Homogenization Buffer (SF9HB) for Sf9 Microsomal Preparation

2013 ◽  
Vol 2013 (11) ◽  
pp. pdb.rec076729-pdb.rec076729
Keyword(s):  
Microsomal Preparation

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 specificity of binding of growth hormone and prolactin to purified plasma membranes from pregnant-rabbit liver

1986 ◽  
Vol 236 (3) ◽  
pp. 657-663 ◽  
Author(s):  
C F Webb ◽  
H F Cadman ◽  
M Wallis
Keyword(s):  
Growth Hormone ◽  
Plasma Membranes ◽  
Human Growth ◽  
Rabbit Liver ◽  
Complex Nature ◽  
Binding Properties ◽  
Microsomal Preparation ◽  
Pregnant Rabbit ◽  
Specificity Of Binding ◽  
Triton X

The binding of 125I-labelled human growth hormone (hGH) to a purified plasma membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction with Triton X-100, was dependent on time, temperature, the cations used and the receptor concentration. Solubilization did not affect the binding properties of the receptors at low concentrations of Triton X-100. Some somatogenic hormones, such as bovine GH, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled hGH from purified plasma membranes and solubilized receptor preparations, but GHs and prolactins from various other species were rather ineffective. The results indicate that although there are binding sites for hGH in these pregnant rabbit liver membranes, few of these are specifically somatogenic or lactogenic. The binding properties of the purified plasma membranes are similar to those of a microsomal preparation studied previously, suggesting that the complex nature of the binding of hGH is not due to the heterogeneity of cellular membranes used to study binding, but is a property of the receptors associated with plasma membranes.

scholarly journals The vectorial release of nascent immunoglobulin peptides

1971 ◽  
Vol 122 (1) ◽  
pp. 5-11 ◽  
Author(s):  
Michael J. Bevan
Keyword(s):  
Immunoglobulin A ◽  
Specific Antiserum ◽  
Microsomal Preparation ◽  
Radioactive Label ◽  
Large Background

A microsomal preparation from a mouse plasmacytoma, MOPC 47A, that secretes immunoglobulin A was used to study the release of nascent immunoglobulin peptides in vitro. Nascent chains were released with puromycin and characterized with specific antiserum against the immunoglobulin product of the tumour. When the tissue had been prelabelled with [3H]leucine the experiments were complicated by the large background of completed radioactive polypeptides in the microsomal preparation. Up to one-third of the released radioactivity in the microsomal preparation could be recognized as immunoglobulin. With [3H]-puromycin as the radioactive label, however, the results are much easier to interpret, although the proportion of released radioactivity that can be identified as immunoglobulin is lower (up to one-tenth). Both types of experiment demonstrate that all of the recognizable nascent immunoglobulin chains remain in association with the microsomal vesicles after release from the ribosomes.

3,5-Diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine inactivates rat liver cytochrome P-450c, but not its orthologue, rabbit lung form 6

1990 ◽  
Vol 68 (3) ◽  
pp. 370-373 ◽  
Author(s):  
D. S. Riddick ◽  
J. E. Mackie ◽  
T. E. Massey ◽  
G. S. Marks
Keyword(s):  
Key Words ◽  
Rat Liver ◽  
Rabbit Lung ◽  
Microsomal Preparation ◽  
Liver Cytochrome ◽  
Cytochrome P 450

Various rat liver cytochrome P-450 (P-450) isozymes are targets for mechanism-based inactivation by 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (4-ethyl DDC). Unlike rat liver, which contains multiple P-450 isozymes, rabbit lung contains only three major isozymes referred to as forms 2, 5, and 6. We have examined the ability of 4-ethyl DDC to destroy P-450 heme in hepatic and pulmonary microsomes from untreated and β-naphthoflavone (βNF)-treated rabbits. This compound destroyed 31% of the P-450 in either hepatic microsomal preparation, but was ineffective at lowering P-450 and heme levels in pulmonary microsomes when examined at a range of concentrations (0.45 – 5.0 mM). These data suggest that rabbit pulmonary P-450 forms 2, 5, and 6 are not targets for destruction by 4-ethyl DDC, despite the ability of this compound to inactivate rat liver P-450c, the orthologue of rabbit lung form 6.Key words: 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine, cytochrome P-450, mechanism-based inactivation, rabbit pulmonary microsomes.

scholarly journals Synthesis and application of photoaffinity analogues of inositol 1,4,5-trisphosphate selectively substituted at the 1-phosphate group

1990 ◽  
Vol 272 (3) ◽  
pp. 817-825 ◽  
Author(s):  
R Schäfer ◽  
M Nehls-Sahabandu ◽  
B Grabowsky ◽  
M Dehlinger-Kremer ◽  
I Schulz ◽  
...  
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Inositol Phosphates ◽  
Specific Binding ◽  
Acinar Cells ◽  
Phosphate Group ◽  
Pancreatic Acinar Cells ◽  
Maximal Effect ◽  
Microsomal Preparation ◽  
Specific Binding Sites

We have synthesized two photolabile arylazido-analogues of Ins(1,4,5)P3 selectively substituted at the 1-phosphate group for determination of Ins(1,4,5)P3-binding proteins. These two photoaffinity derivatives, namely N-(4-azidobenzoyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AbaIP3) and N-(4-azidosalicyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AsaIP3), bind to high affinity Ins(1,4,5)P3-specific binding sites at a 9-fold lower affinity (Kd = 66 and 70 nM) than Ins(1,4,5)P3 (Kd = 7.15 nM) in a fraction from rat pancreatic acinar cells enriched in endoplasmic reticulum (ER). Other inositol phosphates tested showed comparable (DL-myo-inositol 1,4,5-trisphosphothioate, Kd = 81 nM) or much lower affinities for the binding sites [Ins(1,3,4,5)P4, Kd = 4 microM; Ins(1,4)P2, Kd = 80 microM]. Binding of AbaIP3 was also tested on a microsomal preparation of rat cerebellum [Kd = 300 nM as compared with Ins(1,4,5)P3, Kd = 45 nM]. Ca2+ release activity of the inositol derivatives was tested with AbaIP3. It induced a rapid and concentration-dependent Ca2+ release from the ER fraction [EC50 (dose producing half-maximal effect) = 3.1 microM] being only 10-fold less potent than Ins(1,4,5)P3 (EC50 = 0.3 microM). From the two radioactive labelled analogues ([3H]AbaIP3 and 125I-AsIP3) synthesized, the radioiodinated derivative was used for photoaffinity labelling. It specifically labelled three proteins with apparent molecular masses of 49, 37 and 31 kDa in the ER-enriched fraction. By subfractionation of this ER-enriched fraction on a Percoll gradient the 37 kDa Ins(1,4,5)P3 binding protein was obtained in a membrane fraction which showed the highest effect in Ins(1,4,5)P3-inducible Ca2+ release (fraction P1). The other two Ins(1,4,5)P3-binding proteins, of 49 and 31 kDa, were obtained in fraction P2, in which Ins(1,4,5)P3-induced Ca2+ release was half of that obtained in fraction P1. We conclude from these data that the 37 kDa and/or the 49 and 31 kDa proteins are involved in Ins(1,4,5)P3-induced Ca2+ release from the ER of rat pancreatic acinar cells.

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