scholarly journals Praline metabolism by germinating Lilium longiflorum pollen. I. Labelling of cytoplasmic, wall and culture medium molecules

2014 ◽  
Vol 50 (1-2) ◽  
pp. 51-66 ◽  
Author(s):  
W. V. Dashek ◽  
R. R. Mills
Keyword(s):  
Growth Medium ◽  
Culture Medium ◽  
Gel Filtration ◽  
Lilium Longiflorum ◽  
Paper Electrophoresis ◽  
Void Volume ◽  
Molecular Weights ◽  
The Common ◽  
Two Peaks ◽  
Salt Extract

Radioactivity occurs in trithloroacetic acid (TCA)-soluble and precipitable, cytoplasm and salt-washed walls following germination of <em>Lilium longiflorum</em>, cv. 'Ace' pollen in medium containing [<sup>14</sup>C]-proline (Pro). Sephadex gel filtration on G-25 through G-100 was employed to determine whether radioactivity in cytoplasm, wall and growth medium from pollen fed [<sup>14</sup>C]-Pro or [<sup>3</sup>H]=Pro plus [<sup>14</sup>C]-arafbinose (Ara) was contained within molecules possessing molecular weights of 5,000 to 100,000 daltones or greater. G-25 elution profiles of a crude cytoplasmic fraction (15,000 X g supernatant) from [<sup>14</sup>C]-Pro labelled pollen yielded a radioctive void volume peak and a retarded peak. The void volume peak contained hydroxyproline (Hyp), and exhibited a coincidence of [<sup>3</sup>H]-Pro and [<sup>14</sup>C] -Ara labelling when pollen was double labelled with the two isotopes. This peak also contained radioactivity when pollen was germinated in 2-[<sup>3</sup>H]-myo-inositol. Germination in medium supplemented with 100 µM 2,2'-dipyridyl eliminated radioactivity from 2-[<sup>3</sup>H]-myo-inositol or [<sup>14</sup>C]-,Pro in the peak. Filtratian on G-25 of aTCA-soluble fraction of a salt-extract of walls from [<sup>14</sup>C]-Pro labelled pollen resulted in void volume and two retarded peaks. Void volume and two retarded peaks were also obtained upon G-25 filtration of a cellulase-digest of walls from [M]-Pro labeled pollen. The void volume peak contained Hyp, Lys, Gly, Ala, Ser, Glu and Asp acids, Val, Tyr, Leu or lieu and Pro. Sephadex G-90, 75, and 100 elution profiles of cellulasedigests of walls from [<sup>3</sup>H]-,Pro and [<sup>14</sup>C]-Ara labelled pollen yielded radioactive retarded and Hyp-containing void volume peaks with a coincidence of [<sup>3</sup>H] and [<sup>14</sup>C] labelling. Label in the void volume was obtained when either rhozyme P11- or pepsin-digests of walls from [<sup>14</sup>C]-Pro labelled pollen were gel filtered on G-50. Paper electrophoresis coupled with paper chromatography of acid hydrolyzates of salt-washed wall fractions demonstrated 15 of the common amino acids. Gel filtration on G-25 of growth medium in which pollen was germinated resulted in two peaks, one of which eluted in the void volume. contained Hyp and excluded during subsequent gel filtration on G-100.

IDENTIFICATION AND PURIFICATION OF A NEW PROTEIN FROM WATER-SOLUBLE EXTRACTS OF HUMAN ADRENAL GLAND

1979 ◽  
Vol 82 (3) ◽  
pp. 383-NP ◽  
Author(s):  
M. A. AL-AWQATI ◽  
Y. B. GORDON ◽  
T. CHARD
Keyword(s):  
Molecular Weight ◽  
Adrenal Gland ◽  
Gel Filtration ◽  
Water Soluble ◽  
Double Immunodiffusion ◽  
Molecular Weights ◽  
Physicochemical Methods ◽  
Two Peaks ◽  
Sepharose 4B ◽  
New Protein

An homogenate of human foetal adrenal gland was subjected to negative immunoabsorption by column chromatography using anti-whole human serum coupled to Sepharose 4B. Two peaks were eluted and used to immunize rabbits. The antisera produced were absorbed and tested for specificity by double immunodiffusion. Two antigens, which appeared to be specific to the adrenal gland, were identified having molecular weights of 25 000 and 65 000 as determined by gel filtration. The lower molecular weight antigen was isolated by physicochemical methods and found to be a protein. The amino acid composition is reported.

scholarly journals The separation of bovine brain β-N-acetyl-D-hexosaminidases. Abnormal gel-filtration behaviour of β-N-acetyl-D-glucosaminidase C

1975 ◽  
Vol 151 (2) ◽  
pp. 257-261 ◽  
Author(s):  
B Overdijk ◽  
W M J van der Kroef ◽  
W A Veltkamp ◽  
G J M Hooghwinkel
Keyword(s):  
Brain Tissue ◽  
Salt Concentration ◽  
Gel Filtration ◽  
Bovine Brain ◽  
Void Volume ◽  
Electrophoretic Separation ◽  
Molecular Weights ◽  
Fourth Form

Bovine brain tissue was extracted and the 50 000g supernatant was separated by electrophoresis, DEAE-Sephadex chromatography and gel filtration on Sephadex G-200 and Bio-Gel P-200. The electrophoretic separation showed that the β-N-acetyl-D-hexosaminidases (hexosaminidases) of bovine brain tissue were composed of four different fractions. Two fractions (A and B) exerted both glucosaminidase and galactosaminidase activity, a third fraction (C) showed only glucosaminidase activity, whereas a fourth form (D) with specificity towards the galactosaminide moiety was found to be present. DEAE-Sephadex chromatography at pH 7.0 showed that the B form was eluted with the void volume, whereas the A and D forms could be eluted in one peak by raising that salt concentration. The C form could not be detected in the eluate. Gel filtration on Sephadex G-200 showed that the B, A and D forms had almost equal molecular weights. In this case also the C form could not be detected in the column eluates. Gel filtration on Bio-Gel P-200 revealed that the C form was eluted with the void volume.

scholarly journals Plasmin Inhibitors in Human Urine

1977 ◽  
Author(s):  
H. Sumi ◽  
Y. Takada ◽  
A. Takada
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Molecular Form ◽  
Membrane Filter ◽  
Gel Filtration ◽  
Total Activity ◽  
Native Form ◽  
Molecular Weights ◽  
Two Peaks

By the gel filtration of urinary protein on Sephadex G-200, two peaks of activity to hydrolyze Nα-acetylglycyl-L-lysine methyl ester (AGLMe) were detected. One was the native form of urokinase, and the molecular weight was about 54,000. The other was of high molecular weight, and eluted in void fraction. The high molecular form was thought to be a complex of urokinase and urinary plasmin inhibitor (UPI). By using Arg-Sepharose, membrane filter (M.W. 10,000), and pevikon block electrophoresis, we could isolate four types of UPI from normal human urine. One UPI was positively charged at pH 8.6, and of high molecular weight. Other types were negatively charged, and the molecular weights by gel filtration on Sephadex G-200 were about 67,000 (UPI6.7), 45,000 (UPI4.5), and 22, 000 (UPI2.2), respectively. In acrylamide disc gel electrophoresis, UPI57 migrated to serum prealbumin fraction, and UPI45 and UPI? 2 were less anionic. Negative-charged UPIs could be adsorbed on trypsin-Sepharose, and were thought to be identical to urinary trypsin inhibitors. Purified UPIs showed strong inhibition on caseinolytic- and esterolytic-activities of plasmin, and the total activity was about 16 UPIU(inhibited 16 casein U of plasmin)/liter of urine.

Physicochemical and Functional Characterisation of Amaranth (Amaranthus hypochondriacus) Protein Isolates Obtained by Isoelectric Precipitation and Micellisation

2005 ◽  
Vol 11 (4) ◽  
pp. 269-280 ◽  
Author(s):  
M. Y. Cordero-de-los-Santos ◽  
J. A. Osuna-Castro ◽  
A. Borodanenko ◽  
O. Paredes-López
Keyword(s):  
Protein Denaturation ◽  
Gel Filtration ◽  
Isoelectric Precipitation ◽  
Scanning Calorimetry ◽  
Protein Isolates ◽  
Amaranthus Hypochondriacus ◽  
Molecular Weights ◽  
Functional Characterisation ◽  
Definition Of ◽  
Two Peaks

Amaranth protein isolates were obtained by two distinct methods, i.e. alkaline extraction-isoelectric precipitation (IP) and micellisation (MP). IP had a greater protein yield (56.4%) and protein content (93.1%) than MP (15.9 and 80.2%, respectively). The gel filtration chromatogram of IP isolates displayed a single peak of ca. 1,380 kDa, whereas MP isolates showed two peaks at 905kDa and 190kDa. A commercial soybean isolate (CSI), analysed for comparison purposes, presented two peaks with molecular weights of 340kDa and 62kDa. Differential scanning calorimetry showed that amaranth isolates were characterised by two endothermic events, predominating in both isolates the second endotherm with a denaturation temperature of 98.7 °C for IP and 97.2 °C for MP. The better definition of MP endotherms and their higher denaturation enthalpy suggested a more homogenous and less denatured protein population, in comparison to IP and CSI. The amaranth isolates had better solubility at alkaline pHs than the CSI. Foaming and emulsification were better at acidic pH for both IP and MP. Colorimetric evaluations showed that the two amaranth isolates had a higher whiteness index than the CSI. In conclusion, extreme pH treatments in IP resulted in a partial protein denaturation and milder treatments in MP resulted in less protein denaturation and improvement of some functional properties.

Bovine hepatic carboxylesterases chromatographic fractionation, gel filtration, and molecular weight estimation

1969 ◽  
Vol 47 (8) ◽  
pp. 799-805 ◽  
Author(s):  
D. J. Ecobichon
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Bovine Liver ◽  
Weight Estimation ◽  
Molecular Weights ◽  
Starch Gel ◽  
Two Peaks ◽  
Second Peak

The carboxylesterase activity of bovine liver was fractionated by DEAE-cellulose chromatography into two peaks of activity. Electrophoresis in starch gel showed that the peak eluted first was composed of a group of five electrophoretically slow bands while the second peak was a single, rapidly migrating band. Gel filtration of the crude extract on Sephadex G-100 and G-200 yielded a single peak of esterase activity containing both the electrophoretically slow and fast bands. The determination of molecular weights by gel filtration on Sephadex G-200 and G-100 yielded estimates of 52 000 and 55 000, respectively. The molecular weight estimates of the DEAE-cellulose fractionated electrophoretically slow and fast bands on Sephadex G-100 were identical, namely 55 000.

scholarly journals Heat inactivation and Sephadex chromatography of the small-intestine disaccharidases of the chick

1970 ◽  
Vol 116 (1) ◽  
pp. 71-78 ◽  
Author(s):  
R. C. Siddons
Keyword(s):  
Small Intestine ◽  
Gel Filtration ◽  
The Other ◽  
Void Volume ◽  
Heat Inactivation ◽  
Maltase Activity ◽  
Two Peaks ◽  
Hydrolysis Of ◽  
Second Peak ◽  
Intestinal Disaccharidases

1. The maltase, sucrase, isomaltase and palatinase activities of the chick small intestine are localized in particles that sediment when centrifuged at 100000g for 90min. 2. Solubilization of the particle-bound disaccharidases without loss of activity was achieved by digestion with papain. Trypsin was less effective and caused a preferential solubilization of the sucrase, isomaltase and palatinase activities. 3. On Sephadex G-200 columns, the solubilized preparations yielded two disaccharidase peaks. The first peak was eluted close to the void volume of the column and contained all the sucrase, isomaltase and palatinase activities and some of the maltase activity. The remainder of the maltase activity was eluted beyond the total volume of the column. 4. Precipitation with ethanol did not affect the behaviour of the disaccharidases of gel filtration. 5. The maltase activity of the second peak on rechromatography in a buffer containing 0.01m-maltose was eluted close to the void volume. 6. Similar pH optima but different Km values were obtained for the maltase activities of the two peaks. 7. Heat-inactivation studies showed that the first peak contained two disaccharidase enzymes; one hydrolysed sucrose and maltose and the other hydrolysed isomaltose, palatinose and maltose. The second peak contained three disaccharidase enzymes all specific for the hydrolysis of maltose. 8. It is proposed that the intestinal disaccharidases of the chick exist in the form of two complexes: a sucrase–isomaltase complex and a maltase complex.

Chromatographie and Electrophoretic Properties of Synthetic Human Fibrinopeptides

1974 ◽  
Vol 32 (02/03) ◽  
pp. 651-658
Author(s):  
Robin McKenzie ◽  
D. S Pepper ◽  
A. B Kay
Keyword(s):  
Thin Layer ◽  
High Voltage ◽  
Low Voltage ◽  
Gel Filtration ◽  
Paper Electrophoresis ◽  
Molecular Weights ◽  
Electrophoretic Mobilities ◽  
Rf Values ◽  
Layer Chromatography ◽  
High Voltage Electrophoresis

SummarySome properties of synthetic human fibrinopeptides were studied by thin-layer chromatography, thin-layer electrophoresis and low voltage and high voltage paper electrophoresis. The Rf values and electrophoretic mobilities of the peptides in these systems were determined. In high voltage electrophoresis synthetic and natural (fibrinogen-derived) peptides migrated in an identical fashion.When gel filtration was performed in 0.05 M pyridine or 0.1 N ammonia, synthetic fibrinopeptides A and B appeared to be aggregated. In contrast, when filtration was performed in 1.3 M formic acid, the peptides eluted in positions corresponding to their monomeric molecular weights.In addition it was possible to quantitate synthetic fibrinopeptides by two colorimetric assays, the Sakaguchi reaction and the Folin-Ciocalteu method. Ultraviolet extinction coefficients for each peptide were also determined.

scholarly journals Size heterogeneity of rat pituitary prolactin

1980 ◽  
Vol 189 (3) ◽  
pp. 605-614 ◽  
Author(s):  
M Wallis ◽  
M Daniels ◽  
S A Ellis
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Paper Electrophoresis ◽  
Freezing And Thawing ◽  
Molecular Weights ◽  
Size Heterogeneity ◽  
Very High

The occurrence of multiple forms of rat prolactin with different molecular weights (size heterogeneity) was studied with anterior pituitary extracts, purified rat prolactin and 125I-labelled rat prolactin. In each case, three main forms of the hormone were detected by gel filtration on Sephadex G-100: a major one (80–90%) corresponding to monomeric prolactin (mol.wt. 22000–25000), a peak (8–20%) that could be a dimer (mol.wt. 45000–50000) and a small quantity (1–5%) of a component of much greater molecular weight. On freezing and thawing of 125I-labelled rat prolactin, there was little interconversion of monomer and ‘dimer’ peaks, but both were converted substantially to very high-molecular-weight material. All three peaks of 125I-labelled rat prolactin could be precipitated by anti-(rat prolactin) serum and all three gave similar patterns of radioactive peptides after digestion with chymotrypsin followed by high-voltage paper electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the monomer peak of 125I-labelled prolactin migrated as a single component of mol.wt. 22000, the very high-molecular-weight peak largely dissociated to a component running in the same position as the monomer, and the ‘dimer’ peak migrated partly as a component of mol.wt. 45000 and partly as a component migrating with monomeric prolactin. No treatment was found that could dissociate the ‘dimer’ peak completely to monomeric prolactin.

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Human Fibrinogen ◽  
Human Volunteers ◽  
Cnbr Cleavage ◽  
Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

scholarly journals Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

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