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[29] Guanylate cyclase in human platelets
[29]
PL~TELET GUANYLATE CYCLASE
199
pH optimum. The pH optimum for the reaction is distinctly alkaline (pH 7.6-8.0).6,9 Stability. The enzyme is labile to either heat or sulfhydryl reagents, particularly in the presence of Triton X-100. In the absence of substrate or free metal, activity is rapidly lost by incubation at 37 °. Heavy metals, N-ethylmaleimide, or p-hydroxymercuribenzoate rapidly inactivate the enzyme2 Particulate enzyme preparations are stable at --70 ° for at least 3 months at pH 7.0 but should be frozen and thawed only one time, since enzyme affinity for Mn 2+ as well as stimulation by Triton X-100 decrease with successive freeze-thaws. 7,9 Enzyme preparations stored at --70 ° in the presence of Triton X-100 also show decreases in affinity for Mn ~+ with increased storage time2 Kinetics. When Mn-GTP is varied from 6 to 800 ~M with free Mn 2÷ fixed, reciprocal (Lineweaver-Burk) plots are concave upward. Hill plots have a slope greater than 1.0. When Mn-GTP concentration is fixed, and free Mn 2÷ is varied from 400 to 3000 t~/, reciprocal plots are linear2 Inhibitors. At concentrations of Mn-GTP greater than 0.1 raM, dATP, ATP, dGTP, and ADP are inhibitors of the enzyme. With Mn-GTP varied, reciprocal plots change from concave upward with no inhibitor, to linear at high concentrations of these inhibitors2 Activators. At low concentrations of Mn-GTP (less than 10 tdl//) dATP, ATP dGTP, and ADP can activate the enzyme. Ca 2+ can also activate the enzyme, but the mechanism of activation is obscure2 Alternate Substrates. When Mn-dGTP is used as a substrate, deoxycGMP is formed. Although it has not been proved that guanylate cyclase catalyzes this reaction, GTP and ATP are inhibitors, and the enzyme involved in deoxy-cGMP formation is denatured by heat at the same rate as the enzyme catalyzing cGMP formation. TM ~ D. L. Garbers, J. L. Suddath, and J. G. Hardman, unpublished observations.
[29] Guanylate Cyclase in Human Platelets By EYCKE BOHME, REGINE JUNG, and ILSE MECHLE~t
Determination of Ouanylate Cyclase Activity Guanylate cyclase activity was determined by incubation for 15 minutes or longer at 37 ° with 0.5 mM [8-3H]GTP (0.25-1 t~Ci), 3 mM MnCl~, 2 mM unlabeled cGMP, and 100 mM N-Tris(hydroxymethyl)me-
200
BIOSYNTHESIS OF CYCLIC NUCLEOTIDES
[29]
thyl-2-aminoethane sulfonate (TES)-NaOH buffer, pH 7.4, containing 50 mM NaC1, if not otherwise indicated. The product formed and the substrate remaining at the end of the incubation period were isolated by column chromatography on polyethyleneimine cellulose. ~
Distribution of Guanylate Cyclase in Human Blood Cells
Human blood cells were separated by differential centrifugation and by use of Dextran. '~ In plasma and homogenates of erythrocytes, no guanylate cyclase activity was detectable. In homogenates of mixed leukocytes (containing some platelets), the guanylate cyclase activity was about 50 pmoles min -1 mg protein -1. The enzyme activity in platelets varied between 0.2 and 2.0 nmoles min -~ mg protein-i; this is substantially (up to an order of magnitude) higher than in any other mammalian tissue so far studied except outer segments of retinal rods.
Preparation of Platelet Homogenates
Platelets were isolated from human blood by differential centrifugation at 4 ° with 4 mM EDTA added as an anticoagulant. The blood was centrifugated at 400 g for 15 minutes, and platelet-rich plasma was obtained. The platelet-rich plasma was centrifugated at 1500 g for 10 minutes, and the platelets were suspended in 100 mM TES-NaOH-buffer, pH 7.4, containing 90 mM NaC1. The platelet suspension was then centrifugated at 600 g for 10 minutes, and the platelets were resuspended in fresh buffer. The guanylate cyclase activity measured in the platelet homogenates was not significantly affected if EDTA was replaced by 15 mM sodium citrate. The enzyme activity was slightly higher if the platelet isolation procedure was performed at room temperature (20-25°). Suspensions of platelets were usually disintegrated ultrasonically using a MSE 60-W Ultrasonic Disintegrator for 15 seconds. The guanylate cyclase activity was not significantly different if the platelets were disintegrated by hypotonic shock in 5 mM TES-NaOH buffer, pH 7.4, or by repeated freezing and thawing. If the ultrasonic treatment was performed for more than 30 seconds or if the platelets were exposed to the hypotonic medium for more than 30 minutes or if freezing and thawing was repeated more than 3 times, the guanylate cyclase activity was decreased. G. Schultz, E. BShme, and J. G. Hardman, this volume [2]. 2W. A. Skoog and W. S. Beck, Blood ll, 436 (1956).
[29]
PLATELET GUANYLATE CYCLASE
201
Apparent SubceUular Distribution of Guanylate Cyclase More than 90% of the total guanylate cyclase activity measured in subcellular fractions was found in the 250,000 g, 60-minute supernatant fluid. The apparent distribution of guanylate cyclase between particulate and soluble fractions was not affected by the mode of cell disintegration. Unlike the particulate guanylate cyclase activity from a number of other sources, that in platelets was not increased by Triton X-100. The enzyme in platelet homogenates or high speed supernatant fractions was inhibited by 50% by about 1 mg/ml of Triton X-100 in the incubation medium.
Influence of Divalent Cations on Platelet Guanylate Cyclase The formation of cGMP depends on the presence of a divalent cation. If the guanylate cyclase activity was measured with 1 mM GTP and 10 mM Me -~÷,the activity with Mg 2+ was 60%, with Fe 2÷ 35%, and with Ca 2÷ 5% of that observed with Mn ~*. The formation of cGMP measured in the presence of 1 mM GTP and 5 mM Mn 2+ was completely inhibited by Hg 2÷, Zn 2÷ or Cu ~÷ (0.1-10 mM) and partially inhibited by Co s÷, Pb 2+, Fe ~÷, Fe :+, or Ba 2+ (1 or 10 mM). The addition of Mg :÷ and especially of Ca 2+, however, caused an increase in the guanylate cyclase activity measured with 1 mM GTP and 5 mM Mn 2÷. The effect of Ca 2÷ was more than additive while that of Mg ~+ was less than additive. The effect of Ca ~÷ on guanylate cyclase activity highly depended on the respective concentrations of Ca ~÷, Mn 2+, and GTP.
Apparent Activation of Platelet Guanylate Cyclase with Incubation Time The formation of cGMP in assays using crude homogenates or 250,000 g, 30-minute supernatant fractions was not proportional to the incubation time; the enzyme activity increased with the length of the incubation time. A maximal activity was observed after about 60 minutes of incubation at 37 °, then the activity slowly declined. The apparent activation of guanylate cyclase with time was also observed if the enzyme preparations were preincubated at 37 ° in the absence of Mn ~÷, GTP, or cGMP (Fig. 1). Addition of EDTA (1 or 5 mM) to guanylate eyclase preparations preincubated for various lengths of time retarded but did not prevent the enzyme activation with time; in the presence of EDTA, the same maximal enzyme activity was observed as without addition of this agent.
202
BIOSYNTHESIS
O F C Y C L I C NUCLle, O T I D E S
[2
0.9 o L o.
x
0 0.01
*¢E x
0.6
E
o
0.1
N 0.3
o
•
~
•
1.0
•
10.0
*5 o
i
I
I
i
15
30
60
120
time of preincubotion [minl
FI~. 1. Influence of dithiothreitol (DTT) on the apparent activation of humar platelet guanylate cyclase with time. A platelet homogenate was preincubated for the indicated lengths of time at 37° in 100 mM TES-NaOH buffer, pH 7.¢ containing 90 mM NaCI and DTT at the mitlimolar concentrations indicated in the figure. Guanylate cyclase activity was then measured by incubation for 15 minutes at 37° with the addition of 0.5 mM GTP, 3 mM MnCI~, and 2 mM cGMP. Addition of a sulfhydryl group protecting agent, e.g., dithiothreitol ( D T T ) , reduced the a p p a r e n t activation of the platelet guanylate cyelase with time (see Fig. 1). D T T in high concentrations (1 or 10 m M ) completely abolished the effect of time on guanylate cyclase activity. Dithioerythritol, but not the oxidized form of D T T , also prevented the enzyme activation.
Acknowledgment
The authors' studies were supported by the Deutsche Forschungsgemeinschaft.
PL~TELET GUANYLATE CYCLASE
199
pH optimum. The pH optimum for the reaction is distinctly alkaline (pH 7.6-8.0).6,9 Stability. The enzyme is labile to either heat or sulfhydryl reagents, particularly in the presence of Triton X-100. In the absence of substrate or free metal, activity is rapidly lost by incubation at 37 °. Heavy metals, N-ethylmaleimide, or p-hydroxymercuribenzoate rapidly inactivate the enzyme2 Particulate enzyme preparations are stable at --70 ° for at least 3 months at pH 7.0 but should be frozen and thawed only one time, since enzyme affinity for Mn 2+ as well as stimulation by Triton X-100 decrease with successive freeze-thaws. 7,9 Enzyme preparations stored at --70 ° in the presence of Triton X-100 also show decreases in affinity for Mn ~+ with increased storage time2 Kinetics. When Mn-GTP is varied from 6 to 800 ~M with free Mn 2÷ fixed, reciprocal (Lineweaver-Burk) plots are concave upward. Hill plots have a slope greater than 1.0. When Mn-GTP concentration is fixed, and free Mn 2÷ is varied from 400 to 3000 t~/, reciprocal plots are linear2 Inhibitors. At concentrations of Mn-GTP greater than 0.1 raM, dATP, ATP, dGTP, and ADP are inhibitors of the enzyme. With Mn-GTP varied, reciprocal plots change from concave upward with no inhibitor, to linear at high concentrations of these inhibitors2 Activators. At low concentrations of Mn-GTP (less than 10 tdl//) dATP, ATP dGTP, and ADP can activate the enzyme. Ca 2+ can also activate the enzyme, but the mechanism of activation is obscure2 Alternate Substrates. When Mn-dGTP is used as a substrate, deoxycGMP is formed. Although it has not been proved that guanylate cyclase catalyzes this reaction, GTP and ATP are inhibitors, and the enzyme involved in deoxy-cGMP formation is denatured by heat at the same rate as the enzyme catalyzing cGMP formation. TM ~ D. L. Garbers, J. L. Suddath, and J. G. Hardman, unpublished observations.
[29] Guanylate Cyclase in Human Platelets By EYCKE BOHME, REGINE JUNG, and ILSE MECHLE~t
Determination of Ouanylate Cyclase Activity Guanylate cyclase activity was determined by incubation for 15 minutes or longer at 37 ° with 0.5 mM [8-3H]GTP (0.25-1 t~Ci), 3 mM MnCl~, 2 mM unlabeled cGMP, and 100 mM N-Tris(hydroxymethyl)me-
200
BIOSYNTHESIS OF CYCLIC NUCLEOTIDES
[29]
thyl-2-aminoethane sulfonate (TES)-NaOH buffer, pH 7.4, containing 50 mM NaC1, if not otherwise indicated. The product formed and the substrate remaining at the end of the incubation period were isolated by column chromatography on polyethyleneimine cellulose. ~
Distribution of Guanylate Cyclase in Human Blood Cells
Human blood cells were separated by differential centrifugation and by use of Dextran. '~ In plasma and homogenates of erythrocytes, no guanylate cyclase activity was detectable. In homogenates of mixed leukocytes (containing some platelets), the guanylate cyclase activity was about 50 pmoles min -1 mg protein -1. The enzyme activity in platelets varied between 0.2 and 2.0 nmoles min -~ mg protein-i; this is substantially (up to an order of magnitude) higher than in any other mammalian tissue so far studied except outer segments of retinal rods.
Preparation of Platelet Homogenates
Platelets were isolated from human blood by differential centrifugation at 4 ° with 4 mM EDTA added as an anticoagulant. The blood was centrifugated at 400 g for 15 minutes, and platelet-rich plasma was obtained. The platelet-rich plasma was centrifugated at 1500 g for 10 minutes, and the platelets were suspended in 100 mM TES-NaOH-buffer, pH 7.4, containing 90 mM NaC1. The platelet suspension was then centrifugated at 600 g for 10 minutes, and the platelets were resuspended in fresh buffer. The guanylate cyclase activity measured in the platelet homogenates was not significantly affected if EDTA was replaced by 15 mM sodium citrate. The enzyme activity was slightly higher if the platelet isolation procedure was performed at room temperature (20-25°). Suspensions of platelets were usually disintegrated ultrasonically using a MSE 60-W Ultrasonic Disintegrator for 15 seconds. The guanylate cyclase activity was not significantly different if the platelets were disintegrated by hypotonic shock in 5 mM TES-NaOH buffer, pH 7.4, or by repeated freezing and thawing. If the ultrasonic treatment was performed for more than 30 seconds or if the platelets were exposed to the hypotonic medium for more than 30 minutes or if freezing and thawing was repeated more than 3 times, the guanylate cyclase activity was decreased. G. Schultz, E. BShme, and J. G. Hardman, this volume [2]. 2W. A. Skoog and W. S. Beck, Blood ll, 436 (1956).
[29]
PLATELET GUANYLATE CYCLASE
201
Apparent SubceUular Distribution of Guanylate Cyclase More than 90% of the total guanylate cyclase activity measured in subcellular fractions was found in the 250,000 g, 60-minute supernatant fluid. The apparent distribution of guanylate cyclase between particulate and soluble fractions was not affected by the mode of cell disintegration. Unlike the particulate guanylate cyclase activity from a number of other sources, that in platelets was not increased by Triton X-100. The enzyme in platelet homogenates or high speed supernatant fractions was inhibited by 50% by about 1 mg/ml of Triton X-100 in the incubation medium.
Influence of Divalent Cations on Platelet Guanylate Cyclase The formation of cGMP depends on the presence of a divalent cation. If the guanylate cyclase activity was measured with 1 mM GTP and 10 mM Me -~÷,the activity with Mg 2+ was 60%, with Fe 2÷ 35%, and with Ca 2÷ 5% of that observed with Mn ~*. The formation of cGMP measured in the presence of 1 mM GTP and 5 mM Mn 2+ was completely inhibited by Hg 2÷, Zn 2÷ or Cu ~÷ (0.1-10 mM) and partially inhibited by Co s÷, Pb 2+, Fe ~÷, Fe :+, or Ba 2+ (1 or 10 mM). The addition of Mg :÷ and especially of Ca 2+, however, caused an increase in the guanylate cyclase activity measured with 1 mM GTP and 5 mM Mn 2÷. The effect of Ca 2÷ was more than additive while that of Mg ~+ was less than additive. The effect of Ca ~÷ on guanylate cyclase activity highly depended on the respective concentrations of Ca ~÷, Mn 2+, and GTP.
Apparent Activation of Platelet Guanylate Cyclase with Incubation Time The formation of cGMP in assays using crude homogenates or 250,000 g, 30-minute supernatant fractions was not proportional to the incubation time; the enzyme activity increased with the length of the incubation time. A maximal activity was observed after about 60 minutes of incubation at 37 °, then the activity slowly declined. The apparent activation of guanylate cyclase with time was also observed if the enzyme preparations were preincubated at 37 ° in the absence of Mn ~÷, GTP, or cGMP (Fig. 1). Addition of EDTA (1 or 5 mM) to guanylate eyclase preparations preincubated for various lengths of time retarded but did not prevent the enzyme activation with time; in the presence of EDTA, the same maximal enzyme activity was observed as without addition of this agent.
202
BIOSYNTHESIS
O F C Y C L I C NUCLle, O T I D E S
[2
0.9 o L o.
x
0 0.01
*¢E x
0.6
E
o
0.1
N 0.3
o
•
~
•
1.0
•
10.0
*5 o
i
I
I
i
15
30
60
120
time of preincubotion [minl
FI~. 1. Influence of dithiothreitol (DTT) on the apparent activation of humar platelet guanylate cyclase with time. A platelet homogenate was preincubated for the indicated lengths of time at 37° in 100 mM TES-NaOH buffer, pH 7.¢ containing 90 mM NaCI and DTT at the mitlimolar concentrations indicated in the figure. Guanylate cyclase activity was then measured by incubation for 15 minutes at 37° with the addition of 0.5 mM GTP, 3 mM MnCI~, and 2 mM cGMP. Addition of a sulfhydryl group protecting agent, e.g., dithiothreitol ( D T T ) , reduced the a p p a r e n t activation of the platelet guanylate cyelase with time (see Fig. 1). D T T in high concentrations (1 or 10 m M ) completely abolished the effect of time on guanylate cyclase activity. Dithioerythritol, but not the oxidized form of D T T , also prevented the enzyme activation.
Acknowledgment
The authors' studies were supported by the Deutsche Forschungsgemeinschaft.