scholarly journals Lipid requirement of the membrane sodium-plus-potassium ion-dependent adenosine triphosphatase system

1975 ◽  
Vol 146 (3) ◽  
pp. 713-722 ◽  
Author(s):  
K P Wheeler ◽  
J A Walker ◽  
D M Barker
Keyword(s):  
Adenosine Triphosphatase ◽  
Specific Activity ◽  
Total Phospholipid ◽  
Kidney Tissue ◽  
Residual Activity ◽  
Fold Increase ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Phospholipid Content ◽  
Total Phospholipid Content

The dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) on lipid has been examined in a number of different ways, with the use of various preparations from kidney tissue. The main findings were as follows. (1) The ATPase activities of the preparations examined were closely correlated with their total phospholipid content. (2) Extraction of the ATPase with deoxycholate or Lubrol W, combined with suitable salt-fractionation and washing procedures, removed phospholipid, cholesterol and enzymic activity in parallel; but activity was completely lost before all lipid had been removed. (3) The loss of activity could not be attributed to inhibition by residual detergent. (4) No selective removal of any particular phospholipid class by detergent could be detected. (5) Consistent reactivation of the Lubrol-extracted enzymes was obtained by adding dispersions of exogenous phospholipid, but only some, bearing a net negative charge, such as phosphatidylserine and phosphatidylglycerol, were effective. (6) The degree of reactivation was correlated with the amount of residual activity remaining after lipid depletion. (7) Partial purification of the ATPase, giving a 50-fold increase in specific activity, was not accompanied by selective enhancement of any particular class of phospholipid. We conclude that although the ATPase is dependent on phospholipid, only the reactivation results provide evidence for specificity.

Effect of fetal exposure to gentamicin on phosphate transport in young rat kidney

1993 ◽  
Vol 265 (6) ◽  
pp. F807-F812
Author(s):  
M. Lelievre-Pegorier ◽  
S. Euzet ◽  
C. Merlet-Benichou
Keyword(s):  
Brush Border Membrane ◽  
Rat Kidney ◽  
Total Phospholipid ◽  
Phospholipid Composition ◽  
Cholesterol Content ◽  
Phosphate Transport ◽  
Phospholipid Content ◽  
Postnatal Maturation ◽  
Rat Pups ◽  
Total Phospholipid Content

The renal phosphate (Pi)-transporting capacity normally increases, due to increased carrier system affinity, during the third postnatal week in rats. However, the tubular Pi reabsorption of rat pups born from gentamicin-treated mothers does not increase during this period. This study determines whether exposure to gentamicin in utero selectively alters the postnatal maturation of the carrier affinity for Pi. Pi and glucose transports by proximal tubule brush-border membrane (BBM) were studied. The maximal rate of uptake (Vmax) of Na-Pi cotransport was significantly lower (536 +/- 169 pmol.mg protein-1.10 s-1; n = 6, P < 0.01) in gentamicin-exposed rats than in controls (1,021 +/- 167 pmol.mg protein-1.10 s-1, n = 6), whereas the Michaelis constant (Km) values were the same. Gentamicin exposure had no effect on plasma parathyroid hormone concentration or on BBM glucose transport activity. The total phospholipid content of BBM, their phospholipid composition, cholesterol content, and cholesterol-to-total phospholipid mole ratio were unaltered, suggesting that membrane fluidity was unchanged. The Vmax of BBM alkaline phosphatase was lower in gentamicin-exposed rats than in controls.

Metabolic basis of diethylaminoethoxyhexestrol-induced phospholipid fatty liver

1987 ◽  
Vol 252 (3) ◽  
pp. E375-E379 ◽  
Author(s):  
M. Kubo ◽  
K. Y. Hostetler
Keyword(s):  
Fatty Liver ◽  
Foam Cell ◽  
Total Phospholipid ◽  
Phospholipid Content ◽  
Liver Phospholipid ◽  
Phospholipase A1 ◽  
Total Phospholipid Content ◽  
Metabolic Basis ◽  
First Time

Diethylaminoethoxyhexestrol caused a foam cell lipidosis in humans characterized by phospholipid storage in the liver, spleen, and other tissues, and this represents the first description of acquired lipidosis caused by a drug. It has been proposed that diethylaminoethoxyhexestrol causes phospholipid fatty liver by concentrating in lysosomes and inhibiting phospholipases but it has not previously been possible to measure the intralysosomal concentration of diethylaminoethoxyhexestrol. In this paper we report for the first time the intralysosomal concentration of this drug in rats. After a single oral dose of diethylaminoethoxyhexestrol (100 mg/kg) the intralysosomal concentration was 7.9 mM at 2.5 h, 15.6 mM at 12 h, and 20.9 mM at 24 h, respectively. The total phospholipid content of lysosomes in drug-treated rats increased 1.9-, 6.0-, and 7.6-fold over control at 2.5, 12, and 24 h, respectively. Purified lysosomal phospholipase A1 was strongly inhibited by diethylaminoethoxyhexestrol in vitro. In phospholipid fatty liver, phospholipid accumulation in lysosomes appears to be caused by the presence of diethylaminoethoxyhexestrol in lysosomes at concentrations estimated to be 7.9–20 mM, because drug levels above 1 mM completely block the activity of purified lysosomal phospholipase A1 in vitro.

scholarly journals Hepatic microsomal Ca2+-dependent ATPase. Calmodulin-dependence and partial purification

1983 ◽  
Vol 214 (1) ◽  
pp. 69-75 ◽  
Author(s):  
P B Moore ◽  
N Kraus-Friedmann
Keyword(s):  
Affinity Chromatography ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Fold Increase ◽  
Partial Purification ◽  
Dependent Atpase

The hepatic microsomal fraction contains tightly bound calmodulin as demonstrated by affinity chromatography. When this calmodulin was partially removed by EGTA treatment (0.5 mM-EGTA), the uptake of 45Ca2+ by the microsomal vesicles was stimulated by added calmodulin and inhibited by trifluoperazine (TFP). The Ca2+-dependent ATPase was partially purified on a calmodulin column. This partial purification resulted in a 500-fold increase in the specific activity of the enzyme when measured in the presence of added calmodulin. Antibodies prepared against calmodulin prevented this stimulatory effect. The fraction eluted from the calmodulin column contained several protein bands indicating that the specific activity of the Ca2+-dependent ATPase is probably still underestimated. There are likely to be other calmodulin-sensitive processes present in the hepatic microsomal fraction.

STUDIES IN PHOSPHOLIPID METABOLISM: I. EFFECT OF GUANIDOACETIC ACID AND CHOLINE ON LIVER PHOSPHOLIPIDS

1964 ◽  
Vol 42 (8) ◽  
pp. 1183-1194 ◽  
Author(s):  
J. Blumenstein
Keyword(s):  
Total Phospholipid ◽  
Phospholipid Metabolism ◽  
Phospholipid Content ◽  
Subsequent Reduction ◽  
Total Phospholipid Content

An attempt was made to produce 'forced methylation' and subsequent reduction of lecithin content of livers of rats fed a semipurified diet. The addition of guanidoacetic acid to the diet of the rats did not alter either the total phospholipid extracted from their livers or the liver lecithin content significantly. This constant pattern was observed whether choline was included in the diet or not. However, in animals fed a diet deficient in choline, the ratio of lecithin and cephalin extracted from their livers was altered, although the total phospholipid content remained constant.

Partial purification of a hypertensive substance from rat erythrocytes

1985 ◽  
Vol 63 (10) ◽  
pp. 1321-1326 ◽  
Author(s):  
William D. McCumbee ◽  
Gary L. Wright
Keyword(s):  
Blood Pressure ◽  
Systolic Blood Pressure ◽  
Calcium Uptake ◽  
Specific Activity ◽  
Present Report ◽  
Fold Increase ◽  
Partial Purification ◽  
Rat Erythrocytes ◽  
In Vitro Effects

A crude extract prepared from rat erythrocytes was previously shown to contain an active component (hypertensive factor, HF) that stimulates the in vitro uptake of calcium by aortic rings and causes a sustained and severe elevation in systolic blood pressure in normotensive rats. The present report demonstrates that the in vitro effects of HF on calcium uptake are concentration dependent and reversible, and that these effects on calcium uptake provide a convenient bioassay for monitoring the purification of HF. HF was prepared from a diffusate obtained by dialyzing hemolyzed rat erythrocytes. The diffusate was applied successively to molecular sieve and anion exchange columns resulting in a 1500-fold increase in specific activity relative to the starting hemolysate. The compound stimulating calcium uptake was found to be heat stable and resistant to digestion with trypsin and chymotrypsin. In contrast, treatment with pronase E, a nonspecific protease, markedly increased the calcium transport stimulatory activity. The administration of the purified preparation to normotensive rats having a systolic blood pressure of 118 ± 2 Torr (1 Torr = 133.322 Pa) resulted in a significant elevation of blood pressure (171 ± 11 Torr) by day 5. The severity of this increase further suggests that there is a marked enhancement in potency relative to earlier fractions that were examined. Perhaps most importantly, these results indicate that, at this stage of purification, the pressor component and the calcium-stimulatory component of HF appear to co-purify.

708. Phospholipids in New Zealand dairy products: II. Seasonal variations in the phospholipid content of butter and of milk and cream

1958 ◽  
Vol 25 (2) ◽  
pp. 202-214 ◽  
Author(s):  
A. K. R. McDowell
Keyword(s):  
New Zealand ◽  
Seasonal Variations ◽  
Dairy Products ◽  
Total Phospholipid ◽  
Skim Milk ◽  
Phospholipid Content ◽  
Early Lactation ◽  
Fat Globules ◽  
Total Phospholipid Content

Samples of milk, skim-milk and raw cream from one factory and of pasteurized cream, buttermilk and butter from two factories were taken twice monthly for varying periods for estimation of total phospholipids and (in butter only) of lecithin, cephalin and sphingomyelin.Seasonal variations in the phospholipid contents of milk, skim-milk and raw cream over 13 months probably were due to the greater proportion of small fat globules in autumn (late lactation) milk compared with spring (early lactation) milk.There were regularly recurring seasonal variations in total phospholipid content of the butters over 3 years. Maximum results were found during autumn and minimum results in winter. Lecithin, cephalin and sphingomyelin contents followed the same trends as the total phospholipid contents.Seasonal variations in phospholipid contents of buttermilks and butters were due mainly to variations in the amount of phospholipid per unit of fat in the cream. Differences in butter-making technique had little effect on the proportion of phospholipids from the cream retained in the butter.Average results for total phospholipid content of the products examined were: milk, 0·038% (0·031–0·050%); skim-milk, 0·018% (0·014–0·023%); buttermilk, 0·156% (0·103–0·191%); butter, 0·133% (0·099–0·181%). The average result for percentage of total phospholipid in the fat of the raw creams examined was 0·44% (0·38–0·51%); and in the fat of the pasteurized creams 0·41% (0·35–0·49%). Average weighted results for total phospholipid and for lecithin, cephalin and sphingomyelin contents of butter were 0·139, 0·041, 0·051 and 0·047% respectively.

scholarly journals Partial purification and some properties of a cholinesterase from bush bean (Phaseolus vulgaris L.) roots

1978 ◽  
Vol 175 (3) ◽  
pp. 769-777 ◽  
Author(s):  
D H Mansfield ◽  
G Webb ◽  
D G Clark ◽  
I E P Taylor
Keyword(s):  
Phaseolus Vulgaris ◽  
Specific Activity ◽  
Apparent Molecular Weight ◽  
Fold Increase ◽  
Partial Purification ◽  
Phaseolus Vulgaris L ◽  
Native Form ◽  
Bush Bean ◽  
Electric Eel ◽  
Stokes Radius

A cholinesterase was partially purified from bush bean (Phaseolus vulgaris L.) roots by using acridinium-based ligand affinity chromatography. The procedure gave a 78-fold increase in specific activity, although at least three inactive contaminants remained. The enzyme activity was maximal against acetyl esters of choline and was inhibited by neostigmine. Di-isopropyl phosphorofluoridate completely inhibited activity at concentrations greater than 0.1 mM. The catalytic centre activity was 2 × 10(-4) times that of electric eel acetylcholinesterase. Cholinesterase activity appeared as a peak (s = 4.2 +/- 0.1 S) after isokinetic sedimentation. The Stokes radius was 4.00 nm and the apparent molecular weight was 72700 +/- 1900. The smallest active and native form of the enzyme appeared to be a monomer. This contrasts with animal acetylcholinesterases, in which the smallest active and native forms are multimeric.

scholarly journals Partial Purification, Characterization, and Application of Extracellular Aspartic Protease from Lactobacillus casei WSP in Producing the Bioactive Peptides with Antibacterial and Antioxidant Activity

2018 ◽  
Vol 22 (2) ◽  
pp. 47
Author(s):  
Akhmad Solikhin ◽  
Apon Zaenal Mustopa ◽  
Suharsono Suharsono ◽  
Wendry Setiyadi Putranto
Keyword(s):  
Enzyme Activity ◽  
Metal Ion ◽  
Lactobacillus Casei ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Iodoacetic Acid ◽  
Aspartic Protease ◽  
Partial Purification ◽  
Antioxidant Agents

   Lactobacillus casei WSP-derived an aspartic protease was sequentially purified by using chromatography gel filtration sephadex G-50. It resulted in a 22.81-fold increase of specific activity (51.5 U/mg) with a final yield of 1.9%. The estimated molecular weight of the purified enzyme was 37 kDa and showed gelatinolytic activity in zymogram assay. The enzyme exhibited optimum activity at 40ºC and pH 6 with casein as the substrate. Enzyme activity was significantly inhibited by pepstatin A (0.5 mM and 1 mM), confirming that this enzyme is a group of aspartic proteases, while other inhibitors such as EDTA, PMSF and iodoacetic acid showed no inhibition effect on the activity of enzyme. The addition of metal ion to the enzyme decreased enzyme activity, indicating the proteolytic enzyme was metal ion- dependent. Denaturant such as DDT tended to increase caseinolytic activity. Furthermore, this enzyme was capable of generating the new peptides from skimmed milk with the size 8 kDa, 10 kDa and 15 kDa. These peptides have potential as antibacterial and antioxidant agents.

scholarly journals Effect of hepatic injury on prolyl 3-hydroxylase and 4-hydroxylase activities in rat liver and on immunoreactive prolyl 4-hydroxylase concentrations in the liver and serum

1978 ◽  
Vol 170 (1) ◽  
pp. 129-135 ◽  
Author(s):  
J Risteli ◽  
L Tuderman ◽  
K Tryggvason ◽  
K I Kivirikko
Keyword(s):  
Protein Concentration ◽  
Hepatic Injury ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Partial Purification ◽  
Hydroxylase Activity ◽  
Enzyme Protein ◽  
Carbon Tetrachloride Administration ◽  
Total Enzyme

After severe hepatic injury induced by dimethylnitrosamine, approximately a 4-fold increase in hepatic prolyl 4-hydroxylase activity occurred within 4 days, whereas the increases in total immunoreactive prolyl 4-hydroxylase protein and in prolyl 3-hydroxylase activity were only about 1.4-fold. The different magnitudes of the increases in the prolyl 4-hydroxylase and 3-hydroxylase activities were verified after partial purification of the enzymes by gel filtration. The data support previous reports indicating differential increases in the activities of individual enzymes of collagen biosynthesis in hepatic injury. Separation of prolyl 4-hydroxylase tetramers from the monomer-size protein by gel filtration indicated that the increase in enzyme activity was similar to that in enzyme tetramers, and an increase had also occurred in the ratio of enzyme tetramers to total enzyme protein. Thus the specific activity of the tetramers had remained unchanged in liver injury. The administration of dimethylnitrosamine was also accompanied by a marked increase in the immunoreactive prolyl 4-hydroxylase protein concentration in the serum, and a similar effect was also noted after carbon tetrachloride administration, results suggesting that the increases originated in the liver.

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