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Isolated Membranes and Organelles from Vascular Smooth Muscle

Bonnie F. Sloane

10.1002/cphy.cp020205

Source: Supplement 7: Handbook of Physiology, The Cardiovascular System, Vascular Smooth Muscle

Originally published: 1980

Published online: January 2011

Full Article on Wiley Online Library



Abstract

The sections in this article are:

1 Isolation Techniques
1.1 Homogenization
1.2 Fractionation
2 Identification of Isolated Fractions of Vascular Smooth Muscle
2.1 Mitochondria
2.2 Microsomal and Plasma Membrane Fractions
3 Functional Studies of Isolated Fractions of Vascular Smooth Muscle
3.1 Mitochondria
3.2 Microsomal and Plasma Membrane Fractions
4 Summary
Figure 1. Figure 1.

Fractionation scheme for isolation of mitochondria from bovine vascular smooth muscle. MPA, main pulmonary artery; MV, mesenteric vein; MSE, 225 mM mannitol, 75 mM sucrose, 5 mM EDTA, pH 7.2.

Adapted from Vallières et al. 36
Figure 2. Figure 2.

A: mitochondria isolated from main pulmonary artery. B: mitochondria isolated from mesenteric vein as described in Figure 1. × 15,800.

From Vallières et al. 36
Figure 3. Figure 3.

Distribution of markers and proteins in different fractions of rat myometrium. WGA, wheat germ agglutinin; PNS, postnuclear supernatant; MITO, mitochondria; SOL, soluble; MIC, microsomes; F1–F5, fractions isolated on sucrose density gradient of from 28% to 45% sucrose.

From Daniel et al. 8
Figure 4. Figure 4.

Rates of Ca2+ uptake by bovine main pulmonary artery and mesenteric vein mitochondria at various Ca2+ concentrations. Mitochondria (0.2–2.7 mg/ml) were incubated at 26°C in 100 mM sucrose, 50 mM KCl, 2 mM MgCl2, 5 mM sodium succinate, 3 μM rotenone, 5 mM sodium acetate, 20 mM 4‐morpholinopropanesulfonate (pH 7.2), and 100 μM murexide or 40 μM arsenazo. Rates were measured with arsenazo for Ca2+ concentrations ranging from 1 to 25 μM and with murexide for higher concentrations. A: mitochondria were isolated from main pulmonary artery as described in Figure 1; •, in the presence of 0.2% bovine serum albumin; □, in the absence of albumin; ▵, after addition of Nagarse proteinase. Each point represents the average of two or three experiments on five preparations (•) or on two preparations (□, ▵). B: all points were obtained from two experiments with four preparations of mesenteric vein mitochondria.

From Vallières et al. 36
Figure 5. Figure 5.

Initial rate of Mg2+ release by mitochondria of bovine main pulmonary artery at various external [Mg2+]. Reaction mixtures contained 1.0–1.5 mg/ml of mitochondria. Transport of Mg2+ was measured at 25°C in 200 mM sucrose, 20 mM KCl, 30 mM 4‐morpholinopropanesulfonate, 3 μM rotenone (pH 7.1), and 30 μM eriochrome blue. Each point represents the average of three experiments on three mitochondrial preparations.

From Sloane et al. 33
Figure 6. Figure 6.

Calcium uptake of microsomal vesicles of rabbit aorta in a representative experiment. Incubation was at 37°C in 30 μmol tris‐HCl, 300 μmol KCl, 9 μmol Mg‐ATP, 15 μmol ammonium oxalate, 15 μmol sodium azide, 0.4 μCi 45CaCl2, and 0.3 or 0.06 μmol CaCl2. Curve a, 100 μM calcium, complete medium; curve b, 20 μM calcium, complete medium; curve c, 100 μM calcium, oxalate omitted; curve d, 100 μM calcium, Mg‐ATP omitted.

From Fitzpatrick et al. Science 176(4032): 305–306, 1972. Copyright 1972 by the American Association for the Advancement of Science
Figure 7. Figure 7.

A: effect of varying initial free calcium level on calcium uptake by vesicular fraction of vascular smooth muscle. Calcium uptake was determined at 37°C in an incubation solution consisting of 5 mM ATP, 5 mM MgCl2, 104 mM KCl, 18 mM imidazole (pH 7.0), 10 mM sodium azide, 10 mM potassium oxalate, 6 mM creatine phosphate, 0.1 mg/ml creatine phosphokinase, and 0.05 μCi/ml 45CaCl2; 0.040 mM ethylene glycol‐bis(β‐aminoethyl ether)‐N,N′‐tetraacetic acid (EGTA) plus various amounts of CaCl2 were added to give the initial free calcium levels shown. B: Woolf plot of the vesicular calcium uptake shown in A. The average Km and Vmax for seven such determinations is given.

From Ford and Hess 14, by permission of the American Heart Association, Inc
Figure 8. Figure 8.

Distribution of calcium uptake activity and enzyme markers in six fractions obtained by centrifuging microsomal vesicles in a sucrose density gradient. A: representative data from guinea pig intestinal smooth muscle. B: representative data from rabbit aorta. Specific activity of calcium uptake was measured from 5 to 15 min after start of incubation. Specific activity of sodium potassium adenosine triphosphatase (ATPase) is in μmol inorganic phosphate (Pi)/mg protein per 15 min. Specific activity of 5′‐nucleotidase is in μmol Pi/mg protein per 30 min. Specific activity of NADH oxidase is in μmol NADH/mg protein per min.

From Hurwitz et al. Science 179 (71): 384–386, 1973. Copyright 1973 by the American Association for the Advancement of Science


Figure 1.

Fractionation scheme for isolation of mitochondria from bovine vascular smooth muscle. MPA, main pulmonary artery; MV, mesenteric vein; MSE, 225 mM mannitol, 75 mM sucrose, 5 mM EDTA, pH 7.2.

Adapted from Vallières et al. 36


Figure 2.

A: mitochondria isolated from main pulmonary artery. B: mitochondria isolated from mesenteric vein as described in Figure 1. × 15,800.

From Vallières et al. 36


Figure 3.

Distribution of markers and proteins in different fractions of rat myometrium. WGA, wheat germ agglutinin; PNS, postnuclear supernatant; MITO, mitochondria; SOL, soluble; MIC, microsomes; F1–F5, fractions isolated on sucrose density gradient of from 28% to 45% sucrose.

From Daniel et al. 8


Figure 4.

Rates of Ca2+ uptake by bovine main pulmonary artery and mesenteric vein mitochondria at various Ca2+ concentrations. Mitochondria (0.2–2.7 mg/ml) were incubated at 26°C in 100 mM sucrose, 50 mM KCl, 2 mM MgCl2, 5 mM sodium succinate, 3 μM rotenone, 5 mM sodium acetate, 20 mM 4‐morpholinopropanesulfonate (pH 7.2), and 100 μM murexide or 40 μM arsenazo. Rates were measured with arsenazo for Ca2+ concentrations ranging from 1 to 25 μM and with murexide for higher concentrations. A: mitochondria were isolated from main pulmonary artery as described in Figure 1; •, in the presence of 0.2% bovine serum albumin; □, in the absence of albumin; ▵, after addition of Nagarse proteinase. Each point represents the average of two or three experiments on five preparations (•) or on two preparations (□, ▵). B: all points were obtained from two experiments with four preparations of mesenteric vein mitochondria.

From Vallières et al. 36


Figure 5.

Initial rate of Mg2+ release by mitochondria of bovine main pulmonary artery at various external [Mg2+]. Reaction mixtures contained 1.0–1.5 mg/ml of mitochondria. Transport of Mg2+ was measured at 25°C in 200 mM sucrose, 20 mM KCl, 30 mM 4‐morpholinopropanesulfonate, 3 μM rotenone (pH 7.1), and 30 μM eriochrome blue. Each point represents the average of three experiments on three mitochondrial preparations.

From Sloane et al. 33


Figure 6.

Calcium uptake of microsomal vesicles of rabbit aorta in a representative experiment. Incubation was at 37°C in 30 μmol tris‐HCl, 300 μmol KCl, 9 μmol Mg‐ATP, 15 μmol ammonium oxalate, 15 μmol sodium azide, 0.4 μCi 45CaCl2, and 0.3 or 0.06 μmol CaCl2. Curve a, 100 μM calcium, complete medium; curve b, 20 μM calcium, complete medium; curve c, 100 μM calcium, oxalate omitted; curve d, 100 μM calcium, Mg‐ATP omitted.

From Fitzpatrick et al. Science 176(4032): 305–306, 1972. Copyright 1972 by the American Association for the Advancement of Science


Figure 7.

A: effect of varying initial free calcium level on calcium uptake by vesicular fraction of vascular smooth muscle. Calcium uptake was determined at 37°C in an incubation solution consisting of 5 mM ATP, 5 mM MgCl2, 104 mM KCl, 18 mM imidazole (pH 7.0), 10 mM sodium azide, 10 mM potassium oxalate, 6 mM creatine phosphate, 0.1 mg/ml creatine phosphokinase, and 0.05 μCi/ml 45CaCl2; 0.040 mM ethylene glycol‐bis(β‐aminoethyl ether)‐N,N′‐tetraacetic acid (EGTA) plus various amounts of CaCl2 were added to give the initial free calcium levels shown. B: Woolf plot of the vesicular calcium uptake shown in A. The average Km and Vmax for seven such determinations is given.

From Ford and Hess 14, by permission of the American Heart Association, Inc


Figure 8.

Distribution of calcium uptake activity and enzyme markers in six fractions obtained by centrifuging microsomal vesicles in a sucrose density gradient. A: representative data from guinea pig intestinal smooth muscle. B: representative data from rabbit aorta. Specific activity of calcium uptake was measured from 5 to 15 min after start of incubation. Specific activity of sodium potassium adenosine triphosphatase (ATPase) is in μmol inorganic phosphate (Pi)/mg protein per 15 min. Specific activity of 5′‐nucleotidase is in μmol Pi/mg protein per 30 min. Specific activity of NADH oxidase is in μmol NADH/mg protein per min.

From Hurwitz et al. Science 179 (71): 384–386, 1973. Copyright 1973 by the American Association for the Advancement of Science
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Article Title: Isolated Membranes and Organelles from Vascular Smooth Muscle
 

How to Cite

Bonnie F. Sloane. Isolated Membranes and Organelles from Vascular Smooth Muscle. Compr Physiol 2011, Supplement 7: Handbook of Physiology, The Cardiovascular System, Vascular Smooth Muscle: 121-132. First published in print 1980. doi: 10.1002/cphy.cp020205