Characterization of human angiotensinogen

In this study of the physical and chemical properties of human angiotensinogen were determined. Human angiotensinogen is a glycoprotein containing 14% carbohydrate. The molecular weight as determined by sedimentation equilibrium studies was 56,800. A higher molecular weight was obtained on sodium dodecyl sulfate electrophoresis. Ferguson-type plots indicated that angiotensinogen is another glycoprotein which behaves anomalously on sodium dodecyl sulfate electrophoresis. The COOH-terminal amino acid was found to be serine while two NH2-terminal amino acids, alanine and aspartic acid (or asparagine), were detected. The specific angiotensin I content of angiotensinogen preparations can vary considerably with no effect on the apparent homogeneity of the isolated protein. A protein with negligible angiotensin I content has been obtained from a preparation of human angiotensinogen. The COOH-terminal amino acid of this protein was serine while the only NH2-terminal amino acid detected was alanine.     

Conformational changes expected in endogenous opioid peptides upon their interaction with acidic lipids

The amino terminal amino acid sequence of purified human angiotensinogen has been determined. The first 25 residues with the exception of number 14 were identified. The sequence of the first ten amino acids is that of angiotensin I. The sequence surrounding the renin cleavage site in this protein is Leu-¹⁰Val-¹¹Ile-¹²His-¹³. Thus, human renin must cleave a Leu-Val peptide bond in human angiotensinogen to release angiotensin I, rather than a Leu-Leu bond as reported for other species. The differences between the sequence of human angiotensinogen and that previously reported for a tetradecapeptide derived from equine and porcine angiotensinogen may be responsible for the known species specificity of renin.     

Expression of human angiotensinogen cDNA in Escherichia coli

Human angiotensinogen cDNA clones were isolated from a human liver library. Nucleotide sequence analysis of these cDNA clones revealed that position 1075 in the messenger RNA, which is part of a PstI recognition sequence, is different from the published sequence (Kageyama, R., Ohkubo, H., and Nakanishi, S. (1984) Biochemistry 23, 3603-3609). This change results in an altered amino acid at this position in the corresponding protein sequence and suggests possible restriction fragment length polymorphism. The full length human angiotensinogen cDNA was constructed from partial cDNA clones and ligated into an isopropyl-1-thio-beta-D-galactopyranoside inducible bacterial expression vector pUC9 to develop expression plasmid pUCHAG27. This plasmid permitted the synthesis of human angiotensinogen in Escherichia coli. The recombinant bacteria overproduced a 53-kDa protein which was recognized by anti-human angiotensinogen antibodies. The synthesis of this protein was greatly increased upon induction with isopropyl-1-thio-beta-D-galactopyranoside. The chimeric protein, almost identical to human angiotensinogen, was partially purified by ammonium sulfate fractionation and gel filtration on Sephadex G-100. Human kidney renin was shown to enzymatically cleave this recombinant protein to produce des-(angiotensin I)-angiotensinogen and a small polypeptide. Thus, we provide evidence that recombinant human angiotensinogen synthesized through E. coli is biologically active and serves as a substrate for human renin.     

Chemical characterization of angiotensin I in porcine follicular fluid

In the search for novel neuropeptides in porcine follicular fluid (pff) using smooth muscle contractile activity as a response parameter, a substance with a marked activity was isolated in a pure form. By amino acid analysis and sequential study, this substance has been chemically revealed to be angiotensin I. A much smaller amount of additional activity was isolated and found to be angiotensin II, as determined by radioimmunoassay. Radioimmunoassays for angiotensin I and II confirmed that the amount of angiotensin I determined was much greater than that of angiotensin II. A comparative study of the extractions, however, indicated a large amount of angiotensin I had been generated from angiotensinogen by endogenous renin in the follicular fluid which could be activated during extraction and ultrafiltration at a low pH. These findings are consistent with the previous reports that described a high concentration of prorenin in the follicular fluid, acid activation of prorenin to renin and the subsequent generation of angiotensin I from endogenous angiotensinogen.     

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.     

Purification, amino acid sequence and immunological characterization of Ole e 6, a cysteine-enriched allergen from olive tree pollen

The Ole e 6 allergen from olive tree pollen has been isolated by combining gel permeation and reverse-phase chromatographies. It is a single and highly acidic (pI 4.2) polypeptide chain protein. Its NH₂-terminal amino acid sequence has been determined by Edman degradation. Total RNA from the olive tree pollen was isolated, and a specific cDNA was amplified by the polymerase chain reaction using a degenerate oligonucleotide primer designed according to the NH₂-terminal sequence of the protein. The nucleotide sequencing of the cDNA rendered an open reading frame encoding a 50 amino acid polypeptide chain, in which two sets of the sequential motif Cys-X₃-Cys-X₃-Cys are present. No sequence similarity has been found between this protein and other previously described polypeptides.     

呼吸道合胞病毒B亚型分离株的G蛋白基因分析

对一株长春地区B亚型分离株(CC169)的G蛋白基因进行了 序列分析,结果表明：我国呼吸道合胞病毒(RSV)分离株CC169同RSV B亚型原型株CH18537的 核苷酸同源性为94%,核苷酸的有义突变率达65%。由核苷酸推导出氨基酸序列的同源性为894%,氨基酸的变异全部发生在胞外区,并主要集中在一个高度保守区的两端,胞内区和跨 膜区保守不变。氨基酸的变异导致了分离株既有糖基化位点的改变,又有蛋白长度的变异。 此外还初步探讨了我国RSV B 亚型分离株CC169的G蛋白基因同原型株之间的变异与疫苗研制 中的意义。     

The primary structure of bovine cellular retinoic acid-binding protein

The complete amino acid sequence of bovine adrenal gland cellular retinoic acid-binding protein (CRABP) has been determined. The primary structure was established by analyses of cyanogen bromide fragments and peptides obtained by trypsin and Staphylococcus aureus protease digestions. The polypeptide chain of bovine CRABP comprises 136 amino acid residues. From partial sequence information, CRABP has been shown to be homologous to cellular retinol-binding protein, myelin protein P2, and the fatty acid-binding Z-protein. A comparison of the complete amino acid sequences of the members of this protein family, which also includes the rat intestinal fatty acid-binding protein, shows that CRABP is more similar to cellular retinol-binding protein and protein P2 than to the fatty acid-binding proteins. All five proteins are very similar in their NH2-terminal regions, suggesting that this part is important for a property common to the members of this protein family. This is the first report of a complete amino acid sequence of a CRABP.     

Die Proteinstruktur des RNS-Bakteriophagen fr

Summary The coat protein of the RNA containing bacteriophage f_r has been hydrolyzed and its amino acid composition determined (Table 1). Furthermore, the protein was split with trypsin and the tryptic peptides were separated by column chromatography on Dowex 1 (Figure 1) and purified by paper chromatography and electrophoresis.The amino acid composition of all but one tryptic peptide are given in Table 2. The large peptide T₁₃ which is much more difficult to purify than all other peptides, was isolated by several methods. Its amino acid composition is shown in Table 3. All tryptic peptides are compiled in Table 4.Amino acid sequences have been fully or partially determined for 9 tryptic peptides (Table 5) and the others are presently being investigated.These findings are compared with the results from other RNA phages, especially f₂. It is concluded from the available data that the relationship between the coat proteins of the RNA phages is similar to that between the various naturally occurring strains of tobacco mosaic virus whose amino acid sequences are known.

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Herrn Prof.G. Melchers zum 60. Geburtstag gewidmet.     

Computer graphics modelling and the specificity of renins

Mouse submaxillary, mouse kidney and human renins have been modelled using the three-dimensional structure of the homologous endothiapepsin, which has been defined by high-resolution X-ray analysis, and the amino acid sequences derived from protein, complementary DNA or gene sequencing. These computer graphics studies have shown that all renins can adopt three-dimensional structures similar to those of other aspartic proteinases with small insertions and deletions at the surface and often at beta-turns. The catalytically essential aspartates lie at the centre of a deep and extended cleft, and differences in the subsites, especially at S3' have been correlated with the known specificities of the renins. The models have shown that various residues on the surfaces of the renins adjacent to the active site cleft may play a part in recognizing and binding angiotensinogen. Reasons for a neutral pH optima have also been suggested.     

Phosphoenolpyruvate-dependent protein kinase enzyme I of Streptococcus faecalis: purification and properties of the enzyme and characterization of its active center

Enzyme I, the phosphoenolpyruvate:protein phosphotransferase (EC 2.7.3.9), which is part of the bacterial phosphoenolpyruvate- (PEP) dependent phosphotransferase system, has been purified from Streptococcus faecalis by using a large-scale preparation. Size exclusion chromatography revealed a molecular weight of 140 000. On sodium dodecyl sulfate gels, enzyme I gave one band with a molecular weight of 70 000, indicating that enzyme I consists of two identical subunits. The first 59 amino acids of the amino-terminal part of the protein have been sequenced. It showed some similarities with enzyme I of Salmonella typhimurium. The active center of enzyme I has also been determined. After phosphorylation with [32P]PEP, the enzyme was cleaved by using different proteases. Labeled peptides were isolated by high-performance liquid chromatography on a reversed-phase column. The amino acid composition or amino acid sequence of the peptides has been determined. The largest labeled peptide was obtained with Lys-C protease and had the following sequence: -Ala-Phe-Val-Thr-Asp-Ile-Gly- Gly-Arg-Thr-Ser-His*-Ser-Ala-Ile-Met-Ala-Arg-Ser-Leu-Glu-Ile-Pro-Ala- Ile-Val-Gly-Thr-Lys-. It has previously been shown that the phosphoryl group is bound to the N-3 position of a histidyl residue in phosphorylated enzyme I. The single His in position 12 of the above peptide must therefore carry the phosphoryl group.     

Rule of antibody structure. Primary structure of a human monoclonal IgAl-immunoglobulin (myeloma protein Tro). VI. Amino acid sequence of the L-chain, lambda-type, subgroup II]

The primary structure of the L-chain of an IgA1-immunoglobulin (Myeloma protein Tro) has been determined by means of cleavage with trypsin and, if necessary, with alpha-chymotrypsin. The tryptic peptides of the variable part were characterized by amino acid analysis, Dansyl-Edman degradation and cleavage with carboxypeptidase; the peptides of the constant part were identified by amino acid analyses and determination of its N- and C-terminal residues. The sequence of the remaining amino acids and the arrangement of the peptides were established in homology to known structures. The protein comprises 216 amino acids. The homology of the variable part clearly characterizes it as belonging to subgroup II of lambda-chains. In positions 27a, b and c, there are the subgroup-specific additional residues and in position 96 is the characteristic deletion. The constant part of the chain is Kern- and Oz- which indicates that it has serine in position 154 and arginine in position 191.     

The structure of rice storage protein glutelin precursor deduced from cDNA

The complete amino acid sequence of rice storage protein glutelin was determined by the sequencing of the corresponding cDNA. The deduced glutelin precursor has a 37 amino acid signal peptide sequence at the NH₂ terminus, which is followed by a 269 amino acid acidic subunit (M_r = 32 489) and a 193 amino acid basic subunit (M_r = 19 587). The glutelin precursor sequence is homologous to those of pea legumin and soybean glycinin.     

Purification and characterization of metallothionein from liver of cadmium exposed Rhesus monkeys (Macaca mulatta)

Metallothionein (MT) a low molecular weight, Cd-binding, cysteine rich, cytosolic protein has been isolated, purified and characterized from cadmium exposed Rhesus monkeys maintained on protein calorie malnourished (PCM) diet. Metallothionein was resolved into three isoforms i.e. MT_a, MT_b and MTV_c. The ratio of Cd, Zn and Cu varied in these isometallothioneins. MT_c was the major isometallothionein. UV Spectra of MT_c revealed the presence of mercaptide bonds and absence of aromatic amino acids. These observations were further confirmed by amino acid analysis of MT_c which demonstrated high cysteine content (22.6) followed by serine, glycine and lysine. The molecular weight of MT_c as determined by gel filtration and amino acid analysis was 13000 and 6398 daltons respectively. This demonstrates that MT_c is a non-globular ellipsoid polypeptide. MT_c showed a unique property of binding selenium. Monkey liver metallothionein was immunologically identical with human metallothionein. All the characteristics of MT_c obtained in the present study reveal a similarity between monkey and human metallothionein probably due to closer phylogenetic relationship between the two species.     

The flow of a viscous liquid in a converging tube

The problem of the viscous flow of an incompressible Newtonian liquid in a converging tapered tube has been solved in spherical polar coordinates. The method of the solution involves the Stokes' stream function and a technique introduced by Stokes in the study of a sphere oscillating in a fluid. The theory for the flow in a rigid tube includes: (1) the pulsatile flow with both radial and angular velocity components; (2) the steady state flow with both radial and angular velocity components and (3) the very slow steady state flow with only a radial velocity component present. For a tapered elastic tube, the velocity of the propagated pulse wave is determined. The solution given is in terms of the elastic constants of the system and the coordinates for this type of geometry. The pulse velocity is then related to the velocity in an elastic cylindrical tube with the necessary correction terms to account for the tapered tube. Supported in part by the American Heart Association (No. 62F4EG). This work was done during the tenure of an Established Investigatorship of the American Heart Association.     

gamma-Carboxyglutamic acid in invertebrates: its identification in hermatypic corals

The presence of a γ-carboxyglutamate-containing protein in hermatypic corals has been established. γ-Carboxyglutamate has been isolated from the alkaline hydrolysate of protein extracted from the coral Lobophyllia corymbosa, by chromatography of the hydrolysate on Dowex AG 1-X8 (formate form), followed by chromatography on an amino acid analyzer column. This procedure achieves complete separation of γ-carboxyglutamate from an acid-stable compound which is also present in the alkaline hydrolysate of coral protein, and which has proved difficult to separate from γ-carboxyglutamate by other methods. The identity of the γ-carboxyglutamate thus isolated has been established unequivocally by determining the yield of glutamic acid after acid treatment (2 M HCl, 6 h, 110°C). The color factor for the γ-carboxyglutamate isolated from Lobophyllia is 0.45₄ times the value for glutamic acid, in good agreement with the value determined for authentic γ-carboxyglutamic acid (0.45₈) under exactly the same conditions. Virtually identical results have been obtained for the coral, Acropora cuneata. These experiments provide the first secure evidence for the presence of γ-carboxyglutamate in an invertebrate species, and they clearly have important implications for our understanding of invertebrate mineralization.     

Specific labeling of the thyroxine binding site in thyroxine-binding globulin: determination of the amino acid composition of a labeled peptide fragment isolated from a proteolytic digest of the derivatized protein

[125I] Thyroxine has been covalently bound to the thyroxine binding site in thyroxine-binding globulin by reaction with the bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. An average of 0.47 mol of [125I] thyroxine was incorporated per mol protein; nonspecific binding amounted to 8%. A labeled peptide fragment was isolated from a proteolytic digest of the derivatized protein by HPLC and its amino acid composition was determined. Comparison with the amino acid sequence of thyroxine-binding globulin indicated partial correspondence of the labeled peptide with two possible regions in the protein. These regions also coincide with part of the barrel structure present in the closely homologous protein, alpha 1-antitrypsin.     

Porcine liver low Mr phosphotyrosine protein phosphatase: The amino acid sequence

Porcine low M_r phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH₂-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH₂-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low Mr PTPases. This PTPase is strongly inhibited by pyridoxal 5′-phosphate (K=21ΜM) like the low Mr PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40–73 sequence with the corresponding sequence of other low Mr PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low M_r PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5?-phosphate inhibition has been proposed.     

Tetrahymena HMG nonhistone chromosomal protein. Isolation and amino acid sequence lacking the N- and C-terminal domains of vertebrate HMG 1

The complete amino acid sequence of a high mobility group (HMG) nonhistone chromosomal protein of the ciliated protozoan Tetrahymena pyriformis (GL strain) was determined. This protein was extracted with 0.5 M HClO4 together with histone H1 (molar ratio 1:1) from the whole histone extract, then purified by gel filtration and reverse-phase HPLC. The HMG protein showed a single electrophoretic band on SDS gel electrophoresis. The amino acid sequence was determined by Edman degradation of intact protein, BrCN fragments, and their staphylococcal protease and tryptic peptides. Thus the total sequence, consisting of 99 amino acid residues and having a molecular weight of 11,626, was completely determined. Phosphorus analysis of the tryptic peptides, containing serine or threonine, showed that this HMG protein was phosphorylated at two positions, each 6-7%, and contained 0.15 mol phosphate/mol protein. This Tetrahymena HMG is rather similar to the central part of vertebrate HMG 1 in terms of the amino acid sequence and the hydropathy profile.     

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.