γ-Carboxyglutamate analysis by selective anion exchange elution and fluorescent detection

A rapid, sensitive method for the determination of free γ-carboxyglutamic acid (γ-CG) in urine and in the alkaline hydrolysates of proteins is presented. An aliquot of urine or protein hydrolysate containing added γ-[¹⁴C]CG is chromatographed on Dowex 1 employing stepwise treatments in N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid buffers and final selective elution of γ-CG with MgCl₂. Aliquots of the eluted γ-CG fractions are counted to determine percentage recovery and assayed for γ-CG content by fluorescence employing o-phthalaldehyde. This procedure correlates well with direct determination of γ-CG by the established procedure of automatic amino acid analysis.     

Vitamin K-dependent gamma-carboxyglutamic acid formation by kidney microsomes in vitro.

γ-Carboxyglutamic acid has been identified as a constituent of renal tissue in chicken, rat, and rabbit and is depressed by vitamin K-deficiency or dicoumarol diets. Thorough perfusion of rat and rabbit kidneys to remove blood contamination does not remove the γ-carboxyglutamate containing protein(s), which appear to be localized in the cortex. Incubation of kidney microsomes with [¹⁴C]NaHCO₃in vitro results in the post-translational formation of protein bound $\text{[}{}^{14}\text{C]-γ-carboxyglutamic}$ acid. Incorporation is stimulated 1.6- to 34-fold by addition of the active vitamin K 2-methyl, 3-farnesyl, 1,4-naphthoquinone. About 80% of incorporated, non-dialyzable ¹⁴C is situated in the γ-carboxyl group of γ-carboxyglutamic acid.     

Vitamin K dependent formation of gamma-carboxyglutamate residues in tumor microsomes

Vitamin K stimulated the incorporation of ¹⁴C into proteins when microsomes from melanoma, mammary gland, mast cell and lymphoma tumors were incubated with Na₂¹⁴CO₃. The ¹⁴C label in the [¹⁴C] proteins was identified as [¹⁴C] γ-carboxyglutamate (Gla), which is formed by carboxylation of glutamic acid residues. Carboxylation in tumor microsomes ranged from 2 to 19% of the carboxylation in normal liver microsomes per mg of microsomal protein. Carboxylation in microsomes was completely blocked by 10 μM Warfarin. SDS-polyacrylamide gel analysis of the melanoma [¹⁴C] Gla protein(s) revealed one major peak of ¹⁴C with an apparent MW of less than 6,000.     

Structure primaire de l'anhydrase carbonique érythrocytaire B humaine: II. Clivage par le bromure de cyanogène et séquence des résidus 1–148

The amino acid sequence of the IICNBr fragment of the human erythrocyte carbonic anhydrase B has been determined. This fragment contains the first 148 of the 260 residues of the N-acetylated single polypeptide chain of the protein. After tryptic hydrolysis of this fragment, eleven peptides have been isolated by gel filtration and chromatography on Dowex 50 W-X₂ or DEAE-Sephadex. Eight of them were identified with already sequenced peptides previously isolated from tryptic hydrolysate of the whole protein. The other three ones were obtained in pure form and sequenced. The combined amino acid content of these eleven peptides only account for 124 of the 148 amino acid residues in the IICNBr fragment. The tryptic attack of the maleylated IICNBr fragment gave three peptides as was expected from the number of arginine residues (2) in this fragment: two arginyl peptides (II₁, II₃) and one homoseryl peptide (II₂). They were purified by gel filtration. The unidentified 24 residue tryptic peptide has been isolated from the demaleylated II₂ tryptic hydrolysate and sequenced. The order of the twelve tryptic peptides of IICNBr fragment has been obtained by study of chymotrypsic peptides isolated from II₁ and IICNBr fragment.     

Interaction between gamma-carboxyglutamic acid and glutamate dehydrogenase

The amino acid γ-carboxyglutamic acid, recently discovered in some vitamin K-dependent blood-clotting factors, shows interesting kinetic effects on glutamate dehydrogenase. It is not metabolized by the enzyme; it is a powerful competitive inhibitor (K_i = 3.8 × 10^?4 m) with respect to NAD⁺ and glutamate. On the other hand the reverse reaction is activated by γ-carboxyglutamate, both K_m and V being altered; this effect is additive with the well-known activating effect of ADP.     

Glutamic acid independent production of poly-γ-glutamic acid by Bacillus amyloliquefaciens LL3 and cloning of pgsBCA genes

A new glutamic acid independent poly-γ-glutamic acid (γ-PGA) producing strain, which was identified as Bacillusamyloliquefaciens LL3 by analysis of 16S rDNA and gyrase subunit A gene (gyrA), was isolated from fermented food. The product had a molecular weight of 470, 801 and l-glutamate monomer content of 98.47%. The pre-optimal medium, based on single-factor tests and orthogonal design, contained 50 g/L sucrose, 2 g/L (NH₄)₂SO₄, 0.6 g/L MgSO₄, and provided well-balanced changes in processing parameters and a γ-PGA yield of 4.36 g/L in 200 L system. The γ-PGA synthetase genes pgsBCA were cloned from LL3, and successfully expressed by pTrcLpgs vector in Escherichia coli JM109, resulting the synthesis of γ-PGA without glutamate. This study demonstrates the designedly improved yield of γ-PGA in 200 L system and the first report of pgsBCA from glutamic acid independent strain, which will benefit the metabolized mechanism investigation and the wide-ranging application of γ-PGA.     

A comparative review of the structure and biosynthesis of thyroglobulin

Thyroglobulin, the major iodoglycoprotein of the thyroid (M_r 669 kDa) has a sedimentation coefficient of 19 S and an isoelectric point (pI) of 4.4–4.7. The protein has been isolated and purified from saline extracts of the gland of several animal species, by methods such as ammonium sulfate fractionation, DEAE-cellulose chromatography and Sepharose 4B/6B gel-filtration. DEAE-cellulose chromatography of thyroglobulin from many species, by linear gradient, yielded a complex elution pattern, while camel thyroglobulin showed only a major and minor peak. As an iodoprotein, the protein has 0.1–2.0% iodine. The amino acid and iodoamino acid composition of thyroglobulins, in general, is similar. However, a high thyroxine content (15 mol/mol protein) has been noted for buffalo species. Asparagine or aspartic acid has been reported as the major N-terminal amino acid for thyroglobulins of several animal species whereas glutamic acid is the sole N-terminal amino acid for buffalo thyroglobulin. As a glycoprotein, thyroglobulin contains 8–10% total carbohydrate with galactose, mannose, fucose, N-acetyl glucosamine and sialic acid residues. The carbohydrate in the protein is distributed as two distinct units, A and B. In addition, human thyroglobulin has carbohydrate unit C. The occurrence of sulfate and phosphate as Gal-3-SO₄ and Man-6-PO₄, respectively, has been reported in few species. The quaternary structure of native thyroglobulin is comprised of two equal sized subunits of 330 kDa. However, the protein appears to contain 4–8 non-identical units in few species. The synthesis of thyroid hormones occurs in the matrix of the protein and is regulated by pituitary thyrotropin. The role of tyrosine residues 5 and 130 in thyroxine synthesis has been well documented.     

Isolation and characterization of a small molecular weight protein of linseed meal

A small M_r, protein from linseed meal has been isolated by CM-Sephadex chromatography. The protein was found to be homogeneous by the techniques of gel filtration, polyacrylamide gel electrophoresis and ultracentrifugation. It had S_20,w value of 1.6S. Amino acid composition of the protein revealed a high amount of glutamic acid, cystine, arginine and glycine. The absorption spectrum of the protein consisted of a peak at 280 nm with a shoulder at 290 nm. The fluorescence emission maximum was at 340 nm. The protein contained large amounts of α-helix and β-structure. SDS-PAGE showed the protein to consist of a single polypeptide chain. The M_r estimated by Archibald's method, sedimentation-diffusion method and gel filtration was 17 000,16 000 and 15 000 respectively. Difference spectra studies as a function of pH and temperature showed no variation in the conformation of the protein, probably due to disulphide bridges.     

Isolation,primary structure and synthesis of leucomyosuppressin,an insect neuropeptide that inhibits spontaneous contractions of the cockroach hindgut

1. A neuropeptide that inhibits spontaneous contractions of the isolated cockroach hindgut was purified from head extracts of the cockroach, Leucophaea maderae.2. The inability of aminopeptidase M to degrade the peptide and the presence of glutamic acid in the hydrolysate suggested N-terminal blocking by pyroglutamic acid. The N-terminal pGlu was removed enzymatically and the unblocked fragment was sequenced with an automated peptide sequencer.3. The structure determined (pGlu-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH₂) was synthesized and shown to be both chemically and biologically identical with the natural product.4. Leucomyosuppressin is the first inhibitory neuropeptide isolated and structurally identified from an insect source.     

The occurrence of γ-carboxyglutamate in a protein isolated from ox liver mitochondria

A calcium-binding protein has been isolated from ox liver mitochondria. The purification procedure includes ammonium sulfate fractionation and chromatographies on Sephadex G-100 and DEAE-Sephadex, respectively. The isolated protein contains γ-carboxyglutamate, has a molecular weight of 59,000, and an isoelectric point of about 3.8. By submitochondrial fractionation it was shown that the calcium-binding protein is located between the inner and outer mitochondrial membranes.     

Rhipicephalus sanguineus: amino acid composition of egg shell

Acid hydrolysis of the protein fraction of a batch of egg shells of Rhipicephalus sanguineus was followed by determination of the amino acids in the hydrolysate. Using thin layer chromatography, the amino acids—lysine, arginine, aspartic acid, serine, glycine, glutamic acid, alanine, threonine, valine, tyrosine, isoleucine, and leucine were identified. Alkaline hydrolysis of the fraction followed by TLC revealed the presence of tryptophane. Chitin was revealed utilizing a chitosan test.     

Purification and characterization of angiotensin I converting enzyme inhibitory peptides from the rotifer, Brachionus rotundiformis

Angiotensin I converting enzyme (ACE) inhibitory peptide was isolated from the marine rotifer, Brachionus rotundiformis. ACE inhibitory peptides were separated from rotifer hydrolysate prepared by Alcalase, α-chymotrypsin, Neutrase, papain, and trypsin. The Alcalase hydrolysate had the highest ACE inhibitory activity compared to the other hydrolysates. The IC₅₀ value of Alcalase hydrolysate for ACE inhibitory activity was 0.63 mg/ml. We attempted to isolate ACE inhibitory peptides from Alcalase prepared rotifer hydrolysate using gel filtration on a Sephadex G-25 column and high performance liquid chromatography on an ODS column. The IC₅₀ value of purified ACE inhibitory peptide was 9.64 μM, and Lineweaver–Burk plots suggest that the peptide purified from rotifer protein acts as a competitive inhibitor against ACE. Amino acid sequence of the peptide was identified as Asp-Asp-Thr-Gly-His-Asp-Phe-Glu-Asp-Thr-Gly-Glu-Ala-Met, with a molecular weight 1538 Da. The results of this study suggest that peptides derived from rotifers may be beneficial as anti-hypertension compounds in functional foods resource.     

Sequence determination of eglin c using combined microtechniques of amino acid analysis,peptide isolation,and automatic Edman degradation

The complete amino acid sequence of a proteinase inhibitor, eglin c (M_r 8100), has been determined with less than 150 μg of the protein using the following microtechniques: (a) amino acid analysis with a low-nanogram amount of protein hydrolysate using dimethylaminoazobenzene sulfonyl chloride, (b) peptide isolation at the picomole level using the dimethylaminoazobenzene isothyiocyanate (DABITC) precolumn derivatization method, and (c) automatic Edman degradation. One amino acid residue has been corrected for the previously reported sequence. The Contribution of each technique to the microsequencing is discussed. In addition, a new high-performance liquid chromatography system that gives a complete baseline separation of all phenylthiohydantoin-amino acids is described.     

Separation of the formation of γ-coniceine and aliphatic amines from got activity in Conium maculatum

L-Alanine:5-keto-octanal aminotransferase has been separated from GOT and GPT activities by DEAE-cellulose chromatography. Two transaminase peaks A and B were observed for γ-coniceine formation and assay showed that these peaks also contained the amino acid:aldehyde aminotransferase (AAT) responsible for aliphatic amine biosynthesis. Kinetic studies showed that γ-coniceine formation with transaminase A had K_{m for L-alanine and 5-keto-octanal of 27 mM and 1.6 mM respectively. The Vmaxwas 1.3 nkats/mg protein and the activation energy was 3.0 kJ/mol. For transaminase B the Kinm} for L-alanine and 5-keto-octanal are 55 mM and 0.14 mM respectively with a V_maxof 3.3 nkats/mg protein and an activation energy of 0.33 kJ/mol. Transaminase A and B had the same MW and have distinguishable pH_max. γ-Coniceine formation by transaminase A was inhibited uncompetitively by glyoxalate and competitively by pyruvate, whereas with transaminase B uncompetitive inhibition was also observed with glyoxalate; no inhibition was observed with pyruvate which at low concentration (0.25 M) showed slight stimulation of activity.     

Timed appearance of a calcium-binding protein containing gamma-carboxyglutamic acid in developing chick bone

Bone contains a small protein, rich in the vitamin K-dependent calcium-binding amino acid γ-carboxyglutamate (Gla). This protein, named osteocalcin, is extractable by neutral EDTA demineralization and contains over 80% of the total peptide Gla found in bone. Osteocalcin binds Ca²⁺ ions with moderate affinity (2 moles of Ca²⁺/6500 g of protein; K_d = 0.83 mM). Osteocalcin appears in embryonic chick bones (mandible, calvaria, tibiotarsus, and femur) coincident with the first histologically observable deposition of bone mineral at 8 to 12 days after fertilization. The quantity of this protein increases dramatically during development with characteristic onset and kinetics for each type of bone. In the long bone diaphysis (midshaft), the fraction of noncollagen protein represented by osteocalcin increases 100- to 200-fold between the 8th and 20th day. Relative to total bone protein, the increase is about 35-fold. Osteocalcin may play a role in the development of mineralized tissues and may be a characteristic product of cells differentiated with respect to bone and/or cartilage formation.     

Direct evidence for the presence of beta-hydroxyphenylalanine and beta-hydroxytyrosine in cutinase from Fusarium solani pisi

Extracellular cutinase induced by cutin hydrolysate in glucose-grown Fusarium solani f. pisi was isolated in electrophoretically homogeneous form. This enzyme was similar to cutinase I generated by cutin-grown cells in its catalytic properties such as pH optimum, substrate specificity, and inactivation by “active serine”-directed reagents. Its molecular weight was 26,300 and this enzyme had a much larger content of serine and threonine residues than that found in cutinase from the cutin-grown cells. The hydrolysate-induced enzyme was a glycoprotein containing 6% carbohydrates. Alkaline NaB³H₄ treatment of the protein generated labeled protein and labeled carbohydrates. Analyses of the hydrolysates of these labeled products showed that alanine, α-aminobutyrate, phenylalanine, and tyrosine in the protein were labeled strongly suggesting that serine, threonine, β-hydroxyphenylalanine, and β-hydroxytyrosine were involved in O-glycosidic linkages in this protein. The protein hydrolysate also contained labeled gulonic acid, suggesting that d-glucuronic acid was attached to the protein via a base stable linkage, presumably an amide linkage at the N-terminus. The labeled reduced carbohydrates were identified by ion-exchange, thin-layer, gas-liquid, and high-performance liquid chromatographic techniques as mannitol, arabitol, gulonic acid, and 2-aminosorbitol. Thus mannose, arabinose, glucuronic acid, and glucosamine (possibly N-acetylated) were attached O-glycosidically to the hydroxyamino acids. Induction of cutinase by cutin hydrolysate in the presence of tritiated phenylalanine gave labeled cutinase. Cleavage of the O-glycosidically attached carbohydrates by anhydrous HF, followed by enzymatic hydrolysis of the labeled protein, gave rise to labeled amino acids, which upon analysis with an amino acid analyzer revealed four radioactive components. Two of them were identified as phenylalanine and tyrosine, while the other two cochromatographed with authentic β-hydroxyphenylalanine and β-hydroxytyrosine not only by the amino acid analyzer but also upon thin-layer chromatography. These results constitute the first direct evidence for the presence of the novel β-hydroxyaromatic amino acids in a protein.     

Use of reversed-phase high-performance liquid chromatography for simultaneous determination of glutamine synthetase and glutamic acid decarboxylase in crude extracts

Glutamine and γ-aminobutyric acid (GABA), formed from glutamic acid in crude tissue extracts by glutamine synthetase and glutamic acid decarboxylase respectively, were separated by derivatization with dansyl chloride followed by reversed-phase high-performance liquid chromatography on an Altex Ultrasphere ODS-5 column. The mobile phase was a gradient of 100 mM potassium dihydrogen phosphate (pH 2.1) with 0–40% acetonitrile. The amounts of glutamine and GABA formed from glutamic acid were determined under different reaction conditions.     

Isolation of individual amino acids from various microbiological sources using reversed-phase high-performance liquid chromatography

A new method for the preparative isolation of individual amino acids on a milligram scale based on reversed-phase high-performance liquid chromatography (RP-HPLC) after pre-column derivatization with carbobenzoxychloride (ZCl) has been developed. The chromatographic procedure was tested by the investigation of jack bean urease hydrolysate. The method has been applied to the preparative separation of Z-amino acids (from 10 up to 16) obtained from protein hydrolysates of various sources (green microalgae, blue-green algae, halophilic and methylotrophic microorganisms) and was proved to be reliable by the separation of deuterated amino acids (enrichment 97–99%) from Methylobacillus flagellatum (due to the bioconversion of CD₃OD and D₂O). Independent of the biological source of the protein, the amino acids were isolated with high recovery (from 68% up to 89%) and chromatographic purity (from 96% up to 99%). The method was also applied for the isolation of phenylalanine and leucine excreted by amino-acid overproducing microorganisms.     

The protein sequence homology of γ-crystallins among major vertebrate classes and their DNA sequence homology to heat-shock protein genes

A systematic characterization of lens crystallins from five major classes of vertebrates was carried out by exclusion gel filtration, cation-exchange chromatography and N-terminal sequence determination. All crystallin fractions except that of γ-crystallin were found to be N-terminally blocked. γ-Crystallin is present in major classes of vertebrates except the bird, showing none, or decreased amounts, of this protein in chicken and duck lenses, respectively. N-Terminal sequence analysis of the purified γ-crystallin polypeptides showed extensive homology between different classes of vertebrates, supporting the close relatedness of this family of crystallin even from the evolutionarily distant species. Comparison of nucleotide sequences and their predicted amino acid sequences between γ-crystallins of carp and rat lenses and heat-shock proteins demonstrated partial sequence homology of the encoded polypeptides and striking homology at the gene level. The unexpected strong homology of complementary DNA (cDNA) lies in the regions coding for 40 N-terminal residues of carp γ-II, rat γ2-1, and the middle segments of 23,000- and 70,000-M _r heat-shock proteins. The optimal alignment of DNA sequences along these two segments shows about 50% homology. The percentage of protein sequence identity for the corresponding aligned segments is only 20%. The weak sequence homology at the protein level is also found between the invertebrate squid crystallin and rat γ-crystallin polypeptides. These results pointed to the possibility of unifying three major classes of vertebrate crystallins into one α/β/γ superfamily and corroborated the previous supposition that the existing crystallins in the animal kingdom are probably mutually interrelated, sharing a common ancestry.     

Proline biosynthesis in Escherichia coli kinetic and mechanistic properties of glutamate semialdehyde dehydrogenase

The kinetics of the NADP⁺- and phosphate-dependent oxidation of glutamic acid 5-semialdehyde are consistent with a rapid-equilibrium random order mechanism. The K_m for dl-pyrroline-5-carboxylic acid is 2.5 mM, for NADP⁺ is 0.05 mM and for phosphate is 0.35 mM. TheV_max is approx. 8.0 units per mg protein. The reaction is highly specific for the dl-pyrroline-5-carboxylic acid and NADP⁺, but a number of divalent anions can substitute for phosphate. NADPH is competitive with respect to all three substrates and an analog of γ-glutamyl phosphate, 3-(phosphonoacetylamido)-l-alanine, is competitive with respect to dl-pyrroline-5-carboxylic acid and non-competitive with respect to NADP⁺ and phosphate, suggesting dead-end complex formation.