scholarly journals The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Phospholipid depletion and re-activation of guinea-pig liver microsomal enzyme

1977 ◽  
Vol 163 (1) ◽  
pp. 117-124 ◽  
Author(s):  
A B Graham ◽  
D T Pechey ◽  
K C Toogood ◽  
S B Thomas ◽  
G C Wood
Keyword(s):  
Guinea Pig ◽  
Degradation Products ◽  
Active Form ◽  
Gel Filtration ◽  
Transferase Activity ◽  
Uridine Diphosphate ◽  
Pig Liver ◽  
Highly Active ◽  
Microsomal Membranes ◽  
Guinea Pig Liver

More than 80% of the phospholipid component of guinea-pig liver microsomal membranes (prepared with 154mM-KCl) was removed by treatment with phospholipase A followed by extraction of the lysophosphatides and fatty acids produced with albumin. Delipidation strongly inactivated the highly active UDP-glucuronyltransferase of these preparations and activity was restored by mixtures of phosphatidylcholine and lysophosphatidylchlone. However, small quantities of lysophosphatides were still associated with the delipidated fractions after extraction with albumin and might have influenced the inactivation and re-activation observed. To eliminate these uncertainties, microsomal proteins and phospholipids were separated by gel filtration on Sephadex G-150 in the presence of cholate. This technique also strongly inactivated the enzyme but did not generate membrane-active phospholipid degradation products. High transferase activity was again restored to the delipidated protein by choline glycerophosphatides. These results confirm the view that the fully active form of microsomal UDP-glucuronyltransferase is phospholipid-dependent.

scholarly journals Purification and properties of a new testosterone 17β-dehydrogenase (NADP+) from guinea-pig liver

1977 ◽  
Vol 163 (3) ◽  
pp. 401-407 ◽  
Author(s):  
E Kaguera ◽  
S Toki
Keyword(s):  
Guinea Pig ◽  
Column Chromatography ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Supernatant Fraction ◽  
Pig Liver ◽  
Ring Junction ◽  
Purification And Properties ◽  
Androstane Derivatives ◽  
Guinea Pig Liver

As a result of studies of guinea-pig live testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), a new testosterone 17beta-dehydrogenase was discovered. The new enzyme was purified to a single homogeneous protein from the 105 000 g-supernatant fraction of guinea-pig liver by (NH4)2SO4 fractional precipitation and two gel-filtration stages, DEAE-cellulose column chromatography and hydroxyapatite column chromatography. It was characterized by many properties. The enzyme has almost the same properties as the classical testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), with respect to cofactor requirement, pH optima for dehydrogenation, effect of phosphate ion on the NAD+-dependent reaction and molecular weight, but characteristic differences were observed in substrate-specificity between the two dehydrogenases. With various androstane derivatives, the configuration of the A/B-ring junction was closely connected with enzyme activity. 5alpha-Androstanes, such as 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol and 17beta-hydroxy-5alpha-androstan-3-one, and 5beta-congeners, such as 5beta-androstane-3alpha,17beta-diol, 5beta-androstane-3beta,17beta-diol and 17beta-hydroxy-5beta-androstan-3-one, served as substrates for both the EC 1.1.1.64 enzyme and the new enzyme. The EC 1.1.1.64 enzyme oxidized testosterone more rapidly than did the new enzyme. These comparisons were based on the relative activities, apparent Km values and apparent Vmax values.

Biosynthesis of phosphatidylglycerol and phosphatidylglycerolphosphate in submitoehondrial membranes isolated from guinea pig liver is absolutely dependent on CDP-diglycerides imported from microsomal membranes

1990 ◽  
Vol 68 (1) ◽  
pp. 111-116 ◽  
Author(s):  
Lidija Stuhne-Sekalec ◽  
Nikola Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Microsomal Membranes ◽  
Guinea Pig Liver

The biosynthesis of radioactively labelled phosphatidylglycerol via phosphatidylglycerophosphate in outer and inner mitochondrial membranes isolated from guinea pig liver was found to depend absolutely on CDP-diglycerides, which could not be biosynthesized in these membranes. The requirement for CDP-diglycerides in the biosynthesis of labelled phosphatidylglycerol could be fulfilled by the transfer of biosynthesized [3H]CDP-diglycerides from the microsomal membranes to the outer and inner mitochondrial membranes.Key words: submitoehondrial membranes, transfer, CDP-diglycerides, phosphatidylglycerol, phosphatidylglycerophosphate.

Mitochondrial importation of lipids and liponucleotides from microsomes independent of and facilitated by purified cytosol proteins

1980 ◽  
Vol 58 (10) ◽  
pp. 1082-1090 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
N. Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Neutral Lipids ◽  
Protein S ◽  
Mitochondrial Import ◽  
Pig Liver ◽  
Conventional Procedure ◽  
Independent Mechanism ◽  
Microsomal Membranes ◽  
Experimental Findings ◽  
Guinea Pig Liver

The mitochondrial importation of microsomal lipids and liponucleotides in the presence and in the absence of partially purified cytosol protein(s) isolated from guinea pig liver was studied by the aid of isomeric (5-, 12-, and 16-(N-oxyl-4′,4′-dimethyloxazolidine)stearoyl) spin-labelled radioactive phosphatidic acid, phosphatidylcholine, neutral lipids, and CDP-diglycerides. Using a conventional procedure for the protein purification, cytosol protein(s) was purified approximately 1000-fold in respect to its ability to catalyze the translocation of isomeric spin-labelled lipids and liponucleotides from the microsomal to mitochondrial membranes. The highest activity of this protein was exhibited with biosynthesized spin-labelled lipids and liponucleotides bound to the microsomal membranes as substrates and the lowest, with the synthetic liponucleotides and derived lipids bound to the microsomal membranes. The partially purified protein was active in catalyzing the mitochondrial import of phospholipids from microsomes after heat treatment up to 90 °C.In addition to the cytosol protein catalyzing mechanism of mitochondrial import of lipids and liponucleotides from microsomal membranes, another cytosol protein independent mechanism of the mitochondrial importation of the same lipids and liponucleotides was also demonstrated in an agreement with our previous reports on the existence of cytosol protein independent intermembranous translocation of phospholipids. These experimental findings are discussed in terms of possible physiological significance and reaction mechanisms involved in the mitochondrial import of lipids and liponucleotides from the microsomal membranes of guinea pig liver.

Expression and rapid purification of highly active hexahistidine-tagged guinea pig liver transglutaminase

2004 ◽  
Vol 33 (2) ◽  
pp. 256-264 ◽  
Author(s):  
Steve M.F.G. Gillet ◽  
Roberto A. Chica ◽  
Jeffrey W. Keillor ◽  
Joelle N. Pelletier
Keyword(s):  
Guinea Pig ◽  
Pig Liver ◽  
Highly Active ◽  
Rapid Purification ◽  
Guinea Pig Liver

Biosynthesis, utilization, and translocation of endogenous isomeric spin-labelled radioactive cytidinediphosphodiglycerides from isolated guinea pig liver microsomal to mitochondrial membranes

1979 ◽  
Vol 57 (7) ◽  
pp. 1019-1025 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
N. Z. Stanacev
Keyword(s):  
Cytochrome C ◽  
Guinea Pig ◽  
Reductase Activity ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Cytochrome C Reductase ◽  
Lipid Species ◽  
Microsomal Membranes ◽  
Guinea Pig Liver ◽  
Nadph Cytochrome C Reductase

When isolated guinea pig liver microsomal membranes were incubated with isomeric (5-, 12-, and 16-doxyl stearoyl) spin-labelled sn-3-[2-3H]phospfaatidic acid in the presence of CTP and Mg2+, formation of corresponding CDP-[2-3H]diglycerides (in an amount representing 16.5–17.4% of the labelled lipids), which were acceptable substrates in the microsomal biosynthesis of sn-3-[2-3H]phosphatidyl-myo-[U-l4C]inositols, took place. When microsomal membranes containing known amounts of labelled CDP-diglycerides were incubated with unlabeled mitochondrial membranes, reisolated mitochondria contained labelled lipids in an amount which could not be accounted for by the microsomal contamination of reisolated mitochondria, determined by the assay of NADPH – cytochrome c reductase activity, establishing therefore the translocation of labelled CDP-diglycerides (and other labelled lipids) from microsomal to mitochondrial membranes in an amount of ~50% of microsomal content. The rate of loss of paramagnetic lipid species in microsomal and in reisolated mitochondrial membranes was found to be quite different. When reisolated mitochondria containing trans-located isomeric spin-labelled CDP-[2-3H]diglycerides were further incubated with sn-3-[U-14C]glycerophosphate, the formation of labelled phosphatidylglycerophosphate and phosphatidylglycerol was detected. These findings established that the translocation of endogenously formed CDP-[2-3H]diglycerides occurred from isolated microsomal membranes to both outer and inner mitochondrial membranes.

Reduced Levels of Uridine Diphosphate Glucose Pyrophosphorylase Activity in Fetal and Neonatal Human and Guinea Pig Liver

Neonatology ◽  
1989 ◽  
Vol 56 (3) ◽  
pp. 174-180
Author(s):  
G. Vaca ◽  
L.P. Castro-Félix ◽  
C. Medina ◽  
M.D. Medina ◽  
R. Blancarte ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Uridine Diphosphate ◽  
Pig Liver ◽  
Uridine Diphosphate Glucose ◽  
Diphosphate Glucose ◽  
Guinea Pig Liver

Phosphatidylinositol movement between isolated microsomal and mitochondrial membranes

1983 ◽  
Vol 61 (12) ◽  
pp. 1282-1291 ◽  
Author(s):  
J. Chudzik ◽  
N. Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Direct Contact ◽  
Reaction Medium ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Liver Cytosol ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Phosphatidylinositol Transfer ◽  
Guinea Pig Liver

Transfer of membrane-bound phosphatidyl-[2′-3H]inositol from microsomal to unlabelled mitochondrial and from mitochondrial to unlabelled microsomal membranes was studied using partially purified cytosol proteins isolated from guinea pig liver cytosol. In the absence and presence of these proteins the amounts of phosphatidylinositol transfer from microsomal to mitochondrial membranes were approximately 21 and 33%, respectively, and the amounts from mitochondrial to microsomal membranes were approximately 31 and 39%, respectively. The release of phosphatidyl-[2′-3H]inositol from microsomal membranes in the absence of mitochondria was dependent on concentration of cytosol proteins. Two mechanisms for movement between membranes are proposed. In cytosol-protein-independent movement of phosphatidyl-[2′-3H]inositol from microsomal to mitochondrial membranes, a direct contact between membranes is required, since phosphatidyl-[2′-3H]inositol was not detected in the reaction medium. In the cytosol-protein-catalyzed transfer, formation of phosphatidyl-[2′-3H]inositol – cytosol protein complex is postulated, since phosphatidyl-[2′-3H]inositol was released into the reaction medium and its movement proceeded from mitochondrial to microsomal membranes in the presence of partially purified cytosol proteins. Thus, contact between the two membranes is probably not necessary for this transfer. Implications for the movement of phospholipids between biological membranes are discussed.

Selective degradation with phospholipases D and C of radioactive isomeric spin-labelled lipids bound to guinea pig liver microsomal membranes

1978 ◽  
Vol 56 (10) ◽  
pp. 943-951 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
N. Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Microsomal Membrane ◽  
Spectral Parameters ◽  
Pig Liver ◽  
Selective Degradation ◽  
Bound Lipids ◽  
Before And After ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Guinea Pig Liver

Membrane-bound lipids of isolated guinea pig liver microsomal membranes were selectively enzymatically labelled with isomeric (5-, 12-, and 16-)doxyl stearic acid. After reisolation, the membranes were degraded with phospholipases D and C under conditions not requiring detergents or organic solvent activators. The degradation of membrane-bound lipids occurred according to the recognized specificity of phospholipases D and C. Temperature-induced changes of degraded membranes containing radioactive spin-labelled isomeric lipids were followed by the electron spin resonance and spectral changes correlated with the lipid composition of membranes. Discontinuities in plots of experimental spectral parameters versus temperature detected in the case of microsomal membranes before and after degradation with phospholipases D and C were attributed to lipid–protein and lipid–lipid interaction(s). On the basis of these and control experiments, discontinuity at around 10–12 °C was attributed to the microsomal membrane phosphatidylcholine intrinsic microsomal membrane protein interaction(s), while discontinuities detected at 19–21 °C approximately and at 20–30 °C approximately were attributed to the phase separation of Ca or Zn salts of membranous phosphatidic acid and to the similar phenomenon involving membrane-bound diglycerides respectively.

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