scholarly journals Evolution of phosphagen kinase1: Isolation, characterization and cDNA-derived amino acid sequence of two-domain arginine kinase from the sea anemone Anthopleura japonicus

1997 ◽  
Vol 328 (1) ◽  
pp. 301-306 ◽  
Author(s):  
Tomohiko SUZUKI ◽  
Yoshitada KAWASAKI ◽  
Takahiro FURUKOHRI
Keyword(s):  
Creatine Kinase ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Arginine Kinase ◽  
Gel Filtration ◽  
Sea Anemone ◽  
The Body ◽  
Native Form ◽  
Significant Similarity

Arginine kinase (AK) was isolated from the body wall muscle of the primitive sea anemone Anthopleura japonicus by Ultrogel AcA34 gel filtration, DEAE-32 chromatography and elution on a Cosmogel-SP column. The denatured molecular mass as determined with SDS/PAGE was 80 kDa, twice that of the usual AK subunit, indicating that this AK has an unusual two-domain structure. The native form was eluted on a Superose 12 column with the same retention time as that of rabbit homodimeric creatine kinase, indicating that Anthopleura AK is a monomer of 80 kDa. The isolated enzyme gave a specific activity of 100-120 μmol of Pi/min per mg of protein in the pH range 7.9-9.1 for the forward reaction. The enzyme is fully activated by Ca2+, as it is with Mg2+. The cDNA-derived amino acid sequence of 715 residues of Anthopleura AK was determined. The validity of the sequence was supported by chemical sequencing of internal tryptic peptides. A bridge intron of 686 bp, which separates the two domains of Anthopleura AK, is present between the second and third nucleotide in the codon of Ala-364. This is the first two-domain AK to be sequenced. Anthopleura AK shows 48-54% amino acid sequence identity with known invertebrate AKs, and also shows a lower, but significant, similarity (39-46%) to marine worm glycocyamine kinase and rabbit creatine kinase.

scholarly journals Purification of macrophage deactivating factor.

1990 ◽  
Vol 171 (4) ◽  
pp. 1347-1361 ◽  
Author(s):  
S Srimal ◽  
C Nathan
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fluid Phase ◽  
Protein S ◽  
Peritoneal Macrophages ◽  
Reversed Phase ◽  
Tgf Beta ◽  
Sds Page

Macrophage deactivation factor (MDF) in P815 tumor cell-conditioned medium was assayed by its suppression of the ability of activated mouse peritoneal macrophages to release hydrogen peroxide. MDF displayed properties of a soluble protein(s) associated with both low (8-25,000) and high (greater than 450,000) Mr fractions. MDF was purified 6,140-fold by a seven-step procedure: extraction with acid-ethanol; precipitation with ether; and fractionation on gel filtration, anion-exchange, diphenyl reversed-phase and C4 reversed-phase HPLC columns, the last column twice. The final preparation contained two species: (a) a approximately 13,000 Mr band on reducing or nonreducing SDS-PAGE and on autoradiograms after radioiodination with chloramine T, and (b) a 66,000 Mr species ranging from approximately 5% to approximately 50% of the protein detectable by silver strain. The 66,000 Mr species was identified as albumin from its NH2-terminal amino acid sequence. However, no amino acid sequence could be obtained for the approximately 13,000 Mr species, either in fluid phase or after electroelution of the corresponding SDS-PAGE band. Thus, approximately 13,000 Mr MDF associates tightly with albumin through a variety of separation techniques, and may have a blocked NH2 terminus. Purified MDF afforded 50% inhibition of activated macrophage H2O2 releasing capacity at a concentration of 1-10 nM. Separation of MDF from most higher Mr moieties was associated with disproportionately small increases in specific activity, suggesting MDF might be partially inactivated by purification. As purified, MDF was approximately 1,000-fold less potent at deactivating macrophages than TGF-beta. Antibodies that neutralized the macrophage-deactivating effect of TGF-beta did not inhibit deactivation by MDF.

scholarly journals A cadmium-binding protein in rat liver identified as ornithine carbamoyltransferase

1988 ◽  
Vol 250 (3) ◽  
pp. 735-742 ◽  
Author(s):  
Y Aoki ◽  
H Sunaga ◽  
K T Suzuki
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Dissociation Constant ◽  
Western Blotting ◽  
Rat Liver ◽  
Specific Activity ◽  
Binding Protein ◽  
Ornithine Carbamoyltransferase ◽  
Rabbit Antibody ◽  
Native Form

A cadmium-binding protein of Mr about 40,000 (40K Cd-BPa) was detected in rat liver by Western blotting [Aoki, Kunimoto, Shibata & Suzuki (1986) Anal. Biochem. 157, 117-122]. It was characterized and identified as ornithine carbamoyltransferase (OCTase, EC 2.1.3.3) on the basis of coincidence of their physicochemical and enzymological features. The amino acid sequence of the N-terminal and those of three tryptic digests in 40K Cd-BPa were identical with those of OCTase. The Mr values of the denatured and native forms of 40K Cd-BPa (39,000 and 110,000 respectively) were the same as those of OCTase. 40K Cd-BPa showed, as OCTase activity, a specific activity of 230 mumol/min per mg of protein and Km of 0.6 mM for ornithine, this value also being essentially the same as that for OCTase. A rabbit antibody against OCTase reacted with 40K Cd-BPa. The native form of 40K Cd-BPa bound to 0.8 molar equiv, of cadmium, with a dissociation constant of 7.6 x 10(-6) M.

scholarly journals Arginine kinase evolved twice: evidence that echinoderm arginine kinase originated from creatine kinase

1999 ◽  
Vol 340 (3) ◽  
pp. 671-675 ◽  
Author(s):  
Tomohiko SUZUKI ◽  
Mika KAMIDOCHI ◽  
Naho INOUE ◽  
Hozumi KAWAMICHI ◽  
Yoichi YAZAWA ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Early Stage ◽  
Arginine Kinase ◽  
Phosphagen Kinase ◽  
Flexible Loop ◽  
Loop Region ◽  
Kinase Family ◽  
Phosphagen Kinases

Arginine kinase (AK) was isolated from the longitudinal muscle of the sea cucumber Stichopusjaponicus. Unlike the monomeric 40 kDa AKs from molluscs and arthropods, but like the cytoplasmic isoenzymes of vertebrate creatine kinase (CK), the Stichopus enzyme was dimeric. To explore the evolutionary origin of the dimeric AK, we determined its cDNA-derived amino acid sequence of 370 residues. A comparison of the sequence with those of other enzymes belonging to the phosphagen kinase family indicated that the entire amino acid sequence of Stichopus AK is apparently much more similar to vertebrate CKs than to all other AKs. A phylogenetic tree also strongly suggests that the Stichopus AK has evolved from CK. These results support the conclusion that AK evolved at least twice during the evolution of phosphagen kinases: first at an early stage of phosphagen kinase evolution (its descendants are molluscan and arthropod AKs) and secondly from CK later in metazoan evolution. A comparison of the amino acid sequence around the guanidino specificity (GS) region (which is a possible candidate for the guanidine substrate recognition site in the phosphagen kinase family) of the Stichopus enzyme with those of other phosphagen kinases showed that the GS region of the Stichopus enzyme was of the AK type: five amino acid deletions in the flexible loop region that might help to accommodate larger guanidine substrates in the active site. The presence of the AK-type deletions in the Stichopus AK, even though it seems that the enzyme's most immediate ancestor was probably CK, strongly suggests that the GS region has a role in substrate specificity. Stichopus AK and presumably other echinoderm AKs seem to have evolved from the CK gene; the sequence of GS region might have been replaced by the AK type via exon shuffling. The presence of an intron near the GS region in the Stichopus AK gene supports this hypothesis.

scholarly journals Complete cDNA-derived amino acid sequence of chick muscle creatine kinase.

1984 ◽  
Vol 259 (24) ◽  
pp. 15224-15227
Author(s):  
C P Ordahl ◽  
G L Evans ◽  
T A Cooper ◽  
G Kunz ◽  
J C Perriard
Keyword(s):  
Creatine Kinase ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Muscle Creatine Kinase ◽  
Muscle Creatine

scholarly journals Streptococcus salivarius Fimbriae Are Composed of a Glycoprotein Containing a Repeated Motif Assembled into a Filamentous Nondissociable Structure

2001 ◽  
Vol 183 (9) ◽  
pp. 2724-2732 ◽  
Author(s):  
Céline Lévesque ◽  
Christian Vadeboncoeur ◽  
Fatiha Chandad ◽  
Michel Frenette
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cell Surface ◽  
Polyclonal Antibodies ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Spontaneous Mutant ◽  
Streptococcus Salivarius ◽  
Streptococcal Species ◽  
Repeated Motif

ABSTRACT Streptococcus salivarius, a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of S. salivarius fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of S. salivarius ATCC 25975) with glass beads. Preliminary experiments showed that S. salivariusfimbriae did not dissociate when they were incubated at 100°C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 × 106 to 40 × 106Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 μm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that S. salivarius fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/φ, where X represents a modified amino acid residue and φ represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species, Streptococcus mitis andStreptococcus constellatus.

scholarly journals The amino acid sequence of toxin RpIII from the sea anemone, Radianthus paumotensis

FEBS Letters ◽  
1987 ◽  
Vol 218 (1) ◽  
pp. 59-62 ◽  
Author(s):  
Robert M. Metrione ◽  
Hugues Schweitz ◽  
Kenneth A. Walsh
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Sea Anemone

Physicochemical properties of a novel α-L-arabinofuranosidase fromRhizomucor pusillusHHT-1

2001 ◽  
Vol 47 (8) ◽  
pp. 767-772 ◽  
Author(s):  
A KM Shofiqur Rahman ◽  
Shinya Kawamura ◽  
Masahiro Hatsu ◽  
M M Hoq ◽  
Kazuhiro Takamizawa
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Rhizomucor Pusillus ◽  
Terminal Amino ◽  
Metal Cofactors

The zygomycete fungus Rhizomucor pusillus HHT-1, cultured on L(+)arabinose as a sole carbon source, produced extracellular α-L-arabinofuranosidase. The enzyme was purified by (NH4)2SO4fractionation, gel filtration, and ion exchange chromatography. The molecular mass of this monomeric enzyme was 88 kDa. The native enzyme had a pI of 4.2 and displayed a pH optimum and stability of 4.0 and 7.0–10.0, respectively. The temperature optimum was 65°C, and it was stable up to 70°C. The Kmand Vmaxfor p-nitrophenyl α-L-arabinofuranoside were 0.59 mM and 387 µmol·min–1·mg–1protein, respectively. Activity was not stimulated by metal cofactors. The N-terminal amino acid sequence did not show any similarity to other arabinofuranosidases. Higher hydrolytic activity was recorded with p-nitrophenyl α-L-arabinofuranoside, arabinotriose, and sugar beet arabinan; lower hydrolytic activity was recorded with oat–spelt xylan and arabinogalactan, indicating specificity for the low molecular mass L(+)-arabinose containing oligosaccharides with furanoside configuration.Key words: α-L-arabinofuranosidase, enzyme purification, amino acid sequence, Rhizomucor pusillus.

scholarly journals Unique Presence of a Manganese Catalase in a Hyperthermophilic Archaeon, Pyrobaculum calidifontis VA1

2002 ◽  
Vol 184 (12) ◽  
pp. 3305-3312 ◽  
Author(s):  
Taku Amo ◽  
Haruyuki Atomi ◽  
Tadayuki Imanaka
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Catalase Activity ◽  
Specific Activity ◽  
Homology Search ◽  
Aerobic Conditions ◽  
Hyperthermophilic Archaeon ◽  
Manganese Catalase ◽  
Catalase Gene ◽  
Pyrobaculum Calidifontis

ABSTRACT We had previously isolated a facultatively anaerobic hyperthermophilic archaeon, Pyrobaculum calidifontis strain VA1. Here, we found that strain VA1, when grown under aerobic conditions, harbors high catalase activity. The catalase was purified 91-fold from crude extracts and displayed a specific activity of 23,500 U/mg at 70°C. The enzyme exhibited a Km value of 170 mM toward H2O2 and a k cat value of 2.9 × 104 s−1·subunit−1 at 25°C. Gel filtration chromatography indicated that the enzyme was a homotetramer with a subunit molecular mass of 33,450 Da. The purified catalase did not display the Soret band, which is an absorption band particular to heme enzymes. In contrast to typical heme catalases, the catalase was not strongly inhibited by sodium azide. Furthermore, with plasma emission spectroscopy, we found that the catalase did not contain iron but instead contained manganese. Our biochemical results indicated that the purified catalase was not a heme catalase but a manganese (nonheme) catalase, the first example in archaea. Intracellular catalase activity decreased when cells were grown anaerobically, while under aerobic conditions, an increase in activity was observed with the removal of thiosulfate from the medium, or addition of manganese. Based on the N-terminal amino acid sequence of the purified protein, we cloned and sequenced the catalase gene (katPc ). The deduced amino acid sequence showed similarity with that of the manganese catalase from a thermophilic bacterium, Thermus sp. YS 8-13. Interestingly, in the complete archaeal genome sequences, no open reading frame has been assigned as a manganese catalase gene. Moreover, a homology search with the sequence of katPc revealed that no orthologue genes were present on the archaeal genomes, including those from the “aerobic” (hyper)thermophilic archaea Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii. Therefore, Kat Pc can be considered a rare example of a manganese catalase from archaea.

scholarly journals Primary structure of bovine complement activation fragment C4a, the third anaphylatoxin. Purification and complete amino acid sequence

1982 ◽  
Vol 207 (2) ◽  
pp. 253-260 ◽  
Author(s):  
M A Smith ◽  
L M Gerrie ◽  
B Dunbar ◽  
J E Fothergill
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Primary Structure ◽  
Gel Filtration ◽  
Complete Amino Acid Sequence ◽  
The Third ◽  
Human Sequence ◽  
Bovine Plasma ◽  
Bovine Sequence ◽  
C 50

Purification of C4a from heat-activated bovine plasma by elution from CM-Sephadex C-50 at pH 7.4 and gel filtration on Sephadex G-50 gives a 20% yield of pure C4a. The complete amino acid sequence of bovine C4a has been determined by automatic sequencer degradation of CNBr and enzymic fragments, and by carboxypeptidase digestion. The 77-residue bovine sequence shows 12 differences from the human sequence with five of these differences occurring in the C-terminal 11 residues. The sequence of C4a confirms earlier suggestions of homology with C3a and C5a: the three sequences show an almost equal number of identities with each other. The six cysteine residues of the ‘disulphide knot’ are conserved as well as seven other residues including the C-terminal arginine.

A novel phytase from Citrobacter gillenii: characterization and expression in Pichia pastoris (Komagataella pastoris)

2020 ◽  
Author(s):  
Artur A Tkachenko ◽  
Anna N Kalinina ◽  
Larisa N Borshchevskaya ◽  
Sergey P Sineoky ◽  
Tatiana L Gordeeva
Keyword(s):  
Amino Acid ◽  
Pichia Pastoris ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Gene Encoding ◽  
Maximum Reaction Rate ◽  
Maximum Reaction ◽  
Wide Ph Range ◽  
Recombinant Phytase ◽  
Ph Range

Abstract The phyCg gene encoding a new phytase from C. gillenii was optimized, synthesized, cloned, and expressed in Pichia pastoris. Analysis of the amino acid sequence of the enzyme showed that it belongs to the histidine acid phosphatase family. The amino acid sequence of the PhyCg phytase has the highest homology (73.49%) with a phytase sequence from Citrobacter braakii. The main characteristics for the purified recombinant phytase were established. The optimum pH and temperature were 4.5 and 50°C, respectively. The specific activity of the enzyme was 1577 U/mg. The Michaelis constant (Km) and the maximum reaction rate (Vmax) for sodium phytate were 0,185 mM and 2185 U/mg, respectively. The enzyme showed the pH and trypsin stability and had a high activity over a wide pH range.

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