scholarly journals The two cytochromes c in the facultative methylotroph Pseudomonas AM1

1980 ◽  
Vol 192 (2) ◽  
pp. 411-419 ◽  
Author(s):  
David T. O'Keeffe ◽  
Christopher Anthony
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Isoelectric Point ◽  
Ph Dependence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Redox Potentials ◽  
Midpoint Potential ◽  
Cytochromes C

It was previously suggested that there is only one soluble cytochrome c in Pseudomonas AM1, having a molecular weight of 20000, a redox midpoint potential of about +260mV and a low isoelectric pint [Anthony (1975) Biochem. J.146, 289–298; Widdowson & Anthony (1975) Biochem. J.152, 349–356]. A more thorough examination of the soluble fraction of methanol-grown Pseudomonas AM1 has now revealed the presence of two different cytochromes c. These were both purified to homogeneity by acid treatment, ion-exchange chromatography, gel filtration, chromatography on hydroxyapatite and preparative isoelectric focusing. Molecular weights were determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; midpoint redox potentials were determined directly by using platinum and calomel electrodes; isoelectric points were estimated by electrophoresis and by the behaviour of the two cytochromes on ion-exchange celluloses. The more abundant cytochrome cH (λmax. 550.5nm) had a low molecular weight (11000), a midpoint potential of about +294mV and a high isoelectric point, not being adsorbed on DEAE-cellulose in 20mm-Tris/HCl buffer, pH8.0. The less abundant cytochrome cL (λmax. 549nm) was about 30% of the total; it had a high molecular weight (20900), a midpoint potential of about +256mV and a low isoelectric point, binding strongly to DEAE-cellulose in 20mm-Tris/HCl buffer, pH8.0. The pH-dependence of the midpoint redox potentials of the two cytochromes c were very similar. There were four ionizations affecting the redox potentials in the pH range studied (pH4.0–9.5), two in the oxidized form (pK values about 3.5 and 5.5) and two in the reduced form (pK values about 4.5 and 6.5), suggesting that the ionizing groups involved may be the two propionate side chains of the haem. Neither of the cytochromes c was present in mutant PCT76, which was unable to oxidize or grow on C1 compounds, although still able to grow well on multicarbon compounds such as succinate. Whether or not these two cytochromes c have separate physiological functions is not yet certain.

scholarly journals The purification and properties of the soluble cytochromes c of the obligate methylotroph Methylophilus methylotrophus

1980 ◽  
Vol 192 (2) ◽  
pp. 421-427 ◽  
Author(s):  
A R Cross ◽  
C Anthony
Keyword(s):  
Molecular Weight ◽  
Isoelectric Point ◽  
Low Molecular Weight ◽  
Redox Potentials ◽  
Obligate Methylotroph ◽  
Methylophilus Methylotrophus ◽  
Midpoint Potential ◽  
Purification And Properties ◽  
Cytochromes C ◽  
Cytochrome Cl

The obligate methylotroph Methylophilus methylotrophus contains three distinct soluble cytochromes c. The major cytochromes, cytochrome cH (about 50% of the total) and cytochrome cL (about 42%), were similar in most respects to the cytochromes cH and cL of the facultative methylotroph Pseudomonas AM1 [O'Keeffe & Anthony (1980) Biochem. J. 192, 411-419]. Cytochrome cH had a high isoelectric point, a midpoint redox potential at pH 7.0 of 373 mV and a low molecular weight (8500). The cytochrome cL had a low isoelectric point, a midpoint potential of 310 mV and a molecular weight of 21,000. The third cytochrome, cytochrome cLM, was clearly distinct from cytochromes cH and cL. Like the cytochrome cL of Pseudomonas AM1, the cytochrome cL of M. methylotrophus had the lowest midpoint potential, it reacted most rapidly with methanol dehydrogenase and it combined to the greatest extent with CO. Cytochromes cH and L of M. methylotrophus differed from those from Pseudomonas AM1 in having unusually high midpoint redox potentials for non-photosynthetic bacteria and in exhibiting a split alpha-band at low temperatures.

scholarly journals Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5′-phosphoramidate from adenosine 5′-phosphosulphate and ammonia

1981 ◽  
Vol 195 (3) ◽  
pp. 545-560 ◽  
Author(s):  
Heinz Fankhauser ◽  
Jerome A. Schiff ◽  
Leonard J. Garber
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Low Ph ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Unknown Compound ◽  
Reactive Blue 2

Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5′-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5′-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5′-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5′-phosphoramidate. The enzyme that catalyses the formation of adenosine 5′-phosphoramidate from ammonia and adenosine 5′-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH4)2SO4 precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2–agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000–65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3′-phosphate 5′-phosphosulphate will not replace adenosine 5′-phosphosulphate, and the apparent Km for the last-mentioned compound is 0.82mm. The apparent Km for ammonia (assuming NH3 to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5′-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5′-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme does not catalyse any of the known reactions of adenosine 5′-phosphosulphate, including those catalysed by ATP sulphurylase, adenosine 5′-phosphosulphate kinase, adenosine 5′-phosphosulphate sulphotransferase or ADP sulphurylase. Adenosine 5′-phosphoramidate is found in old samples of the ammonium salt of adenosine 5′-phosphosulphate and can be formed non-enzymically if adenosine 5′-phosphosulphate and ammonia are boiled. In the non-enzymic reaction both adenosine 5′-phosphoramidate and AMP are formed. Thus the enzyme forms adenosine 5′-phosphoramidate by selectively speeding up an already favoured reaction.

scholarly journals The purification and molecular properties of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa

1981 ◽  
Vol 197 (2) ◽  
pp. 427-436 ◽  
Author(s):  
G A Nimmo ◽  
J R Coggins
Keyword(s):  
Molecular Weight ◽  
Neurospora Crassa ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium ◽  
Phosphate Synthase

Neurospora crassa contains three isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, which are inhibited by tyrosine, tryptophan and phenylalanine respectively, and it was estimated that the relative proportions of the total activity were 54%, 14% and 32% respectively. The tryptophan-sensitive isoenzyme was purified to homogeneity as judged by polyacrylamide-gel electrophoresis and ultracentrifugation. The tyrosine-sensitive and phenylalanine-sensitive isoenzymes were only partially purified. The three isoenzymes were completely separated from each other, however, and can be distinguished by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and Ultrogel AcA-34 and polyacrylamide-gel electrophoresis. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the tryptophan-sensitive isoenzyme contained one type of subunit of molecular weight 52000. The molecular weight of the native enzyme was found to be 200000 by sedimentation-equilibrium centrifugation, indicating that the enzyme is a tetramer, and the results of cross-linking and gel-filtration studies were in agreement with this conclusion.

PURIFICATION AND PROPERTIES OF CHORIONIC GONADOTROPHIN FROM TROPHOBLASTIC TISSUE, URINE AND PLASMA OF A PATIENT WITH A HYDATIDIFORM MOLE

1973 ◽  
Vol 56 (3) ◽  
pp. 441-NP ◽  
Author(s):  
A. PALA ◽  
M. MEIRINHO ◽  
G. BENAGIANO
Keyword(s):  
Molecular Weight ◽  
Isoelectric Focusing ◽  
Sodium Dodecyl Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Hydatidiform Mole ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Chorionic Gonadotrophin ◽  
Tissue Preparation ◽  
Trophoblastic Tissue

SUMMARY Purified human chorionic gonadotrophin (HCG) fractions were obtained from urine, peripheral plasma and chorionic tissue of a woman with hydatidiform mole by a combination of DEAE-cellulose and DEAE-Sephadex ion exchange chromatography and gel filtration on Sephadex G-100. Homogeneity of the purified fractions obtained was demonstrated by analytical polyacrylamide gel electrophoresis and immunoelectrophoresis. The preparation from plasma had a higher ratio of biological to immunological activity than the urine or tissue preparation. The biological potency of the plasma HCG was 23000 i.u./mg, whereas the urinary and the trophoblastic tissue preparations showed a potency of 20000 and 6500 i.u./mg, respectively. Further characterization of the three fractions was achieved by analytical isoelectric focusing and determination of the molecular weight. The peak of radioimmunological activity after isoelectric focusing was found to be, in all three cases, at pH 4·90 and the molecular weight obtained after incubation with sodium dodecyl sulphate and urea was around 63000 When the molecular weight was determined after incubation in a medium containing 4 m-urea, sodium dodecyl sulphate and 2-mercaptoethanol two separate fractions were obtained from each of the three preparations. The heavier fraction showed a molecular weight of approximately 35000 and the lighter fraction had a molecular weight of approximately 27000.

scholarly journals Hydrolysed Collagen from Sheepskins as a Source of Functional Peptides with Antioxidant Activity

2019 ◽  
Vol 20 (16) ◽  
pp. 3931 ◽  
Author(s):  
Arely León-López ◽  
Lucía Fuentes-Jiménez ◽  
Alma Delia Hernández-Fuentes ◽  
Rafael G. Campos-Montiel ◽  
Gabriel Aguirre-Álvarez
Keyword(s):  
Molecular Weight ◽  
Antioxidant Activity ◽  
Isoelectric Point ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Content ◽  
Good Alternative ◽  
Hydrolysis Time ◽  
Scanning Calorimetry ◽  
Sem Images

The extraction and enzymatic hydrolysis of collagen from sheepskins at different times of hydrolysis (0, 10, 15, 20, 30 min, 1, 2, 3 and 4 h) were investigated in terms of amino acid content (hydroxyproline), isoelectric point, molecular weight (Mw) by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method, viscosity, Fourier-transform infrared (FTIR) spectroscopy, antioxidant capacity by 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays, thermal properties (Differential Scanning Calorimetry) and morphology by scanning electron microscopy (SEM) technique. The kinetics of hydrolysis showed an increase in the protein and hydroxyproline concentration as the hydrolysis time increased to 4 h. FTIR spectra allowed us to identify the functional groups of hydrolysed collagen (HC) in the amide I region for collagen. The isoelectric point shifted to lower values compared to the native collagen precursor. The change in molecular weight and viscosity from time 0 min to 4 h promoted important antioxidant activity in the resulting HC. The lower the Mw, the greater the ability to donate an electron or hydrogen to stabilize radicals. From the SEM images it was evident that HC after 2 h had a porous and spongy structure. These results suggest that HC could be a good alternative to replace HC from typical sources like pigs, cows and fish.

scholarly journals CO-binding c-type cytochromes and a high-potential cytochrome c in Nitrosomonas europaea

1983 ◽  
Vol 211 (2) ◽  
pp. 503-506 ◽  
Author(s):  
D J Miller ◽  
P M Wood
Keyword(s):  
Cytochrome C ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Nitrosomonas Europaea ◽  
Redox Potentials ◽  
Cytochromes C ◽  
High Redox Potential

The purification of two soluble CO-binding cytochromes c from Nitrosomonas europaea is described. Cytochrome cCO−550 ran on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 32 000, whereas for cytochrome cCO−552 the apparent Mr was 16 000. Redox potentials (Em, 7) were determined as +140 and −50mV respectively. Cytochrome cCO−550 was co-purified with a cytochrome c−553, for which an unusually high redox potential of +450mV was measured. These latter components were not resolved by gel-filtration chromatography or electrophoresis under denaturing conditions.

Purification of proteins similar to HPr and enzyme I from the oral bacterium Streptococcus salivarius. Biochemical and immunochemical properties

1983 ◽  
Vol 29 (12) ◽  
pp. 1694-1705 ◽  
Author(s):  
Christian Vadeboncoeur ◽  
Mario Proulx ◽  
Luc Trahan
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Streptococcus Salivarius ◽  
Dodecyl Sulfate ◽  
Molecular Sieving ◽  
Enzyme I

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is made of several proteins. Two of them are designated general proteins because they are required for the transport and phosphorylation of all sugars of the PTS. These two proteins are found in the soluble fraction of cellular extracts and are termed HPr and enzyme I (EI). We reported in this work the purification and the characterization of these two proteins from Streptococcus salivarius ATCC 25975. HPr was purified by DEAE-cellulose chromatography, molecular sieving on Ultrogel AcA44, and carboxymethylcellulose chromatography. Sodium dodecyl sulfate electrophoresis in the presence of urea revealed a single band with a molecular weight of 6700. The protein contained no tryptophan and had a pi of 4.8. The purification scheme of EI was as follows: DEAE-cellulose chromatography, hydroxylapatite chromatography, DEAE-Sephadex A-50 chromatography, preparative electrophoresis, and molecular sieving on Ultrogel AcA34. The five-step purification for EI produced a 199-fold purified preparation with a specific activity of 530 μmol of HPr phosphorylated per minute per milligram of protein at 37 °C. The fraction obtained after filtration on Ultrogel AcA34 gave one band (68 000) on sodium dodecyl sulfate – polyacrylamide gel electrophoresis. The molecular weight of the native enzyme determined by gel filtration at 4 °C was 135 000, suggesting that it was a dimer. Enzyme I had a pI of 4.2, a pH optimum of 6.7, a Km for HPr of about 27 μM, a Km for phosphoenolpyruvate of 0.48 mM, and kinetics that were consistent with a Ping-Pong mechanism. Evidence had been obtained which indicated that S. salivarius enzyme I was antigenically very similar to enzyme I from various strains of Streptococcus mutans, but not to the enzyme from Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, and Escherichia coli.

scholarly journals Characterization of glyoxalase I purified from pig erythrocytes by affinity chromatography

1977 ◽  
Vol 165 (3) ◽  
pp. 503-509 ◽  
Author(s):  
Anne-Charlotte Aronsson ◽  
Bengt Mannervik
Keyword(s):  
Affinity Chromatography ◽  
Isoelectric Point ◽  
Reduced Glutathione ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Glyoxalase I ◽  
Active Components ◽  
Catalytically Active

Glyoxalase I (EC 4.4.1.5) was purified about 10000-fold from pig erythrocytes in a yield of approx. 20%. The purification included affinity chromatography on S-hexylglutathione coupled to Sepharose 4B. The purified enzyme normally contained two catalytically active components which were resolved by polyacrylamide-gel electrophoresis. After treatment with reduced glutathione only one component was found. The two components were also demonstrable after isoelectric focusing or DEAE-cellulose chromatography and could also in these cases be fused into one species by preincubation with reduced glutathione. It is proposed that the most acidic form of glyoxalase I is a mixed disulphide with glutathione. Except for these interconvertible forms, the purified enzyme was homogeneous, as judged by disc electrophoresis and sodium dodecyl sulphate/polyacrylamidegel electrophoresis. The molecule is a dimer (48000 daltons), composed of apparently identical subunits (24000 daltons). The isoelectric point was 4.8 at 4°C. The amino acid composition was consistent with the low isoelectric point. The enzyme contained about two thiol groups per enzyme molecule. EDTA inactivated the enzyme and bivalent metal ions could restore fully or partially the catalytic activity; Mg2+ and Mn2+ gave highest activity. It is proposed that a major biological function of glyoxalase I is the detoxification of methylglyoxal formed by enterobacteria in the alimentary canal.

scholarly journals Rabbit β-glucuronidase. Purification and properties, and the existence of multiple forms

1974 ◽  
Vol 138 (3) ◽  
pp. 395-405 ◽  
Author(s):  
Roger T. Dean
Keyword(s):  
Molecular Weight ◽  
Isoelectric Focusing ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Multiple Forms ◽  
Hydroxyapatite Gel

1. β-Glucuronidase (EC 3.2.1.31) was purified from rabbit liver by a procedure involving autolysis, (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration, sedimentation in a sucrose gradient, and isoelectric focusing. 2. Electron microscopy revealed ferritin as the major contaminant in later stages of purification and also showed aggregates of enzyme molecules. Particular attention was paid to the removal of ferritin. 3. The purified enzyme was homogeneous in polyacrylamide-gel electrophoresis both in non-dissociating conditions and in the presence of sodium dodecyl sulphate, and in Ouchterlony gel diffusion and immunoelectrophoresis against polyspecific antisera. 4. Sedimentation in sucrose gradients gave a molecular weight of 300000, whereas gel filtration indicated 440000. 5. Subunits of 75000 molecular weight were observed in gel electrophoresis in the presence of sodium dodecyl sulphate and in gel filtration in the presence of urea. 6. The Km value for p-nitrophenyl β-d-glucuronide was 0.6mm, and the enzyme was extremely sensitive to lactone inhibitors. It was also inhibited by Hg2+ ions. 7. Multiple forms were observed in the pure enzyme by isoelectric focusing, with pI values of 4.5–5.8. Subunits showed similar heterogeneity. The origin of the multiple forms was investigated in detail, and the possibility of artifact generation largely excluded. Some of the forms of lowest pI disappeared after neuraminidase digestion. The nature of the residual heterogeneity remains to be elucidated.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

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