Storage Buffer (SB) for Microsomal Preparation

2013 ◽  
Vol 2013 (11) ◽  
pp. pdb.rec076695-pdb.rec076695
Keyword(s):  
Microsomal Preparation ◽  
Storage Buffer

scholarly journals 3-Hydroxy-3-methylglutaryl-coenzyme A reductase. A comparison of the modulation in vitro by phosphorylation and dephosphorylation to modulation of enzyme activity by feeding cholesterol- or cholestyramine-supplemented diets

1980 ◽  
Vol 185 (2) ◽  
pp. 435-441 ◽  
Author(s):  
Konstantinos A. Mitropoulos ◽  
Brian L. Knight ◽  
Bernard E. A. Reeves
Keyword(s):  
Microsomal Fraction ◽  
Coenzyme A ◽  
Maximal Activity ◽  
Kinetic Properties ◽  
Standard Diet ◽  
Microsomal Preparation ◽  
Phosphoprotein Phosphatase ◽  
Coa Reductase ◽  
Hydroxymethylglutaryl Coa Reductase ◽  
Coenzyme A Reductase

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hydroxymethylglutaryl-CoA reductase) was considerably inhibited during incubation with ATP+Mg2+. The inactivated enzyme was reactivated on further incubation with partially purified cytosolic phosphoprotein phosphatase. The inactivation was associated with a decrease in the apparent Km of the reductase for hydroxymethylglutaryl-CoA, and this was reversed on reactivation. The slight increase in activity observed during incubation of microsomal fraction without ATP was not associated with a change in apparent Km and, unlike the effect of the phosphatase, was not inhibited by NaF. Liver microsomal fraction from rats given cholesterol exhibited a low activity of hydroxymethylglutaryl-CoA reductase with a low apparent Km for hydroxymethylglutaryl-CoA. Mícrosomal fraction from rats fed cholestyramine exhibited a high activity with a high Km. To discover whether these changes had resulted from phosphorylation and dephosphorylation of the reductase, microsomal fraction from rats fed the supplemented diets and the standard diet were inactivated with ATP and reactivated with phosphoprotein phosphatase. Inactivation reduced the maximal activity of the reductase in each microsomal preparation and also reduced the apparent Km for hydroxymethylglutaryl-CoA. There was no difference between the preparations in the degree of inactivation produced by ATP. Treatment with phosphatase restored both the maximal activity and the apparent Km of each preparation, but never significantly increased the activity above that observed with untreated microsomal fraction. It is concluded that hydroxymethylglutaryl-CoA reductase in microsomal fraction prepared by standard procedures is almost entirely in the dephosphorylated form, and that the difference in kinetic properties in untreated microsomal fraction from rats fed the three diets cannot be explained by differences in the degree of phosphorylation of the enzyme.

scholarly journals The transfer of mannose to dolichol diphosphate oligosaccharides in pig liver endoplasmic reticulum

1975 ◽  
Vol 152 (2) ◽  
pp. 191-199 ◽  
Author(s):  
Gerard J. A. Oliver ◽  
Frank W. Hemming
Keyword(s):  
Endoplasmic Reticulum ◽  
Periodate Oxidation ◽  
The Other ◽  
Microsomal Preparation ◽  
Pig Liver ◽  
Mannose Residue ◽  
Chromatographic Data ◽  
Endogenous Lipid

The transfer, catalysed by pig liver microsomal preparations, of mannose, from GDP-mannose, to lipid-linked oligosaccharides and the properties of the products are described. Solubility, hydrolytic and chromatographic data suggest that they are dolichol diphosphate derivatives. The presence of two N-acetyl groups in at least part of the heterogenous oligosaccharide portion was tentatively deduced. Reduction with borohydride of the oligosaccharide showed that the newly added mannose residues were not at its reducing end. Periodate oxidation suggested that 60% of these were at the non-reducing terminus and that 40% were positioned internally. T.l.c. showed the presence of seven oligosaccharide fractions with chromatographic mobilities corresponding to glucose oligomers with 7–13 residues. The molar proportions of the oligosaccharide fractions in the mixture were determined by borotritiide reduction and the number of mannose residues added to each oligosaccharide fraction during the incubation was calculated. Two of the oligosaccharide fractions had received on average one, or slightly more than one, mannose residue per chain during the incubation; four of the other fractions were each shown to be a mixture, 20–25% of which had received one mannose residue during the incubation and 75–80% of which had not been mannosylated during the incubation. This supported other evidence for the presence of endogenous lipid-linked oligosaccharides in the microsomal preparation which had been formed before the incubation in vitro. Evidence for the possibility of two pools of dolichol monophosphate mannose, one being more closely associated with mannosyl transfer to dolichol diphosphate oligosaccharides than the other, is also discussed.

REDUCTIVE PATHWAYS OF PROGESTERONE METABOLISM IN THE RAT OVARY

1972 ◽  
Vol 69 (1) ◽  
pp. 141-152 ◽  
Author(s):  
A. Zmigrod ◽  
H. R. Lindner ◽  
S. A. Lamprecht
Keyword(s):  
Hydroxysteroid Dehydrogenase ◽  
Prolactin Secretion ◽  
Ovariectomized Rats ◽  
Corpora Lutea ◽  
Microsomal Preparation ◽  
Progesterone Metabolism ◽  
Rat Ovary ◽  
Reducing Activity ◽  
Pituitary Prolactin

ABSTRACT Progesterone underwent extensive reductive metabolism when incubated with a microsomal preparation from rat ovaries in the presence of NADPH. The major products formed were 3β-hydroxy-5α-pregnan-20-one, 5α-pregnane-3,20-dione and 5α-pregnane-3β,20α-diol. Newly formed corpora lutea of pregnancy were almost devoid of any microsomal A-ring reducing activity (5α-reductase and a 3β-hydroxysteroid dehydrogenase) and of soluble 20α-hydroxysteroid dehydrogenase. The behaviour of the A-ring reducing enzymes paralleled that of 20α-ol dehydrogenase in that their activity (i) was high during the oestrous cycle; (ii) declined between the third and seventh day of pregnancy; and (iii) increased sharply in corpora lutea of pregnancy when ergocornine – a drug blocking pituitary prolactin secretion – was given to the rats, yet remained low when prolactin and ergocornine were administered concurrently. However, the A-ring reducing activity did not show the sharp pre-partum rise exhibited by 20α-ol dehydrogenase, thus deviating from a pattern compatible with a co-ordinate control of the three enzymes involved in the metabolic inactivation of progesterone. Contrary to a report in the literature, 5α-pregnane-3,20-dione (20 mg/rat/day) was found to be ineffective when tested for pregnancy or deciduoma supporting activity in ovariectomized rats. The microsomal reductases, if indeed operative in vivo, may restrict the availability of progesterone as an oestrogen precursor.

scholarly journals The impact of storage buffer, DNA extraction method, and polymerase on microbial analysis

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Luisa K. Hallmaier-Wacker ◽  
Simone Lueert ◽  
Christian Roos ◽  
Sascha Knauf
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Dna Extraction Method ◽  
Microbial Analysis ◽  
Storage Buffer ◽  
The Impact
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