scholarly journals Studies on the soluble phosphodiesterases of chicken gizzard smooth muscle

1983 ◽  
Vol 215 (3) ◽  
pp. 627-636 ◽  
Author(s):  
R J Birnbaum ◽  
J F Head
Keyword(s):  
Smooth Muscle ◽  
Deae Cellulose ◽  
Gel Permeation Chromatography ◽  
Hydrolytic Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Dependent Manner ◽  
Chicken Gizzard ◽  
Calmodulin Binding ◽  
Discrete Band

In this study we describe the identification of four soluble forms of cyclic nucleotide phosphodiesterase from chicken gizzard smooth muscle. These isoenzymes were separated from one another by ion-exchange chromatography on DEAE-cellulose and by calmodulin-Sepharose affinity chromatography. Each form migrates as a single discrete band when it is electrophoresed on non-denaturing polyacrylamide gels and stained for phosphodiesterase activity. Each form is also eluted as a single peak on gel-permeation chromatography, giving apparent Mr values of 114 000, 116 000, 122 000 and 59 000. All four enzymes have apparent Km values in the 0-20 microM range, although their relative specificities for cyclic AMP and cyclic GMP differ. Two of the forms bind to calmodulin in a Ca2+-dependent manner; however, only one is activated by calmodulin. The interaction of the second calmodulin-binding form with calmodulin is disrupted by the papaverine derivative verapamil without significantly altering the hydrolytic activity of the enzyme.

Catabolism of (R)-Amygdalin and (R)-Vicianin by Partially Purified ß-Glycosidases from Prunus serotina Ehrh. and Davallia trichomanoides

1984 ◽  
Vol 39 (3-4) ◽  
pp. 232-239 ◽  
Author(s):  
Gary Kuroki ◽  
Pauline A. Lizotte ◽  
Jonathan E. Poulton
Keyword(s):  
Ion Exchange ◽  
Deae Cellulose ◽  
Hydrolytic Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Prunus Serotina ◽  
Artificial Substrates ◽  
Black Cherry ◽  
Significant Activity ◽  
Optimum Ph

Mature black cherry (Prunus serotina Ehrh.) seeds accum ulate high levels of the cyanogenic disaccharide (R)-amygdalin. Extracts from these seeds contain two β-glycosidases which have been identified and completely resolved by DEAE-cellulose ion-exchange chromatography. Amygdalin hydrolase hydrolyzed (R)-am ygdalin at an optimum pH of 5.5, releasing (R)-prunasin and D-glucose. This enzyme showed highest activity towards (R)-am ygdalin and failed to hydrolyze (R)-prunasin. linamarin, β-gentiobiose and cellobiose. A distinct β-glycosidase, prunasin hydrolase, displayed a pronounced preference for (R)-prunasin, hydrolyzing this cyanogenic monosaccharide at an optimum pH of 6.5 to mandelonitrile and D-glucose. Prunasin hydrolase was inactive towards (R)-am ygdalin, linamarin, and β-gentiobiose. Both enzymes showed significant activity towards the artificial substrates β-ONPGlu and β-PNPGlu but did not hydrolyze α-PNPGlu. In view of the pronounced specificity of these enzymes towards endogenous cyanogens, it is concluded that upon disruption of black cherry seeds (R)- amygdalin is catabolized to mandelonitrile in a stepwise manner (the sequential mechanism) by amygdalin hydrolase and prunasin hydrolase with (R)-prunasin serving as intermediate. Young fronds of Davallia trichomanoides are rich sources of (R)-vicianin (the β-vicianoside of (R)-mandelonitrile). A β-glycosidase, vicianin hydrolase, has been partially purified from frond extracts by ion-exchange chromatography. At the optimum pH of 6.0, this enzyme showed highest hydrolytic activity with (R)-vicianin, although both (R)-am ygdalin and (R)-prunasin could be hydrolyzed at approximately 15% of the rate observed with (R)-vicianin. It failed to hydrolyze β-gentiobiose, cellobiose, linamarin and α-PNPGlu. Closer exam ination revealed that (R)-vicianin and (R)-amygdalin were hydrolyzed at the aglycone-disaccharide bond (the simultaneous mechanism) yielding mandelonitrile and the respective disaccharides vicianose and β-gentiobiose

Quantitative fractionation of casein mixtures by fast protein liquid chromatography

1987 ◽  
Vol 54 (3) ◽  
pp. 369-376 ◽  
Author(s):  
D. Thomas Davies ◽  
Andrew J. R. Law
Keyword(s):  
Liquid Chromatography ◽  
Ion Exchange ◽  
Deae Cellulose ◽  
Gel Permeation Chromatography ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fast Protein Liquid Chromatography ◽  
Good Resolution ◽  
Gel Permeation

SummaryAlkylation of whole casein samples by reaction with cysteamine and cystamine in a bis-tris-propane–urea buffer (pH 7·0) followed by fast protein liquid chromatography (FPLC) at 20°C on a Mono Q HR5/5 column in the same buffer and using a NaCl gradient led to good resolution of the whole casein into fractions representing (i) γ2- plus γ3-caseins, (ii) κ-caseins, (iii) β-casein, (iv) αs2-caseins and (v) αsl-caseins, together with small amounts of unidentified materials. Quantitatively the FPLC values agreed well with those for αs1-, β-, αs2- and γ2- plus γ3-caseins obtained by ion-exchange chromatography on DEAE cellulose, Whatman DE52 and with those for º-caseins obtained by gel-permeation chromatography on Sephadex G–150.

scholarly journals The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide–adenine dinucleotide phosphate-linked isoenzymes

1972 ◽  
Vol 130 (1) ◽  
pp. 211-219 ◽  
Author(s):  
Colin H. Self ◽  
P. David J. Weitzman
Keyword(s):  
Molecular Weight ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Molecular Size ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Low Concentrations ◽  
Stimulation Of

Two isoenzymes of NADP-linked isocitrate dehydrogenase have been identified in Acinetobacter lwoffi and have been termed isoenzyme-I and isoenzyme-II. The isoenzymes may be separated by ion-exchange chromatography on DEAE-cellulose, by gel filtration on Sephadex G-200, or by zonal ultracentrifugation in a sucrose gradient. Low concentrations of glyoxylate or pyruvate effect considerable stimulation of the activity of isoenzyme-II. The isoenzymes also differ in pH-dependence of activity, kinetic parameters, stability to heat or urea and molecular size. Whereas isoenzyme-I resembles the NADP-linked isocitrate dehydrogenases from other organisms in having a molecular weight under 100000, isoenzyme-II is a much larger enzyme (molecular weight around 300000) resembling the NAD-linked isocitrate dehydrogenases of higher organisms.

scholarly journals Proteinaceous Protease Inhibitor from Lawsonia Inermis: Purification, Characterization and Antibacterial Activity

2013 ◽  
Vol 8 (10) ◽  
pp. 1934578X1300801 ◽  
Author(s):  
Arvind Dabhade ◽  
Priti Patel ◽  
Ulhas Patil
Keyword(s):  
Antibacterial Activity ◽  
Protease Inhibitor ◽  
Deae Cellulose ◽  
Gel Permeation Chromatography ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Lawsonia Inermis ◽  
Sds Page ◽  
Wide Range ◽  
And Staphylococcus Aureus

A thermo-stable, proteinaceous protease inhibitor (LPI) from Lawsonia inermis is reported. The LPI was purified from Lawsonia inermis seeds by subsequent ammonium sulfate precipitation, ion exchange chromatography (DEAE-Cellulose) and gel permeation chromatography (Sephadex-50). The purified protease inhibitor is effective against a wide range of proteases viz. papain, trypsin, pepsin and metallo-protease. The apparent molecular weight of the protease inhibitor is 19 kDa, determined by SDS-PAGE electrophoresis. The protease inhibitor was found to be stable at 70 °C for 30 min. It was also examined for antibacterial activity against Pseudomonas aeruginosa MTCC 7926 and Staphylococcus aureus NCIM 2079; the IC50 values of the purified LPI were 11.4 μg/mL and 16.6 μg/mL respectively.

THE SEPARATION AND PURIFICATION OF HUMAN LUTEINIZING AND THYROTROPHIC HORMONES

1964 ◽  
Vol 29 (1) ◽  
pp. 61-69 ◽  
Author(s):  
ANNE STOCKELL HARTREE ◽  
W. R. BUTT ◽  
K. E. KIRKHAM
Keyword(s):  
Luteinizing Hormone ◽  
Ion Exchange ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Separation And Purification ◽  
Hormone Activity ◽  
Human Pituitary ◽  
Thyrotrophic Hormone

SUMMARY Ion-exchange chromatography was used to further purify a human pituitary fraction rich in thyrotrophic and luteinizing hormone activities. Approximately twofold concentration of both activities was obtained by chromatography on IRC-50 at pH 7·5, but the hormones were not separated. Subsequent chromatography on DEAE-cellulose at pH 9·5 led to a tenfold concentration of the luteinizing hormone in a fraction practically free of thyrotrophic activity and to a fourfold concentration of the thyrotrophic hormone in a fraction still exhibiting substantial luteinizing hormone activity.

Isolation of a spermatozoal immobilization factor from Staphylococcus aureus filtrates

2009 ◽  
Vol 55 (7) ◽  
pp. 874-878 ◽  
Author(s):  
Vijay Prabha ◽  
Tanushree Gupta ◽  
Siftjit Kaur ◽  
Navchetan Kaur ◽  
Sushila Kala ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Gel Permeation Chromatography ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Human Spermatozoa ◽  
Infertile Woman ◽  
Ammonium Sulfate Precipitation ◽  
Tail Region ◽  
Sperm Immobilization

Staphylococcus aureus isolated from the cervix of an infertile woman was found to cause complete immobilization of human spermatozoa in vitro. Only the cell culture and cell-free supernatant showed immobilization activity, indicating that the sperm immobilization factor might be released extracellularly by the organism because no activity was observed with the washed cells. Heat treatment of the supernatant at 60 °C for 10 min waived its immobilizing activity, indicating that the active component may be a protein. The bioactive molecule from the supernatant was purified to homogeneity by ammonium sulfate precipitation, gel permeation chromatography, and ion exchange chromatography. Sperm immobilization factor (SIF) was found to be an ~20 kDa protein. SIF at a concentration of 10 µg/mL was required to cause 100% immobilization of human spermatozoa after 30 min of incubation at 37 °C, whereas a concentration of 150 µg/mL caused immediate immobilization, and a concentration of 200 µg/mL resulted in instant loss of viability of human spermatozoa, observed by eosin–nigrosin staining. Scanning electron microscopy showed that the treatment of human spermatozoa with SIF caused multiple defects in the head, midpiece, neck, and tail region of human spermatozoa.

Darstellung und Schwingungsspektren der 35Cl/37Cl-markierten Fluoro-Chloro-Platinate(IV) / Preparation and Vibrational Spectra of 35Cl/37Cl Labelled Fluoro-Chloro-Platinates(IV)

1989 ◽  
Vol 44 (6) ◽  
pp. 619-626 ◽  
Author(s):  
P. Erlhöfer ◽  
W. Preetz
Keyword(s):  
Low Temperature ◽  
Mutual Exclusion ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Vibrational Modes ◽  
Geometric Isomers ◽  
Deformation Bands ◽  
Isotopic Shifts ◽  
Ir And Raman

All components of the two series Cs2[PtF„35Cl6-n] and Cs2[PtFn37Cl6-n], including the pairs of geometric isomers for n = 2,3, 4, have been prepared and isolated by ion exchange chromatography on DEAE cellulose. The highly resolved low temperature (80 K) IR and Raman spectra of the pure isotopomers show distinctive isotopic shifts for different vibrational modes up to 10 cm-1. The excellent agreement of the observed values with Teller-Redlich calculations verifies the assignment of all stretching and especially of the narrow deformation bands. For transcomplexes containing 35Cl-Pt-37Cl axes, the lowered symmetry affects the restriction of the rule of mutual exclusion

Molecular cloning of chicken calcyclin (S100A6) and identification of putative isoforms

1997 ◽  
Vol 75 (6) ◽  
pp. 733-738 ◽  
Author(s):  
Bruce G Allen ◽  
Jacquelyn E Andrea ◽  
Cindy Sutherland ◽  
Brett O Schönekess ◽  
Michael P Walsh
Keyword(s):  
Smooth Muscle ◽  
Amino Acid ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Chain Reaction ◽  
Proteolytic Fragment ◽  
Chicken Gizzard ◽  
Muscle Tissues ◽  
Bona Fide ◽  
Polymerase Chain

A full-length cDNA encoding smooth muscle calcyclin (S100A6) was cloned from chicken gizzard, using reverse transcription - polymerase chain reaction techniques. The deduced amino acid sequence contains 92 residues with 12 substitutions and a 2 amino acid C-terminal extension when compared with human calcyclin. Calcyclin was purified from chicken gizzard by Ca2+-dependent hydrophobic chromatography, heat treatment, and anion-exchange chromatography. N-terminal sequencing of two CNBr peptides confirmed its identity as calcyclin. Two isoforms of calcyclin (A and B), which differ with respect to the presence or absence of a C-terminal lysine, were identified and the native protein was shown to exist as noncovalently associated homodimers (AA and BB) and heterodimers (AB). Incubation of purified calcyclin AA with an extract of chicken gizzard did not result in degradation of calcyclin A or appearance of calcyclin B, suggesting that calcyclin B is a bona fide isoform rather than a proteolytic fragment generated during purification. Western blotting of chicken tissues with anti-(gizzard calcyclin) indicated abundant expression of calcyclin in smooth muscle tissues, including esophagus, large intestine, and trachea, with lower levels in lung, heart, kidney, and brain, and none detectable in liver or skeletal muscle.Key words: Ca2+-binding proteins, calcyclin, smooth muscle, cDNA cloning, isoforms.

scholarly journals Aspergillus oryzae acid proteinase. Purification and properties, and formation of π-chymotrypsin

1975 ◽  
Vol 147 (1) ◽  
pp. 45-53 ◽  
Author(s):  
R Davidson ◽  
A Gertler ◽  
T Hofmann
Keyword(s):  
Aspergillus Oryzae ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Molecular Forms ◽  
Acid Proteinase ◽  
Sedimentation Analysis ◽  
Simple Tool ◽  
Purification And Properties ◽  
Yield And Purity

An acid proteinase from Aspergillus oryzae was isolated from a commercial powder by successive (NH4)2SO4 fractionation, acetone precipitation, and ion-exchange chromatography on phosphate- and DEAE-cellulose columns. The purified enzyme was found to be homogeneous by ultracentrifuge-sedimentation analysis (S20, W equal 3.63S), but electrofocusing in polyacrylamide gels and electrophoresis at pH 3.2 revealed that it consists of two very closely migrating bands. No difference in the amino acid composition and enzymic activities of the two partially separated bands could be detected, and it was concluded that the acid proteinase exists in two molecular forms. The enzyme activates bovine trypsinogen and chymotrypsinogen at pH 3.5 (the kappacat. and Km values at 35degrees C are 11.3S- minus 1, 0.10mM and 1.14S- minus 1, 0.18mM respectively). It hydrolyses the Phe-Phe bond of the synthetic pepsin substrates Z-His-Phe-Phe-OEt (kappacat. equal 1.65S- minus 1, Km equal 0.640mM at pH 3.5, 30degrees C) and Z-Ala-Ala-Phe-Phe-OPy4Pr (kappacat. equal 0.37S- minus 1, Km equal 0.037 mM at pH2.9, 39degrees C), where Z represents benzyloxycarbonyl and OPy4Pr represents 3-(4-pyridyl)-propyl 1-ester. Activation of bovine chymotrypsinogen results from the cleavage of the Arg(15)-Ile(16) bond in the zymogen. No other cleavages were observed. The use of A. oryzae proteinase provides a simple tool for the production of pi-chymotrypsin in good yield and purity.

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