Synthesis of albumin via a precursor protein in cell suspensions from rat liver.

The mechanism of the biosynthesis of albumin was studied in cell suspensions from rat liver. The cells were prepared by continuous perfusion of the liver in situ with 0.05% collagenase and 0.10% hyaluronidase and incubated under conditions optimized for the incorporation of amino acids into protein. Seven minutes after starting the incubation L-[1-14C]leucine was added, followed after 25 min by a 15 or 30-min chase with an 830-fold excess of non-radioactive L-leucine. Total protein, an albumin-like protein, and albumin were isolated from samples withdrawn immediately of total protein was found to remain constant after addition of the non-radioactive L-leucine, whereas that of the albumin-like protein decreased and that of albumin increased with incubation time. The increase in albumin radioactivity accounted for the decrease in radioactivity of the albumin-like protein, suggesting that the latter is a precursor of albumin. The precursor protein differed from albumin by an oligopeptide extension at the N-terminal end.     

Isolation and properties of a fatty acid-binding protein from the Pacific lamprey (Lampetra tridentata).

1. A survey of 12 vertebrate species showed that palmitate was bound by an albumin-like serum protein in all classes tested except the dogfish and the lamprey. 2. The major palmitate-binding protein of the Pacific lamprey was isolated and found to be of molecular mass 19,000. 3. The amino acid composition of this protein indicates that it is not a member of the albumin superfamily. 4. The 19-kDa lamprey protein binds bilirubin, cortisol and tryptophan only weakly, but binds palmitate with KA = 25 microM-1, comparable to the first long-chain fatty acid site of bovine albumin (KA = 34 microM-1).     

Purification and characterization of a novel 70-kDa brain protein associated with seizure activities

Using ion exchange HPLC and ammonium sulfate precipitation, we have purified a 70-kDa protein (P70) specific to the cobalt-induced epileptogenic cortex of rat cerebrum and determined certain of its biochemical properties. P70 has a similar isoelectric point (pI; 4.6–4.8), amino acid composition and N-terminal amino acid sequence to rat serum albumin (RSA). Intracortical application of purified P70 to the motor area of normal rat cerebrum induces both ECoG seizure discharges and behavioral seizures. The data suggest that P70 is a novel albumin-like protein linked to the generation of seizure activities. However, it can be clearly distinguished from RSA, since it is able to produce seizure, is a glycoprotein and can be readily separated from RSA by 2-dimensional electrophoresis.     

Cell attachment peptide of C-reactive protein: critical amino acids and minimum length

Human C-reactive protein (CRP) is an acute phase blood component that accumulates at sites of tissue damage and necrosis and is degraded by neutrophils to biologically active peptides. A dodecapeptide composed of amino acids 27–38 of CRP mediates cell attachment in vitro. This peptide was designated the cell-binding peptide (CB-Pep) of CRP. Characterization of the interaction between fibroblasts and modified synthetic peptides with sequential deletions from either the N-terminus or C-terminus revealed that the minimal sequence for cell attachment or inhibition of cell attachment to the CB-Pep was Phe-Thr-Val-Cys-Leu , which corresponds to residues 33–37 within each of the five 206 amino acid subunits of CRP. The pentapeptide by itself mediated cell attachment. Substitutions for each residue within the CB-Pep indicated that the critical residues for activity were Phe-33 and Thr-34. This cell-binding pentapeptide represents a recognition motif for cell adhesion not found in other proteins.     

Xenopus laevis serum albumin: sequence of the complementary deoxyribonucleic acids encoding the 68- and 74-kilodalton peptides and the regulation of albumin gene expression by thyroid hormone during development

In adult Xenopus serum, albumin gene expression is regulated by estrogen through the selective destabilization of its mRNA during the vitellogenic response. The present study reports the cDNA sequence of both the 68K and 74K Xenopus albumin mRNAs, their derived amino acid sequence, and the regulation of albumin gene expression during embryogenesis. Albumin mRNA has a 39 nucleotide 5' untranslated region terminating in a consensus translation initiation site. The derived amino acid sequence yields a 24-amino acid hydrophobic leader sequence (terminating in Lys-Arg) that shares significant homology with the leader peptide of rat albumin. Overall there is 37% sequence identity between rat and frog albumin, with exact conservation of all but one Cys residue and the Pro residues responsible for the three domain structure of the mature protein. The 74K albumin (unlike the 68K albumin) is glycosylated; a point mutation converting Lys256 to Asn introduces an N-linked glycosylation site that is similar to one found in the sequence of mammalian alpha-fetoproteins. A larval albumin-like protein was not detectable by silver staining in serum of tadpoles before the beginning of metamorphosis at stage 48. Albumin mRNA is absent from early tadpoles (stages 22-47); however, it is rapidly induced at stage 48 as one of the earliest manifestations of metamorphosis. Exposure of embryos to 10(-8) M T3, which regulates amphibian metamorphosis, resulted in the premature induction of albumin mRNA, such that it is evident by stage 43.     

Biological evidence for a precursor protein of serum albumin.

Radioactive leucine was injected into the portal vein of rats followed after 15 seconds by a 180 fold excess of nonradioactive leucine. An albumin-like protein in the liver became highly labelled within 15 minutes after injection. After 150 minutes, the radioactivity in the albumin-like protein had decreased to one tenth. In the serum, radioactively labelled albumin started to appear after 15 minutes and increased there-after up to 150 minutes after injection. Radioactivity in albumin within the liver remained constant at a low level. These results suggest that the albumin-like protein is a biological precursor protein of serum albumin, i.e. a proalbumin.     

Neurotensin-like immunoreactivity generated by pepsin from human plasma and gastric tissue

The present study demonstrates precursors of neurotensin-like immunoreactivity (NTLI) endogenous to human gastric tissue and plasma, and the existence of a gastric NTLI-generating enzyme system. The molecular size of the NTLI-precursors in plasma and gastric tissue were estimated by gel permeation chromatography to be ca 50,000-60,000 and 60,000-70,000 Da, respectively. The neurotensin-like peptide generated from the precursor was detected with a carboxyl-terminally directed antiserum but did not cross-react with an amino-terminally directed antiserum. A neurotensin-like peptide isolated from pepsin-treated human plasma was characterized by mass spectrometry and its amino acid sequence determined. This novel nonapeptide, referred to as kinetensin, failed to affect pentagastrin-stimulated acid secretion or blood pressure in the rat. Sequence homologies between neurotensin, kinetensin and proteins of the serum albumin family suggest a common evolutionary origin and raise questions regarding albumin-like proteins as precursors of regulatory peptides.     

Albumin-like proteins from the pituitary glands of Dahl salt-resistant rats.

Dahl selectively bred rats for susceptibility (S strain) or resistance (R strain) to the hypertensive effect of high salt (NaCl) diet. Pituitary glands of R rats accumulate large amounts of four unique proteins not seen in S rats. These proteins were called R1, R2, R3, and R4 in order of decreasing electrophoretic mobility. Albumin, R4, R2, and R1 all bound to an affinity column for albumin (Cibacron blue 3G-A dye coupled to agarose) and were eluted in that order by a KSCN gradient. It was shown by crossed immunoelectrophoresis that R1 and R2 cross-react with plasma albumin. Peptide maps or tryptic digest of R1 and albumin showed that the majority of peptides generated were identical. It was not possible to incorporate labeled amino acid into albumin or the albumin-like R proteins with pituitary incubates, indicating that albumin-like proteins were not synthesized de novo by pituitary glands. R rat pituitary glands showed much greater protease (arginine esterase) activity than did S. This suggests that R proteins are formed locally in the pituitary gland of R proteins are formed locally in the pituitary gland of R rats by cleavabe of specific peptide bonds in albumin. The function of these endogenous albumin fragments is unknown, but albumin fragments produced in vitro by other investigators are known to potentiate bradykinin, a hypotensive peptide.     

Characters of Albumin-like Polypeptides from Wheat Glutenin

The albumin-like polypeptides obtained from wheat glutenin were compared with albumins extracted from wheat flour. Similarity between the albumin-like polypeptides and the albumins was observed in the mobilities on SDS-polyacrylamide gel electrophoresis, in the amino acid compositions, isoelectric focusing profiles, and elution patterns from a Sephadex G–150 column. These results suggested that the albumin-like polypeptides from glutenin were not the ordinary components of glutenin molecules, but contaminants to glutenin.     

Insights into the structural variation between pentapeptide repeat proteins--crystal structure of Rfr23 from Cyanothece 51142

Cyanothece sp. PCC 51142 contains 35 pentapeptide repeat proteins (PRPs), proteins that contain a minimum of eight tandem repeated five-residues (Rfr) of the general consensus sequence A[N/D]LXX. Published crystal structures of PRPs show that the tandem pentapeptide repeats adopt a type of right-handed quadrilateral beta-helix called an Rfr-fold. To characterize how structural features of Rfr-folds might vary with different amino acid sequences, the crystal structure of Cyanothece Rfr23 (174 residues) was determined at 2.4A resolution. The structure is dominated by an Rfr-fold capped at the N-terminus with a nine-residue alpha-helix (M26(*)-E34). The Rfr-fold of Rfr23 contains four structural features previously unobserved in Rfr-folds. First, Rfr23 is composed entirely of type II beta-turns. Second, the pentapeptide repeats are not consecutive in the primary amino acid sequence. Instead, Rfr23 contains 24-residues protruding outside one corner of the first complete N-terminal coil of the Rfr-fold (L56-P79) (24-residue insertion). Third, a disulfide bond between C39 and C42 bridges the beta-turn between the first and second pentapeptide repeats in the first coil (disulfide bracket). NMR spectroscopy indicates that the reduction of the disulfide bracket with the addition of DTT destroys the entire Rfr-fold. Fourth, a single-residue perturbs the Rfr-fold slightly in the last coil between the C-terminal two pentapeptide repeats (single-residue bulge).     

Spatial organization and conformational peculiarities of the callatostatin family of neuropeptides.

The structures and conformational peculiarities of five members of the callatostatin family of neuropeptides, i.e. Leu- and Met-callatostatins, ranging in size from 8 to 16 amino acid residues have been investigated by a theoretical conformational analysis method. A comparative analysis of the conformational flexibilities of Met-callatostatin with those of the hydroxylated analogues, [Hyp2]- and [Hyp3]-Met-callatostatin has been carried out. Helically packed C-terminal pentapeptide in the structure of all investigated Leu-callatostatins are shown to be possible. The reason for the great number low-energy conformers for the callatostatin N-terminus is discussed.     

Quantitative identification of N-terminal amino acids in proteins by radiolabeled reductive methylation and amino acid analysis: application to human erythrocyte acetylcholinesterase

A novel method of determining N-terminal amino acids in proteins is introduced. Reductive methylation of a protein with radiolabeled formaldehyde methylates both the alpha-amino group of the N-terminal amino acid and the epsilon-amino groups of Lys residues. The radiomethylated amino acids are stable to acid hydrolysis, and each of 16 possible hydrolysis-stable N-terminal amino acids can be identified by the unique elution positions of its N alpha-methyl and N alpha,N alpha-dimethyl derivatives with an appropriate amino acid analyzer elution schedule. The technique is at least as sensitive as other N-terminal amino acid determinations and, in addition, permits a quantitative evaluation of the number of N-terminal groups in a sample. Reductive methylation of bovine serum albumin revealed N-terminal Asp at a stoichiometry of 0.97 amino acid residue per polypeptide, while methylation of prolactin resulted in 0.86 residue of N-terminal Thr per polypeptide. Human erythrocyte acetylcholinesterase contained two N-terminal amino acids with stoichiometries of 0.66 Glu and 0.34 Arg per 70-kDa subunit. Identification of Glu as the principal N-terminus of acetylcholinesterase was confirmed by Edman sequencing.     

Biosynthesis of serum albumin in rat liver. Evidence for the existence of `proalbumin''

1. A protein(s) of rat liver (precipitated from soluble extracts of the microsomal fraction by anti-albumin) yields albumin after limited hydrolysis by trypsin. 2. Evidence that the product of limited tryptic hydrolysis is albumin, is based upon ion-exchange chromatography, electrofocusing and peptide `mapping'. 3. The albumin `precursor' is recognized by anti-albumin and is apparently not distinguished from albumin by anti-albumin. 4. A small peptide is liberated from the presumptive albumin precursor during limited tryptic hydrolysis. This peptide is labelled by arginine, but not by leucine, lysine or methionine. 5. These results support our previous suggestion based on kinetic evidence that the albumin-like protein(s), in the anti-albumin precipitate from rat liver, is an albumin precursor.     

Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit.

The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1.     

Chemical and immunological characterizations of equine infectious anemia virus gag-encoded proteins.

The viral core proteins (p15, p26, p11, and p9) of equine infectious anemia virus (EIAV) (Wyoming strain) were purified by reverse-phase high-pressure liquid chromatography. Each purified protein was analyzed for amino acid content, N-terminal amino acid sequence, C-terminal amino acid sequence, and phosphoamino acid content. The results of N- and C-terminal amino acid sequence analysis of each gag protein, taken together with the nucleotide sequence of the EIAV gag gene (R. M. Stephens, J. W. Casey, and N. R. Rice, Science 231:589-594, 1986), show that the order of the proteins in the precursor is p15-p26-*-p11-p9, where a pentapeptide also found in the virus is represented by the asterisk. The data are in complete agreement with the predicted structure of the gag polyprotein and show the peptide bonds cleaved during proteolytic processing. The N-terminus of p15 is blocked to Edman degradation. The p11 protein is identical to the nucleic acid-binding protein of EIAV previously isolated (C. W. Long, L. E. Henderson, and S. Oroszlan, Virology 104:491-496, 1980). High-titer rabbit antiserum was prepared against each purified protein. These antisera were used to detect the putative gag precursor (Pr55gag) and intermediate cleavage products designated Pr49 (p15-p26-*-p11), Pr40 (p15-p26), and Pr35 (p26-*-p11) in the virus and in virus-infected cells. High-titer antisera to EIAV p15 and p26 showed cross-reactivity with the homologous protein of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus.     

Amino-terminal nucleotide-binding sequences of a Lactobacillus deoxynucleoside kinase complex isolated by novel affinity chromatography

A highly efficient new affinity medium for deoxycytidine kinase, deoxycytidine 5'-tetraphosphate-Sepharose (dCp4-Sepharose), has been constructed. A dCp4-Sepharose column effects a one-step, 19,000-fold, purification to homogeneity of dCyd kinase from the ammonium sulfate fraction of Lactobacillus acidophilus R-26 extract, with 60% recovery. dCTP, a potent end-product inhibitor, is used as an eluent, and it also stabilizes the extremely labile purified enzyme. A noncompeting deoxyadenosine kinase activity accompanies the deoxycytidine kinase activity eluted. Native polyacrylamide gel electrophoresis shows a single protein band, which coincides with both deoxycytidine kinase and deoxyadenosine kinase activities at several gel concentrations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a single polypeptide band of 26,000 daltons. Since the native enzyme is known to have an Mr of 50,000, it appears that the enzyme is composed of two subunits of similar size. Sequence analysis of the intact protein from the N-terminus reveals but a single amino acid species per residue up to the 17th residue; at the 18th, 21st, 26th, and 27th residue positions of the sequence, however, there appear to be two different amino acids in almost equal amounts. This may indicate that the enzyme is composed of two nonidentical subunits having the same amino acid sequence near the N-terminus. Residues 6-13 contain the highly conserved Gly-X-X-Gly-X-Gly-Lys sequence found at the active sites of kinases and other nucleotide-binding proteins.     

Isolation of a trypsin inhibitor with deletion of N-terminal pentapeptide from the seeds of Momordica cochinchinensis, the Chinese drug mubiezhi.

A trypsin inhibitor, MCCTI-1, with a molecular weight of 3479 Da as determined by mass spectrometry, was isolated from Momordica cochinchinensis seeds with a procedure involving extraction with 5% acetic acid, ammonium sulfate precipitation, ion exchange chromatography on CM-Sepharose and reverse-phase high performance liquid chromatography. The sequence of its first 13 N-terminal amino acid residues was ILKKCRRDSDCPG which was about 85% identical with the sequence of trypsin inhibitor MCTI-1 from Momordica charantia Linn. When compared with the sequences of most other squash family trypsin inhibitors, the sequence of MCCTI-1 was characterized by the deletion of a pentapeptide from the N-terminus. Trypsin inhibitors also existed in seeds of some hitherto uninvestigated Cucurbitaceae species.     

Amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum

The amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum has been determined. It is composed of 260 amino acid residues and its N-terminus is acetylated. The molecular mass is calculated to be 29 851 Da. The presence of six calcium-binding sites (I-VI) has been proposed, two of them (sites II and VI) have lost their calcium-binding function through amino acid replacements, and the other four are able to bind calcium. Six calcium-binding domains are supposed to be derived from two gene duplications of the two ancestral calcium-binding domains. In comparison with the sequence of chick intestinal calcium-binding protein deduced from a cDNA sequence [(1985) Nucleic Acids Res. 13, 8867-8881], the bovine calcium-binding protein is two amino acid residues shorter at the N-terminus and the other parts show 78.5% identity.     

Characterization and molecular cloning of a glutathione S-transferase gene from the tick, Boophilus microplus (Acari: Ixodidae).

A glutathione S-transferase (GST) was purified from the larval cattle tick, Boophilus microplus (Acari: Ixodidae), by glutathione-affinity chromatography. The purified enzyme appeared as a single band on SDS-PAGE and has a molecular mass of 25.8 kDa determined by mass spectrometry. The N-terminus of the purified enzyme was sequenced. The full-length cDNA of the enzyme was isolated by RT-PCR using degenerate oligonucleotides derived from the N-terminal amino acid sequence. The cDNA contains an open reading frame encoding a 223-amino-acid protein with the N-terminus identical to the purified GST. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian mu class GST.     

Leucyl/phenylalanyl(L/F)-tRNA-protein transferase-mediated aminoacyl transfer of a nonnatural amino acid to the N-terminus of peptides and proteins and subsequent functionalization by bioorthogonal reactions

We report here a new strategy for derivatizing peptides and proteins at the N-terminus. To achieve this, a nonnatural amino acid was charged onto a tRNA and then enzymatically transferred to a lysine (Lys) unit at the N-terminus of a peptide or a protein by using L/F-tRNA-protein transferase. By using the chemoenzymatic technique, beta-(2-quinolyl)-L-alanine, p-azido-L-phenylalanine, and p-acetyl-L-phenylalanine were introduced to the N-terminus. The latter two nonnatural amino acids possess bioorthogonal functional groups to which artificial tags can be introduced. Actually, a biotin tag was coupled to the bioorthogonal ketone group of acetylphenylalanine at the N-terminus of a peptide. N-terminal-specific biotinylation and fluorescence derivatization of the bioorthogonal azido-containing protein or peptide was also carried out based on a [3 + 2] cycloaddition. The enzymatic transfer of a nonnatural amino acid to the N-terminus of target peptides or proteins was also successfully achieved in the presence of other peptides or crude protein mixtures.