scholarly journals Identification and measurement of the folates in sheep liver

1973 ◽  
Vol 136 (2) ◽  
pp. 265-278 ◽  
Author(s):  
William S. Osborne-White ◽  
Richard M. Smith
Keyword(s):  
Ion Exchange ◽  
Lactobacillus Casei ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Microbiological Assay ◽  
Similar Relationship ◽  
Stock Diet ◽  
Sheep Liver ◽  
Extraction Separation ◽  
Before And After

1. Methods are described for the extraction, separation by ion-exchange chromatography and estimation by microbiological assay of the folates in sheep liver. 2. Injection of [2-14C]-pteroylglutamate into a sheep fed on a stock diet led to extensive labelling of chromatographically separable liver folates. About 12% of the label in the liver could not be extracted by the method used. 3. Liver folates were examined in five ewes fed on restricted amounts of a diet of wheaten hay-chaff and gluten and injected weekly with vitamin B12. Chromatographic separation was followed by microbiological assay with Lactobacillus casei, Streptococcus faecalis R. and Pediococcus cerevisiae both before and after treatment of fractions with conjugase (γ-glutamylcarboxypeptidase). Evidence was obtained that the folates present were predominantly polyglutamate forms of tetrahydropteroylglutamate, 5-methyltetrahydropteroylglutamate and 5- (and 10-) formyltetrahydropteroylglutamates. Differences in the responses of the assay organisms permitted quantitative distinction between these three main classes of folates. 4. Methyltetrahydrofolates were eluted in seven successive peaks that were separated by constant increments in the logarithm of eluant [Pi]. A similar relationship existed for seven successive peaks of tetrahydrofolate and may also have existed for each of the two series of formyltetrahydrofolates. 5. Based on these and other observations it is proposed that sheep liver folates consist predominantly of the mono- to hepta-glutamates of each of the reduced pteroates identified. The methods employed allowed quantitative determinations to be made of most of the folates present. The predominant forms were hexaglutamates. 6. Four components active for L. casei were detected that could not be identified. Three of them were polyglutamates.

scholarly journals Folic acid metabolism in vitamin B12-deficient sheep. Effects of injected methionine on liver constituents associated with folate metabolism

1974 ◽  
Vol 142 (1) ◽  
pp. 105-117 ◽  
Author(s):  
Richard M. Smith ◽  
William S. Osborne-White ◽  
Jeffrey M. Gawthorne
Keyword(s):  
Folic Acid ◽  
Ion Exchange ◽  
Vitamin B12 ◽  
Lactobacillus Casei ◽  
Acid Metabolism ◽  
Major Effect ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Folate Metabolism ◽  
Folic Acid Metabolism

1. The effects of injected l-methionine (2g every second day for 28 days) on liver folates and other constituents of liver associated with folate metabolism were studied in vitamin B12-deficient ewes and their pair-fed controls receiving vitamin B12. The dose rate of methionine used was sufficient to restore almost to normal the elevated excretion in the urine of formiminoglutamate in the deficient animals. 2. Liver folates active for Lactobacillus casei, Streptococcus faecalis R and Pediococcus cerevisiae were severely depressed in deficient livers and were partly restored by methionine. Analysis of the folates after ion-exchange chromatography showed that the major effect of methionine was to increase the concentrations of tetrahydrofolates and formyltetrahydrofolates. Methyltetrahydrofolates were also increased, but there was no effect of methionine on the small amounts of incompletely reduced folates present in deficient livers. The folates present were predominantly penta-, hexa- and hepta-glutamates whether or not animals received vitamin B12 or methionine. 3. Concentrations of ATP, NAD+, NADH and NADPH were lower in freeze-clamped liver from vitamin B12-deficient sheep than in liver from pair-fed, vitamin B12-treated sheep. These changes were not affected by methionine which was also without effect on the elevated K+/Na+ ratios found in deficient livers. 4. The livers of vitamin B12-deficient animals contained lower concentrations of choline and higher concentrations of lipid than their pair-fed controls. These effects were reversed by methionine.

scholarly journals The use of pH-gradient ion-exchange chromatography to separate sheep liver cytoplasmic aldehyde dehydrogenase from mitochondrial enzyme contamination, and observations on the interaction between the pure cytoplasmic enzyme and disulfiram

1981 ◽  
Vol 199 (3) ◽  
pp. 573-579 ◽  
Author(s):  
F M Dickinson ◽  
G J Hart ◽  
T M Kitson
Keyword(s):  
Ion Exchange ◽  
Aldehyde Dehydrogenase ◽  
Active Sites ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Gradient ◽  
Original Activity ◽  
Covalent Interaction ◽  
Cytoplasmic Enzyme ◽  
Sheep Liver

1. Sheep liver cytoplasmic aldehyde dehydrogenase can be purified from contamination with the mitochondrial form of the enzyme by pH-gradient ion-exchange chromatography. The method is simple, reproducible and efficient. 2. The purified cytoplasmic enzyme retains about 2% of its original activity in the presence of a large excess of disulfiram. This suggests that the disulfiram-reactive thiol groups are not essential for covalent interaction with the aldehyde substrate during catalysis, as has sometimes been suggested. 3. Between 1.5 and 2.0 molecules of disulfiram per tetrameric enzyme molecule account for the observed loss of activity, suggesting that the enzyme may have only two functional active sites. 4. Experiments show that disulfiram-modified enzyme retains the ability to bind NAD+ and NADH.

Determination of Urinary Creatine

1961 ◽  
Vol 7 (4) ◽  
pp. 284-288 ◽  
Author(s):  
Samuel S Kurohara
Keyword(s):  
Ion Exchange ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Small Contribution ◽  
Guanidino Compounds ◽  
Before And After ◽  
X Irradiation ◽  
Color Value

Abstract A method of separation of creatine using ion-exchange chromatography is described. This method is suitable for serial determinations of urinary creatine despite a small contribution by creatinine and guanidino compounds to the color value of the α-naphthol and diacetyl reaction. The creatine coefficients of four patients, determined in duplicates, before and after the induction of creatinuria by X-irradiation are presented to illustrate the applicability of the method described.

Studies on an Inhibitor of Plasminogen Activation in Human Serum

1973 ◽  
Vol 30 (02) ◽  
pp. 414-424 ◽  
Author(s):  
Ulla Hedner
Keyword(s):  
Ion Exchange ◽  
Human Serum ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Specific Adsorption ◽  
Partial Purification ◽  
Plasminogen Activation ◽  
Preparative Electrophoresis

SummaryA procedure is described for partial purification of an inhibitor of the activation of plasminogen by urokinase and streptokinase. The method involves specific adsorption of contammants, ion-exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and preparative electrophoresis. The inhibitor fraction contained no antiplasmin, no plasminogen, no α1-antitrypsin, no antithrombin-III and was shown not to be α2 M or inter-α-inhibitor. It contained traces of prothrombin and cerulo-plasmin. An antiserum against the inhibitor fraction capable of neutralising the inhibitor in serum was raised in rabbits.

Review on the Application of Mixed-mode Chromatography for Separation of Structure Isoforms

2018 ◽  
Vol 20 (1) ◽  
pp. 56-60 ◽  
Author(s):  
Tsutomu Arakawa
Keyword(s):  
Ion Exchange ◽  
Mixed Mode ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Charged State ◽  
Partial Structure ◽  
Reverse Phase ◽  
Oligomer Formation ◽  
Structure Rearrangement ◽  
Disulfide Scrambling

Proteins often generate structure isoforms naturally or artificially due to, for example, different glycosylation, disulfide scrambling, partial structure rearrangement, oligomer formation or chemical modification. The isoform formations are normally accompanied by alterations in charged state or hydrophobicity. Thus, isoforms can be fractionated by reverse-phase, hydrophobic interaction or ion exchange chromatography. We have applied mixed-mode chromatography for fractionation of isoforms for several model proteins and observed that cation exchange Capto MMC and anion exchange Capto adhere columns are effective in separating conformational isoforms and self-associated oligomers.

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