scholarly journals The relationship between palmitoyl-coenzyme A synthetase activity and esterification of sn-glycerol 3-phosphate by the microsomal fraction of guinea-pig intestinal mucosa

1973 ◽  
Vol 132 (4) ◽  
pp. 707-715 ◽  
Author(s):  
David N. Brindley
Keyword(s):  
Guinea Pig ◽  
Intestinal Mucosa ◽  
Specific Activity ◽  
Synthetase Activity ◽  
Glycerol Phosphate ◽  
Acyltransferase Activity ◽  
Low Concentrations ◽  
System 2 ◽  
The Mean ◽  
Glycerolipid Synthesis

1. With microsomal fractions of guinea-pig intestinal mucosa the mean specific activity of palmitoyl-CoA synthetase was approx. 1.3-fold the esterification of sn-glycerol 3-phosphate with palmitoyl-CoA generated by the endogenous synthetase. The latter activity was approx. 2.5- and 5-fold that when palmitoyl-CoA was generated from palmitoylcarnitine or when it was added directly to the assay system. 2. There were significant correlations (P<0.001) between the specific activities of palmitoyl-CoA synthetase and glycerolipid synthesis from either palmitate or palmitoylcarnitine. 3. The mean molar composition of glycerolipid synthesized from palmitate or palmitoylcarnitine was approx. 18% lysophosphatidate, 75% phosphatidate and 7% neutral lipid. 4. Glycerolipid synthesis from palmitate was inhibited by 80–90% after preincubation of microsomal fractions at 37°C for 40min and was caused by inactivation of palmitoyl-CoA synthetase. 5. Addition of 100–400mm-KCl inhibited palmitoyl-CoA synthetase activity and glycerolipid synthesis from palmitate but stimulated glycerol phosphate acyltransferase activity. 6. Diversion of palmitoyl-CoA synthesized by the endogenous synthetase to palmitoylcarnitine resulted in an almost stoicheiometric decrease in glycerolipid synthesis. 7. Addition of rac-1-monopalmitin promoted utilization of palmitoyl-CoA by the monoglyceride pathway but did not inhibit phosphatidate biosynthesis. 8. With rate-limiting concentrations of CoA and Mg2+ the relative decreases in velocity for palmitoyl-CoA synthetase and glycerolipid synthesis from palmitate were almost identical. However, low concentrations of palmitate and ATP produced greater decreases in synthetase activity than in glycerolipid synthesis. 9. There appears to be a fine balance between the activities of palmitoyl-CoA synthetase and glycerol phosphate acyltransferase, with neither activity being in excess with respect to phosphatidate synthesis.

scholarly journals The relationship between palmitoyl-coenzyme A synthetase activity and esterification of sn-glycerol 3-phosphate in rat liver mitochondria

1973 ◽  
Vol 132 (4) ◽  
pp. 697-706 ◽  
Author(s):  
Mariana Sánchez ◽  
David G. Nicholls ◽  
David N. Brindley
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Synthetase Activity ◽  
Palmitoyl Carnitine ◽  
The Mean ◽  
Glycerolipid Synthesis ◽  
Rate Limiting

1. The specific activities for palmitoyl-CoA synthetase and for sn-glycerol 3-phosphate esterification, with palmitoyl-CoA generated either by the endogenous synthetase or from palmitoyl-(-)-carnitine, CoA and excess of carnitine palmitoyltransferase, were measured with rat liver mitochondria. 2. The mean specific activity of palmitoyl-CoA synthetase was approximately five- and seven-fold the rates of sn-glycerol 3-phosphate esterification from palmitate and palmitoyl-(-)-carnitine respectively. No significant correlation was found in different rats between the activities of palmitoyl-CoA synthetase and sn-glycerol 3-phosphate esterification from either acyl precursor. However, there was a significant correlation (r=0.83, P<0.001) between the rates of glycerolipid synthesis from palmitate and palmitoyl-(-)-carnitine. 3. The mean molar composition of the glycerolipid synthesized from palmitate was 58% lysophosphatidate, 31% phosphatidate and 11% neutral lipid. With palmitoyl-(-)-carnitine the equivalent values were 70, 23 and 7%, which were significantly different. 4. When palmitoyl-CoA synthetase had been inactivated by 60–70% after preincubation of mitochondria at 37°C, it became rate-limiting in glycerolipid biosynthesis. Additions of 1–5mm-ATP prevented inactivation of palmitoyl-CoA synthetase. 5. Preincubation also inhibited the oxidation of palmitate, palmitoyl-CoA, palmitoyl-(-)-carnitine and malate plus glutamate. These inhibitions could not be prevented by addition of ATP. 6. Diversion of palmitoyl-CoA to form palmitoyl-(-)-carnitine did not inhibit sn-glycerol 3-phosphate esterification. 7. The palmitoyl-CoA pool synthesized by the palmitoyl-CoA synthetase was augmented by adding partially purified synthetase or carnitine palmitoyltransferase and palmitoyl-(-)-carnitine. No stimulation of palmitate incorporation into glycerolipids occurred. 8. At low concentrations of Mg2+, palmitate, ATP and CoA the velocity with palmitoyl-CoA synthetase decreased more than that of glycerolipid synthesis from palmitate. 9. It is concluded that in the presence of optimum substrate concentrations the activity of sn-glycerol 3-phosphate acyltransferase and not of palmitoyl-CoA synthetase is rate-limiting in the synthesis of phosphatidate and lysophosphatidate in isolated rat liver mitochondria.

scholarly journals A new assay procedure for monoglyceride acyltransferase

1974 ◽  
Vol 141 (2) ◽  
pp. 407-411 ◽  
Author(s):  
Verena J. Short ◽  
David N. Brindley ◽  
Raymond Dils
Keyword(s):  
Enzyme Activity ◽  
Guinea Pig ◽  
Intestinal Mucosa ◽  
Microsomal Fraction ◽  
Assay System ◽  
Acyltransferase Activity ◽  
Palmitoyl Carnitine ◽  
Assay Procedure ◽  
Acyl Acceptors

1. A new assay system is described for monoglyceride acyltransferase (acylglycerol palmitoyltransferase, EC 2.3.1.22) in which palmitoyl-CoA is generated from palmitoyl-(-)-carnitine. 2. With the microsomal fraction from homogenates of guinea-pig intestinal mucosa, the Vmax. of this enzyme decreased with different acyl acceptors in the order 2-monopalmitoylglycerol>2-hexadecylglycerol>rac-1-monopalmitoylglycerol. 3. There were highly significant correlations between the monoglyceride acyltransferase activity as measured with these three substrates. This demonstrates that each of these substrates can be used to measure the same enzyme activity. 4. The advantages of using generated palmitoyl-CoA with 2-hexadecylglycerol and rac-1-monopalmitoylglycerol as model substrates for this enzyme are discussed.

Über die Verwendung von 86Rb als Indikator für Kalium, Untersuchungen am lichtgeförderten 42K/K- und 86Rb/Rb-Influx bei Elodea densa/ On the Application of 86Rb as a Tracer for Potassium, Measurements of the Lightdependent 42K/K- and 86Rb/Rb-Influx in Elodea densa

1970 ◽  
Vol 25 (6) ◽  
pp. 624-630 ◽  
Author(s):  
Wolf Dietrich Jeschke
Keyword(s):  
Competitive Inhibition ◽  
Specific Activity ◽  
Elodea Densa ◽  
Low Concentrations ◽  
System 2 ◽  
Experimental Values ◽  
System 1 ◽  
Menten Kinetic ◽  
Lower Capacity ◽  
Relative Similarity

1. The light-dependent influx of K⊕ and of Rb⊕ into isolated leaves of Elodea densa was measured by using 42K⊕ and 86Rb⊕ as tracers.2. If 86Rb was used as a tracer for K⊕ and then the K⊕ influx was determined from the specific activity σ86Rb/K an influx vK(86Rb) was obtained which was lower than the K(42K) influx (fig. 1). The ratio of vK(42K)/vK(86Rb) = SK,Rb increased with increasing [Rb⊕]0 and decreasing [K⊕]0 (figs. 1 and 7). The depression of vK(86Rb) as compared to vK(42K) was not a consequence of the competitive inhibition of K⊕ influx by Rb⊕ (cf. fig. 1).3. As a working hypothesis it is proposed that 86Rb⊕ functions only as a tracer of Rb⊕ and that vK(84Rb) can be calculated from the parameters of the Rb (86Rb) influx and its competitive inhibition by K⊕.4. By using the Michaelis - Menten - Kinetics a formula was derived for vK(86Rb), the Michaelis constant KmK(86Rb), and for VmaxK(86Rb). From these calculations it may be predicted a) that vK(86Rb) will show a Michaelis - Menten - Kinetic only if [Rb⊕] is kept constant at all K⊕ concentrations; b) that VmaxK(86Rb) is independent of [Rb⊕] and not necessarily equal to VmaxK(42K); and c) that KmK(86Rb) is greater than KmK(42K) and dependent on [Rb⊕].5. In order to test the calculations the mutual competitive inhibition of K⊕ - and Rb⊕ -influx (figs. 2, 3) was measured. From the Michaelis - Menten - Parameters of the Rb (86Rb) influx and the inhibition constant KiK of potassium vK(86Rb) was calculated. The predictions mentioned above were verified. The calculated depression of vK(86Rb) as compared to vK(42K) corresponded satisfactorily to the experimental values (fig. 7).6. The isotherms of K (42K) and of Rb (86Rb) influx were measured (fig. 4). In the range of low concentrations (system 1) VmaxK(42K) greatly exceeded VmaxRb(86Rb), the same was true of KmK(42K) as compared to KmRb(86Rb). Thus in system 1 the Rb⊕ influx proceeded with much greater affinity but lower capacity than the K⊕ influx. Thus, although with regard to competitive inhibition K⊕ and Rb⊕ seem to use the same transfort site the relative similarity of vK(42K) and vK(86Rb) must be termed rather incidental though predictable.7. At higher concentrations (system 2) the influx of K⊕ and Rb⊕ was less different with respect to Km and Vmax (fig. 7), an observation which was confirmed by competitive inhibition on almost equal terms.8. It must be stated that the value of 86Rb⊕ as a tracer instead of 42K⊕ is limited. However, the metabolic linkages of K⊕ influx can also be detected by the use of 86Rb⊕.

scholarly journals β-Carotene-15,15′-dioxygenase (EC 1.13.11.21) isolation reaction mechanism and an improved assay procedure

1994 ◽  
Vol 72 (3) ◽  
pp. 397-414 ◽  
Author(s):  
Jannine Devery ◽  
B. V. Milborrow
Keyword(s):  
Guinea Pig ◽  
Intestinal Mucosa ◽  
Specific Activity ◽  
Gel Filtration ◽  
Tween 80 ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Protein Aggregate ◽  
Β Carotene ◽  
Dioxygenase Activity

βCarotene-15,15′-dioxygenase (EC 1.13.11.21; β-carotene dioxygenase) activity in extracts from guinea-pig intestinal mucosa was assayed by supplying [15,15′-14C2l- or [15,15′-3H2]β-carotene dissolved in Tween 80. Methods were developed to minimize the breakdown of labelled β-carotene and β-carotene cleavage products during the isolation procedure. Antioxidants and unlabelled carriers were added to extracting solvents and C18 Sep-Pak cartridges were used to isolate the remaining β-carotene and retinaldehyde, which was the only cleavage product detected. The labelled material produced by the enzyme was analysed by either normal-phase TLC or reversed-phase HPLC and characterized chemically as retinaldehyde. The lack of other labelled up-carotenals isolated in these experiments and the formation of between 1.5 and 2 mol retinaldehyde/mol β-carotene consumed confirm the central cleavage mechanism for the enzyme's action. More β-carotene dioxygenase activity was obtained from guinea-pig mucosa than from chicken or pig intestinal mucosa. The β-carotene dioxygenase was obtained as a soluble enzyme which was partially purified by gel filtration and ion-exchange chromatography to a specific activity of 0.6 nmol retinaldehyde formedlmg protein per h. The formation of a lipid-protein aggregate containing the β-carotene dioxygenase activity, which has been reported to be present in the exclusion volume of Sephadex columns, was avoided if the mucosal serapings were homogenized in buffer at a proportion of 1:4 (w/v).

scholarly journals A trypsin-sensitive, heat-labile, N-ethylmaleimide-sensitive factor in adipocyte post-microsomal supernatant which affects the assay of adipocyte glycerol phosphate acyltransferase activities

1983 ◽  
Vol 214 (1) ◽  
pp. 247-255 ◽  
Author(s):  
M H Rider ◽  
E D Saggerson
Keyword(s):  
Trypsin Treatment ◽  
Glycerol Phosphate ◽  
Acyltransferase Activity ◽  
Isolated Mitochondria ◽  
Low Concentrations ◽  
Equivalent Quantity ◽  
Sensitive Factor ◽  
Labile N ◽  
Z Protein ◽  
Coa Hydrolase

Addition of adipocyte 100 000 g post-microsomal supernatant to assays of glycerol phosphate acyltransferase in isolated mitochondria or microsomal fractions decreased activity at lower concentrations of palmitoyl-CoA. At higher concentrations of palmitoyl-CoA, activation was observed on addition of post-microsomal supernatant. The effect of post-microsomal supernatant to decrease activity at lower [palmitoyl-CoA] was abolished by heating or by trypsin treatment, and was also abolished by addition of N-ethylmaleimide to assays or by pretreatment of post-microsomal supernatant with N-ethylmaleimide. The stimulatory effect seen at higher [palmitoyl-CoA] was not sensitive to heat or trypsin treatment. The effect of post-microsomal supernatant at lower [palmitoyl-CoA] cannot be attributed to palmitoyl-CoA hydrolase activity. It was found that brief treatment of adipocyte mitochondria with low concentrations of trypsin was an effective way to remove contaminating microsomal glycerol phosphate acyltransferase activity. Adipocyte post-microsomal supernatant was more effective than an equivalent quantity of liver post-microsomal supernatant protein in decreasing adipocyte microsomal glycerol phosphate acyltransferase activity. The effects of the supernatants from both tissues were decreased by flavaspidic acid. Semi-purified Z-protein fraction from rat liver did not mimic the effect of adipocyte post-microsomal supernatant to decrease glycerol phosphate acyltransferase at lower [palmitoyl-CoA]. Post-microsomal supernatants obtained from noradrenaline-treated adipocytes were less effective than those from control cells in decreasing glycerol phosphate acyltransferase activity in microsomal fractions at lower [palmitoyl-CoA]. It is suggested that adipocyte cytosol may contain an acyl-CoA-binding protein or proteins differing from Z-protein in some respects. The physiological significance of the findings is briefly discussed.

scholarly journals Enzymes of glycerolipid synthesis in small-intestinal mucosa of foetal and neonatal guinea pigs

1975 ◽  
Vol 152 (3) ◽  
pp. 675-679 ◽  
Author(s):  
V J Short ◽  
R Dils ◽  
D N Brindley
Keyword(s):  
Cell Proliferation ◽  
Intestinal Mucosa ◽  
Guinea Pigs ◽  
Protein Concentration ◽  
Small Intestinal Mucosa ◽  
Phosphatidate Phosphatase ◽  
Glycerol Phosphate ◽  
Specific Activities ◽  
Glycerolipid Synthesis ◽  
Small Intestinal

1. The activities of some enzymes of glycerolipid synthesis were measured in homogenates obtained from the intestinal scrapings of 62-66-day foetuses and 2- and 8-day-old guinea pigs. 2. The ratio of protein concentration/DNA concentration was significantly higher (P greater than 0.001) in homogenized tissue from the neonatal compared with the foetal guinea pigs. Enzyme activities were therefore expressed relative to both protein and to DNA. 3. The specific activities (relative to DNA) of palmitoyl-CoA synthetase, glycerol phosphate acyltransferase and phosphatidate phosphatase were higher in homogenized tissues from neonatal than in those from the foetal guinea pigs. These activities are probably involved more in cell proliferation than in the absorption and transport of triacylglycerol. Its activity was not significantly different in the foetal and neonatal guinea pigs when expressed relative to DNA but it was lower in the neonatal guinea pigs when expressed relative to protein. The entry of food into the intestine after birth is therefore not necessary for its activity.

scholarly journals Palmitate activation and esterification in microsomal fractions of rat liver

1975 ◽  
Vol 152 (1) ◽  
pp. 39-49 ◽  
Author(s):  
K A Lloyd-Davies ◽  
D N Brindley
Keyword(s):  
Rat Liver ◽  
Maximum Rate ◽  
Microsomal Fraction ◽  
Synthetase Activity ◽  
Optimum Conditions ◽  
Glycerol Phosphate ◽  
Glycerolipid Synthesis ◽  
The Relationship ◽  
Rate Limiting ◽  
Substrate Concentrations

1. Palmitoyl-CoA synthetase activity in the microsomal fraction of rat liver was measured directly by palmitoyl-CoA production, and indirectly by converting the palmitoyl-CoA into palmitoylcarnitine under optimum conditions. Even in the latter system, palmitoyl-CoA accumulated. The rate of palmitoyl-CoA hydrolysis and the inhibition of palmitoyl-CoA synthetase by palmitoyl-CoA were each estimated to be less than 10% of the maximum rate of palmitoyl-CoA production. The concentration of palmitoyl-CoA present in the assay systems used for measuring palmitate esterification to glycerol phosphate and the activity of palmitoyl-CoA synthetase by using the carnitine-linked determination were measured. These concentrations were not altered by the addition of glycerol phosphate, or of carnitine plus carnitine palmitoyltransferase. The relationship between the activity of palmitoyl-CoA synthetase and the rate of glycerolipid synthesis was investigated. The latter activity was measured by using palmitoyl-CoA generated from palmitate, palmitoyl-AMP or palmitoylcarnitine. It is concluded that, at optimum substrate concentrations, the activity of glycerol phosphate acyltransferase is rate-limiting in the synthesis of phosphatidate by rat liver microsomal fractions. The implications of these results in the measurement of palmitoyl-CoA synthetase and in the control of glycerolipid synthesis are discussed.

Localization of calcium and zinc in the sperm storage tubules of chicken, quail and turkey using X-ray microanalysis

Reproduction ◽  
2000 ◽  
pp. 331-336 ◽  
Author(s):  
L Holm ◽  
H Ekwall ◽  
GJ Wishart ◽  
Y Ridderstrale
Keyword(s):  
Calcium Concentration ◽  
Sperm Storage ◽  
X Ray ◽  
Elemental Concentrations ◽  
Low Concentrations ◽  
Intracellular Content ◽  
Wet Weight ◽  
The Mean ◽  
Sperm Storage Tubules ◽  
Scanning Electron

Sperm storage tubules from the utero-vaginal junction of chickens, quails and turkeys were analysed for calcium and zinc using X-ray microanalysis of ultra-rapidly frozen tissue in a scanning electron microscope. This technique enabled the tubular fluid surrounding the stored spermatozoa and the intracellular content of the cells of the sperm storage tubules to be analysed separately and, by using standards with known concentrations, their elemental concentrations were estimated. The mean (+/- SEM) concentration of calcium in the tubular fluid from chickens, quails and turkeys was 17 +/- 3, 19 +/- 3 and 17 +/- 4 mmol kg(-1) wet weight, respectively. The intracellular calcium concentration of the cells of the tubules did not differ significantly from these values and was also similar in the mucosal epithelial cells of the utero-vaginal junction. Zinc was localized in the cells of turkey sperm storage tubules and tubular fluid, but at low concentrations. No zinc could be detected in corresponding structures from chickens and quails. The concentration of calcium in the tubular fluid is within the range known to inhibit the motility of spermatozoa, supporting this function for calcium during storage. Zinc is known to depress turkey sperm metabolism and it may also be involved in inducing quiescence of spermatozoa during storage in this species.

scholarly journals SYNTHESIS AND MIGRATION OF PROTEINS IN THE CELLS OF THE EXOCRINE PANCREAS AS REVEALED BY SPECIFIC ACTIVITY DETERMINATION FROM RADIOAUTOGRAPHS

1963 ◽  
Vol 16 (1) ◽  
pp. 1-23 ◽  
Author(s):  
H. Warshawsky ◽  
C. P. Leblond ◽  
B. Droz
Keyword(s):  
Specific Activity ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
The Mean ◽  
And Migration ◽  
Rats And Mice ◽  
Silver Grains ◽  
Golgi Zone

Radioautographs of pancreatic acinar cells were prepared in rats and mice sacrificed at various times after injection of leucine-, glycine-, or methionine-H3. Measurements of radioactivity concentration (number of silver grains per unit area) and relative protein concentration (by microspectrophotometry of Millon-treated sections) yielded the mean specific activity of proteins in various regions of the acinar cells. The 2 to 5 minute radioautographs as well as the specific activity time curves demonstrate protein synthesis in ergastoplasm. From there, most newly synthesized proteins migrate to and accumulate in the Golgi zone. Then they spread to the whole zymogen region and, finally, enter the excretory ducts. An attempt at estimating turnover times indicated that two classes of proteins are synthesized in the ergastoplasm: "sedentary" with a slow turnover (62.5 hours) and "exportable" with rapid turnover (4.7 minutes). It is estimated that the exportable proteins spend approximately 11.7 minutes in the Golgi zone where they are built up into zymogen granules, and thereafter 36.0 minutes as fully formed zymogen granules, before they are released outside the acinar cell as pancreatic secretion. The mean life span of a zymogen granule in the cell is estimated to be 47.7 minutes.

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