The Amino Acid Sequence of Glucagon. IV. The Hydrolysis of Glucagon with Subtilisin

Extracellular exo-inulinase has been isolated from a solid-phase culture of the filamentous fungus Aspergillus awamori var. 2250. The apparent molecular mass of the monomer enzyme was 69±1kDa, with a pI of 4.4 and a pH optimum of 4.5. The enzyme hydrolysed the β-(2 → 1)-fructan (inulin) and β-(2 → 6)-fructan (levan) via exo-cleavage, releasing fructose. The values for the Michaelis constants Km and Vmax in the hydrolysis of inulin were 0.003±0.0001mM and 175±5μmol·min−1·mg−1. The same parameters in the hydrolysis of levan were 2.08±0.04mg/ml and 1.2±0.02μmol/min per mg, respectively. The gene and cDNA encoding the A. awamori exo-inulinase were cloned and sequenced. The amino acid sequence indicated that the protein belongs to glycoside hydrolase family 32. A surprisingly high similarity was found to fructosyltransferase from Aspergillus foetidus (90.7% on the level of the amino acid sequence), despite the fact that the latter enzyme is unable to hydrolyse inulin and levan. Crystals of the native exo-inulinase were obtained and found to belong to the orthorhombic space group P212121 with cell parameters a = 64.726 Å (1Å = 0.1 nm), b = 82.041 Å and c = 136.075 Å. Crystals diffracted beyond 1.54 Å, and useful X-ray data were collected to a resolution of 1.73 Å.

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Die Aminosäuresequenz des Asparaginsäure enthaltenden Mureins von Lactobacillus coryniformis und Lactobacillus cellobiosus

Zeitschrift für Naturforschung B ◽

10.1515/znb-1967-1015 ◽

1967 ◽

Vol 22 (10) ◽

pp. 1062-1067 ◽

Cited By ~ 10

Author(s):

Roland Plapp ◽

Otto Kandler

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Aspartic Acid ◽

Cell Walls ◽

Peptide Bond ◽

Partial Hydrolysis ◽

Cross Linking ◽

Peptide Moiety ◽

Lactobacillus Coryniformis ◽

Hydrolysis Of

The amino acid sequence of the peptide moiety of the mureins of Lactobacillus coryniformis and Lactobacillus cellobiosus cell walls was determined. This was accomplished by the identification of peptides obtained after partial hydrolysis of purified cell walls and by the identification of UDP-activated murein precursors accumulated by ᴅ-cycloserine inhibition. The amino acid sequence proved to be : ʟ-ala-ᴅ-glu-ʟ-lys-ᴅ-ala for L. coryniformis and L-ala-D-glu-L-orn-D-ala for L. cellobio-.D-asp D-aspsus. Aspartic acid is involved in the cross-linking of the mureins by forming a peptide bond with the C-terminal D-alanine of an adjacent muropeptide. Glutamic acid as well as aspartic acid are present as amides.

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Partial amino acid sequence and mRNA analysis of cytosolic pyridoxine-beta-d-glucoside hydrolase from porcine intestinal mucosa: proposed derivation from the lactase-phlorizin hydrolase gene

Biochemical Journal ◽

10.1042/bj20031416 ◽

2004 ◽

Vol 380 (1) ◽

pp. 211-218 ◽

Cited By ~ 4

Author(s):

Chi-Wah TSEUNG ◽

Laura G. McMAHON ◽

Jorge VÁZQUEZ ◽

Jan POHL ◽

Jesse F. GREGORY

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Northern Blot ◽

Sequence Data ◽

Cdna Probe ◽

Protein Mass ◽

Sds Page ◽

Mrna Analysis ◽

Hydrolysis Of

We have previously identified and purified a novel β-glucosidase, designated PNGH (pyridoxine-5´-β-d-glucoside hydrolase), from the cytosolic fraction of pig intestinal mucosal. PNGH catalyses the hydrolysis of PNG (pyridoxine-5´-β-d-glucoside), a plant derivative of vitamin B6 that exhibits partial nutritional bioavailability in humans and animals. Preliminary amino acid sequence analysis indicated regions of close similarity of PNGH to the precursor form of LPH (lactase–phlorizin hydrolase), the β-glucosidase localized to the brush-border membrane. We report in the present study amino acid sequence data for PNGH and results of Northern blot analyses, upon which we propose a common genomic origin of PNGH and LPH. Internal Edman sequencing of the PNGH band isolated by SDS/PAGE yielded data for 16 peptides, averaging 10.8 amino acids in length. These peptides from PNGH (approx. 140 kDa) were highly similar to sequences existing over most of the length of the >200 kDa precursor of rabbit LPH; however, we found no PNGH sequences that corresponded to approx. 350 amino acids between positions 463 and 812 of the LPH precursor, a region encoded by exon 7 of the LPH precursor gene (amino acids 568–784), and no sequences that corresponded to regions near the N-terminus. MS analysis of tryptic peptides yielded 25 peptides, averaging 15 amino acids, with masses that matched segments of the rabbit LPH precursor. Northern blot analysis of pig and human small intestinal polyadenylated mRNA using a non-specific LPH cDNA probe showed an expected approx. 6 kb transcript of the LPH precursor, but also an approx. 4 kb transcript that was consistent with the size predicted from the PNGH protein mass. Using a probe specific to the region encoded by exon 7, hybridization occurred only with the 6 kb transcript. Based on these observations, we propose that both PNGH and LPH enzymes have the same genomic origin, but differ in transcriptional and, possibly, post-translational processing.

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Sorting out theLeMANs: endo-β-mannanase genes and their encoded proteins in tomato

Seed Science Research ◽

10.1017/s0960258507786233 ◽

2007 ◽

Vol 17 (3) ◽

pp. 143-154 ◽

Cited By ~ 12

Author(s):

Xuemei Gong ◽

J. Derek Bewley

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Gibberellic Acid ◽

Cell Walls ◽

Plant Cell Walls ◽

Tomato Seeds ◽

C Terminus ◽

Inactive Form ◽

Genes Encoding ◽

Hydrolysis Of

AbstractEndo-β-mannanase (EC 3.2.1.78) is involved in the hydrolysis of mannan-type polysaccharides that are present in plant cell walls, especially those of the seed endosperm. The genes encoding the endo-β-mannanases have been studied extensively in tomato (Solanum lycopersicum), and five genes (LeMAN1,LeMAN2,LeMAN3,LeMAN4andLeMAN5) and/or their products have been isolated and characterized.LeMAN1,LeMAN2andLeMAN3are expressed in tomato seeds,LeMAN4in the fruit andLeMAN5in the flower.LeMAN5andLeMAN2are now considered to be the same gene, and the former is re-designated asLeMAN2*. Transcripts ofLeMANs1, 2and3are detected only in the endosperm of tomato seeds, and their synthesis is promoted by gibberellic acid.LeMAN4, in the fruit, occurs asLeMAN4aandLeMAN4igenes that encode an active or inactive form of endo-β-mannanase, respectively.LeMAN1–4 enzymes encoded by these genes share 80% similarity in amino acid sequence. In tomato, the leucine amino acid present near to the C-terminus of the endo-β-mannanase is the most important for achieving full activity of the enzyme.

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The Amino Acid Sequence of Glucagon. II. The Hydrolysis of Glucagon with Chymotrypsin

Journal of the American Chemical Society ◽

10.1021/ja01568a035 ◽

1957 ◽

Vol 79 (11) ◽

pp. 2798-2801 ◽

Cited By ~ 26

Author(s):

W. W. Bromer ◽

L. G. Sinn ◽

Otto K. Behrens

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Hydrolysis Of

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The Amino Acid Sequence of Glucagon. III. The Hydrolysis of Glucagon by Trypsin

Journal of the American Chemical Society ◽

10.1021/ja01568a036 ◽

1957 ◽

Vol 79 (11) ◽

pp. 2801-2805 ◽

Cited By ~ 41

Author(s):

W. W. Bromer ◽

A. Staub ◽

L. G. Sinn ◽

Otto K. Behrens

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Hydrolysis Of

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IMMUNOLOGICAL STUDIES ON PARATHYROID HORMONE: CHARACTERIZATION OF ANTISERA AGAINST SYNTHETIC 1–34 HUMAN PARATHYROID HORMONE AND EVIDENCE THAT POSITION 30 IN HUMAN PARATHYROID HORMONE IS ASPARTIC ACID

Journal of Endocrinology ◽

10.1677/joe.0.0740461 ◽

1977 ◽

Vol 74 (3) ◽

pp. 461-466 ◽

Cited By ~ 2

Author(s):

T. J. VISSER ◽

C. J. BUURMAN ◽

J. C. BIRKENHÄGER

Keyword(s):

Parathyroid Hormone ◽

Amino Acid ◽

Amino Acid Sequence ◽

Aspartic Acid ◽

Cross Reactivity ◽

Partial Hydrolysis ◽

Human Parathyroid Hormone ◽

Aspartic Acid Residue ◽

Hydrolysis Of

SUMMARY Radioimmunoassays for the measurement of the 1–34 human parathyroid hormone fragment (1–34 hPTH) were developed using antisera raised in rabbits against synthetic 1–34 hPTH-N (amino acid sequence proposed by Niall). Binding of 125I-labelled 1–34 hPTH-N to these antisera was optimal at pH 5·5. Limits of detection varied between 25 and 200 pg/ml. Cross-reactivity of 1–34 bovine PTH was substantial in all assays; 1–34 hPTH-B (structure proposed by Brewer), 1–84 hPTH and 1–29 hPTH cross-reacted only with antisera from one animal. 1–29 Human PTH was obtained from partial hydrolysis of both 1–84 hPTH and 1–34 hPTH-N. Production of 1–29 hPTH from 1–84 hPTH was demonstrated by comparison of the elution profiles of the reaction product and 1–29 bovine PTH on Sephadex G-50. Thus, evidence was obtained that position 30 in native hPTH is occupied by an aspartic acid residue.

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Cold-Active Serine Alkaline Protease from the Psychrotrophic Bacterium Shewanella Strain Ac10: Gene Cloning and Enzyme Purification and Characterization

Applied and Environmental Microbiology ◽

10.1128/aem.65.2.611-617.1999 ◽

1999 ◽

Vol 65 (2) ◽

pp. 611-617 ◽

Cited By ~ 73

Author(s):

Ljudmila Kulakova ◽

Andrey Galkin ◽

Tatsuo Kurihara ◽

Tohru Yoshimura ◽

Nobuyoshi Esaki

Keyword(s):

Activation Energy ◽

Molecular Weight ◽

Amino Acid ◽

Amino Acid Sequence ◽

Alkaline Protease ◽

Catalytic Triad ◽

Psychrotrophic Bacterium ◽

Highly Active ◽

Serine Alkaline Protease ◽

Hydrolysis Of

ABSTRACT The gene encoding serine alkaline protease (SapSh) of the psychrotrophic bacterium Shewanella strain Ac10 was cloned in Escherichia coli. The amino acid sequence deduced from the 2,442-bp nucleotide sequence revealed that the protein was 814 amino acids long and had an estimated molecular weight of 85,113. SapSh exhibited sequence similarities with members of the subtilisin family of proteases, and there was a high level of conservation in the regions around a putative catalytic triad consisting of Asp-30, His-65, and Ser-369. The amino acid sequence contained the following regions which were assigned on the basis of homology to previously described sequences: a signal peptide (26 residues), a propeptide (117 residues), and an extension up to the C terminus (about 250 residues). Another feature of SapSh is the fact that the space between His-65 and Ser-369 is approximately 150 residues longer than the corresponding spaces in other proteases belonging to the subtilisin family. SapSh was purified to homogeneity from the culture supernatant of E. colirecombinant cells by affinity chromatography with a bacitracin-Sepharose column. The recombinant SapSh (rSapSh) was found to have a molecular weight of about 44,000 and to be highly active in the alkaline region (optimum pH, around 9.0) when azocasein and synthetic peptides were used as substrates. rSapSh was characterized by its high levels of activity at low temperatures; it was five times more active than subtilisin Carlsberg at temperatures ranging from 5 to 15°C. The activation energy for hydrolysis of azocasein by rSapSh was much lower than the activation energy for hydrolysis of azocasein by the subtilisin. However, rSapSh was far less stable than the subtilisin.

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The staining pattern of the tropomyosin sequence is displayed in a new paracrystal

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142372 ◽

1986 ◽

Vol 44 ◽

pp. 150-151

Author(s):

M.K. Lamvik ◽

L.L. Klatt

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Staining Pattern ◽

Banding Patterns ◽

Mass Thickness ◽

New Form

Tropomyosin paracrystals have been used extensively as test specimens and magnification standards due to their clear periodic banding patterns. The paracrystal type discovered by Ohtsuki1 has been of particular interest as a test of unstained specimens because of alternating bands that differ by 50% in mass thickness. While producing specimens of this type, we came across a new paracrystal form. Since this new form displays aligned tropomyosin molecules without the overlaps that are characteristic of the Ohtsuki-type paracrystal, it presents a staining pattern that corresponds to the amino acid sequence of the molecule.

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Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646610 ◽

1989 ◽

Vol 61 (03) ◽

pp. 437-441 ◽

Cited By ~ 28

Author(s):

Cindra Condra ◽

Elka Nutt ◽

Christopher J Petroski ◽

Ellen Simpson ◽

P A Friedman ◽

...

Keyword(s):

Molecular Weight ◽

Salivary Gland ◽

Amino Acid ◽

Amino Acid Sequence ◽

Western Blot ◽

Potent Inhibitor ◽

Factor Xa ◽

Coagulation Factor ◽

Sds Page ◽

Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

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