichia coli.

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus. No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin. The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N-terminal methylation has been observed previously for native E. coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.     

Study of fibrinogen-fibrin cryocomplexes by a quantitative analysis of N-terminal amino acids

The reaction between fibrinogen (F) and thrombin (0.003 NIH/ml) has been investigated under physiological conditions. The action of thrombin was inhibited in various time intervals including gel point (4 h), and the reaction mixtures were allowed to stand 0 degrees C. The F-to-des-AA-fibrin (f) ratios were determined both in the initial reaction mixtures and in corresponding cryoprecipitants by the method of N-terminal amino acids quantitative analysis. It is found that the F/f ratio in the cryoprecipitant depends on the F/f ratio in the initial mixture at 37 degrees C. The F/f = 1 ratio in cryoprecipitant previously found by Shainoff and Page is valid only for the initial F/f = 7 ratio. But the F/f value in cryoprecipitants varies in favour of F or f components, if the F/f ratio increases or decreases in the initial mixture at 37 degrees C, respectively. A possible mechanism of various fibrinogen-fibrin cryocomplexes formation is discussed.     

Neisseria pili proteins: amino-terminal amino acid sequences and identification of an unusual amino acid.

The amino-terminal amino acid sequences of the pili proteins from four antigenically dissimilar strains of Neisseria gonorrhoeae, from Neisseria meningiditis, and from Escherichia coli were determined. Although antibodies raised to the pili protein from a given strain of gonococcus cross-reacted poorly or not at all with each of the other strains tested, the amino-terminal sequences were all identical. The meningococcal protein sequence was also identical with the gonococcal sequence through 29 residues, and this sequence was highly homologous to the sequence of the pili protein of Moraxella nonliquifaciens determined by other workers. However, the sequence of the pili protein from E. coli showed no similarity to the other sequences. The gonococcal and meningococcal proteins have an unusual amino acid at the amino termini, N-methylphenylalanine. In addition, the first 24 residues of these proteins have only two hydrophilic residues (at positions 2 and 5) with the rest being predominantly aliphatic hydrophobic amino acids. The preservation of this highly unusual sequence among five antigenically dissimilar Neisseria pili proteins implies a role for the amino-terminal structure in pilus function. The amino terminus may be directly or indirectly (through preservation of tertiary structure) important for the pilus function of facilitating attachment of bacteria to human cells.     

N-terminal amino acid sequence of two novel tumor-derived adenylate cyclase-stimulating proteins: identification of parathyroid hormone-like and parathyroid hormone-unlike domains

A 17,000 dalton human adenylate cyclase-stimulating protein has previously been purified from a human tumor associated with humoral hypercalcemia of malignancy. This report describes the purification of a related 7,000-9,000 MW protein from a second tumor, and provides N-terminal amino acid sequence of these two peptides. The sequences of the peptides are identical, defining the smaller peptide as an N-terminal portion of the larger peptide. The two peptides possess one region of strong homology with parathyroid hormone and a second divergent region. These structural similarities and differences may explain the similarities and differences which occur in patients with hyperparathyroidism and humoral hypercalcemia of malignancy.     

Rapid hydrolysis of proteins and peptides by means of microwave technology and its application to amino acid analysis

A rapid heating method of hydrolysis by the use of microwave oven has been applied to amino acid analysis of proteins and peptides. This convenient method has been compared with the conventional 6 N HCl hydrolysis at 110 degrees for 24 h. The advantages of this new method are its expedition and the accurate and comparable results as compared to the tedious conventional technique. The method provides a rapid processing of multiple samples within minutes instead of days and inexpensive access to the important data of amino acid compositions of proteins by the commonly used microwave oven. The necessary change in the design of hydrolysis vials and the safety precautions accompanying this novel use of microwave acid-digestion method are also described.     

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Summary. We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.