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[19] Adenylate cyclase from kidney and bone
150
BIOSYNTHESIS OF CYCLIC NUCLEOTIDES
[19]
[19] A d e n y l a t e C y c l a s e f r o m K i d n e y a n d B o n e
By S. J. MAax and G. D. AURBACH
Preparation of Enzyme Crude Enzyme Male Sprague-Dawley rats, 150-200 g, are killed by decapitation. The kidneys are removed, chilled in 0.25 M sucrose, and the hilar structures are dissected away. If desired, the kidneys can be sectioned further into cortical, corticomedullary, and medullary zones. The three zones contain distinct receptors for parathyroid hormone, calcitonin, and vasopressin. 1,2 The tissue is transferred to 8 volumes (w/v) of Tris.HC1 0.05 M, pH 7.4 containing 10% (v/v) DMSO and homogenized in glass with a motordriven Teflon pestle. The particulate fraction is obtained by centrifugation at 2000 g for 10 minutes; the pellet is resuspended in the original volume of Tris.HC1-DMSO and centrifuged again. The pellet is resuspended in Tris. HC1-DMSO to give a protein concentration of 15 mg/ml. This preparation is stored in small aliquots in liquid nitrogen. The enzyme in this form is stable indefinitely and has been used for in vitro bioassays for parathyroid hormone 3 or calcitonin. 4
Purification by Density Gradient Centri]ugation ~ Materials Dounce homogenizer (8 ml tube with large-clearance pestle), Kontes Male Sprague-Dawley rats (150-200 g) Swinging-bucket ultracentrifuge (Beckman L2-65B SW 40 rotor) Buffers: (A) 0.25 M sucrose, 10 mM Tris, 1 mM NazEDTA, pH 7.5 (B) 10 mM Tris, 1 mM Na2EDTA, pH 7.5 The kidneys from male Sprague-Dawley rats (150-200 g) are removed and chilled in buffer A. Solutions are kept on ice throughout. The renal capsules and perirenal fat pads are removed, and the hilar structures are cut out with a wedge-shaped incision that includes a small amount of the perihilar renal parenchyma. This removes the intrarenal portion L. R. Chase and G. D. Aurbach, Science 159, 545 (1968). ~S. J. Marx, C. J. Woodard, and G. D. Aurbach, Science 178, 999 (1972). R. Marcus and .G.D. Aurbach, Endocrinology 85, 801 (1969). 4j. Heersche, R. Marcus, and G. D. Aurbach. Endocrinology 94, 241 (1974). S. J. Marx, S. A. Fedak, and G. D. Aurbach, J. Biol. Chem. 247, 6913 (1972).
[19]
REN/kL AND SKELETAL CYCLASE
151
of the renal arteries that are difficult to process in the homogenizer. The kidneys are then bisected in a plane through the hilum and both poles. Hydronephrotic kidneys are discarded. Two volumes of buffer A are added to the renal tissue, and it is homogenized with 10 strokes in a Dounce homogenizer. This crude homogenate is centrifuged in the SS-34 rotor of a Sorvall RC-2B centrifuge. As soon as the rotor has reached 4500 rpm (2200 g), approximately 30 seconds, the rotor drive switch is turned off with the brake on. The pellet is discarded, and the supernatant solution is centrifuged twice again, the precipitates being discarded. Then the remaining supernatant fluid is centrifuged at 4500 rpm for 15 minutes at 4 °. The reddish supernatant fluid is gently poured off and discarded, and the upper portion of the resulting double-layered pellet is resuspended in a small excess of buffer A by gentle swirling and then mixing with 10 strokes of the Dounce homogenizer. This material is diluted to 20-40 ml with buffer A and again centrifuged at 4500 rpm for 15 minutes. The resulting pellet is resuspended in a minimal volume of buffer A giving a thick preparation with protein concentration of 15-25 mg/ml. When tissue from a few animals is being processed suspension is accomplished by aspirating several times through a Pasteur pipette. With larger amounts the ]:)ounce homogenizer is used. This partially purified membrane fraction is overlayered onto continuous gradients of 32% to 42% sucrose (w/w) made up in buffer B. Gradients of 11-12 ml are prepared and samples of 0.5 to 1.5 ml are applied. Material from up to 40 animals can be processed in a single ultracentrifuge run. The gradients should be approximately linear. Centrifugation is carried out at 100,000 g (23,000 rpm in the SW 40 rotor of the Beckman L2-65B ultracentrifuge) at 4 ° with the brake on. After centrifugation, two ma)or bands of turbidity as well as a large pellet are obtained. The upper band (corresponding to a density of 38% sucrose) is aspirated with a Pasteur pipette, diluted with 3 volumes of buffer B, and centrifuged 15 minutes at 2200 g. This pellet is the purified membrane fraction and can be resuspended in appropriate buffers for adenylate cyclase assays. The characteristics of the purified membrane fraction have been described in detail. ~ On electron microscopy, it is seen to be composed of vesicles and large sacs, some containing mitochondria. :No brush border elements are detectable in the preparation. It is enriched with respect to both adenylate cyclase and :Na,K-ATPase but depleted of brush border enzymes such as alkaline phosphatase and 5'-nucleotidase. Thus, this preparation of adenylate cyclase represents membranes not derived from the brush border; it is activated by parathyroid hormone, calcitonin, vasopressin, or isoproterenol. Where indicated, the kidneys can be dissected into gross anatomical regions prior to processing. This allows segre-
152
BIOSYNTHESIS OF CYCLIC NUCLEOTIDES
[19]
ADENYLATE CYCLASE ACTIVITY a
Addition Species Rat
Cow Man
None
PTH
SCT
AVP
2.6±0.0 3.8~0.2 2.3~0.1
15.2±2 8.6±0.5 12.1±0.3
24.1±0.3 3.4~0.2 2.9 ~0..1
25.7±0.1 9.3±0.2 2.6±0.1
, Enzyme was prepared as described from renal eorticomedullaryjunction of the indicated species and tested with maximally effective amounts of parathyroid hormone, salmon ealcitonin, or arginine vasopressin. In the bovine and human preparations, similar results were obtained using bovine or human calcitonins, respectively. Results are the mean ___SE of three determinations, expressed as nanomoles of cyclic AMP generated per milligram of protein in 30 minutes at 22°. The abbreviations used are PTH, bovine parathyroid hormone; SCT, salmon ealcitonin; AVP, arginine vasopressin. gation into regions specifically sensitive to particular hormones; the cortical adenylate cyelase is most responsive to parathyroid hormone, the medullary junction most responsive to vasopressin, and that from the eorticomedullary junction most responsive to calcitonin or vasopressin. 1,2 The same method has been used to prepare enzyme from kidneys of the rat, cow, and man, in which a similar distribution of parathyroid hormone and vasopressin-sensitive enzyme has been found. The bovine and human kidneys, however, contain much less calcitonin-sensitive enzyme (see the table). In contrast to the crude enzyme preparation, the stability of the purified (38% sucrose fraction) adenylate cyclase on storage has been variable whether the material has been stored in Tris buffer A, or Tris with 10% DMSO. Thus, it is best to prepare the membranes freshly for use in the adenylate cyclase system. On the other hand, the preparation seems stable (stored in aliquots as pellets) for radioligand studies. Specific binding activity for 12~I-labeled salmon calcitonin was not significantly altered after storage in liquid nitrogen for many months. Preparation of E n z y m e from N e o n a t a l B o n e
Materials Neonatal rats (0-24 hours old) Stainless steel mortar and pestle Electrically driven rotating Teflon pestle (Tri-R $63) Buffer C: 10 mM Tris, 20 mM Na2EDTA, pH 7.5 in 10% DMSO
[20]
VISUAL PHOTORECEPTOR MEMBRANES
153
Solutions are kept on ice throughout the procedure. Calvaria from 20-40 rat fetuses are dissected and suspended in buffe~ C at 4 °. They are then transferred to a stainless steel mortar (prechilled on dry ice) and powdered while frozen using a prechilled stainless steel pestle. The preparation is then suspended in 6 volumes of buffer C and homogenized for 30 seconds with an electrically driven rotating Teflon pestle. The homogenate is then filtered once through glass wool and centrifuged 15 minutes at 2200 g at 4 °. This pellet can then be resuspended in appropriate buffers for adenylate cyclase assays. The adenylate cyclase in this preparation is sensitive to parathyroid hormone and to calcitonin. The calvarial preparation contains calcitoninbinding receptors similar to those in the kidney. 2 Earlier experiments showed no effect of porcine caleitonin on adenylate cyclase in fetal calvaria2 Recent studies, however, using the buffer (C) containing DMSO show marked stimulation of the enzyme by the highly potent molecule salmon calcitonin. 4 In these latter studies, salmon calcitonin in concentrations as low as 1 ng/ml caused activation of the skeletal enzyme. Porcine calcitonin was also active, but at a higher dose range. L. R. Chase, S. A. Fedak, and G. D. Aurbach, Endocrinology 84, 761 (1969).
[20] P r e p a r a t i o n o f V e r t e b r a t e P h o t o r e c e p t o r M e m b r a n e s for Study of Adenylate Cyclase, Guanylate Cyclase, and Cyclic Nucleotide Phosphodiesterase
By J. J. KEIRNS, ~. MIKI, and M. W. BITENSKY
Principle Photoreceptor membranes (vertebrate rod outer segments) are purified by flotation on heavy sucrose. Instructions for Isolation 1,=
All procedures except deliberate light adaptation are carried out in the complete absence of visible light with the aid of infrared light sources (made from ordinary microscope or high intensity tungsten lamps fitted with a Corning CS No. 7-56 infrared filter) and infrared image converters M. W. Bitensky, R. E. Gorman, and W. H. Miller, Proc. Nat. Acad. Sci. U.S. 68, 561 (1971). 2 N. Miki, J. J. Keirns, F. R. Marcus, and M. W. Bitensky, Exp. Eye Re.s. 18 (1974) in press.
BIOSYNTHESIS OF CYCLIC NUCLEOTIDES
[19]
[19] A d e n y l a t e C y c l a s e f r o m K i d n e y a n d B o n e
By S. J. MAax and G. D. AURBACH
Preparation of Enzyme Crude Enzyme Male Sprague-Dawley rats, 150-200 g, are killed by decapitation. The kidneys are removed, chilled in 0.25 M sucrose, and the hilar structures are dissected away. If desired, the kidneys can be sectioned further into cortical, corticomedullary, and medullary zones. The three zones contain distinct receptors for parathyroid hormone, calcitonin, and vasopressin. 1,2 The tissue is transferred to 8 volumes (w/v) of Tris.HC1 0.05 M, pH 7.4 containing 10% (v/v) DMSO and homogenized in glass with a motordriven Teflon pestle. The particulate fraction is obtained by centrifugation at 2000 g for 10 minutes; the pellet is resuspended in the original volume of Tris.HC1-DMSO and centrifuged again. The pellet is resuspended in Tris. HC1-DMSO to give a protein concentration of 15 mg/ml. This preparation is stored in small aliquots in liquid nitrogen. The enzyme in this form is stable indefinitely and has been used for in vitro bioassays for parathyroid hormone 3 or calcitonin. 4
Purification by Density Gradient Centri]ugation ~ Materials Dounce homogenizer (8 ml tube with large-clearance pestle), Kontes Male Sprague-Dawley rats (150-200 g) Swinging-bucket ultracentrifuge (Beckman L2-65B SW 40 rotor) Buffers: (A) 0.25 M sucrose, 10 mM Tris, 1 mM NazEDTA, pH 7.5 (B) 10 mM Tris, 1 mM Na2EDTA, pH 7.5 The kidneys from male Sprague-Dawley rats (150-200 g) are removed and chilled in buffer A. Solutions are kept on ice throughout. The renal capsules and perirenal fat pads are removed, and the hilar structures are cut out with a wedge-shaped incision that includes a small amount of the perihilar renal parenchyma. This removes the intrarenal portion L. R. Chase and G. D. Aurbach, Science 159, 545 (1968). ~S. J. Marx, C. J. Woodard, and G. D. Aurbach, Science 178, 999 (1972). R. Marcus and .G.D. Aurbach, Endocrinology 85, 801 (1969). 4j. Heersche, R. Marcus, and G. D. Aurbach. Endocrinology 94, 241 (1974). S. J. Marx, S. A. Fedak, and G. D. Aurbach, J. Biol. Chem. 247, 6913 (1972).
[19]
REN/kL AND SKELETAL CYCLASE
151
of the renal arteries that are difficult to process in the homogenizer. The kidneys are then bisected in a plane through the hilum and both poles. Hydronephrotic kidneys are discarded. Two volumes of buffer A are added to the renal tissue, and it is homogenized with 10 strokes in a Dounce homogenizer. This crude homogenate is centrifuged in the SS-34 rotor of a Sorvall RC-2B centrifuge. As soon as the rotor has reached 4500 rpm (2200 g), approximately 30 seconds, the rotor drive switch is turned off with the brake on. The pellet is discarded, and the supernatant solution is centrifuged twice again, the precipitates being discarded. Then the remaining supernatant fluid is centrifuged at 4500 rpm for 15 minutes at 4 °. The reddish supernatant fluid is gently poured off and discarded, and the upper portion of the resulting double-layered pellet is resuspended in a small excess of buffer A by gentle swirling and then mixing with 10 strokes of the Dounce homogenizer. This material is diluted to 20-40 ml with buffer A and again centrifuged at 4500 rpm for 15 minutes. The resulting pellet is resuspended in a minimal volume of buffer A giving a thick preparation with protein concentration of 15-25 mg/ml. When tissue from a few animals is being processed suspension is accomplished by aspirating several times through a Pasteur pipette. With larger amounts the ]:)ounce homogenizer is used. This partially purified membrane fraction is overlayered onto continuous gradients of 32% to 42% sucrose (w/w) made up in buffer B. Gradients of 11-12 ml are prepared and samples of 0.5 to 1.5 ml are applied. Material from up to 40 animals can be processed in a single ultracentrifuge run. The gradients should be approximately linear. Centrifugation is carried out at 100,000 g (23,000 rpm in the SW 40 rotor of the Beckman L2-65B ultracentrifuge) at 4 ° with the brake on. After centrifugation, two ma)or bands of turbidity as well as a large pellet are obtained. The upper band (corresponding to a density of 38% sucrose) is aspirated with a Pasteur pipette, diluted with 3 volumes of buffer B, and centrifuged 15 minutes at 2200 g. This pellet is the purified membrane fraction and can be resuspended in appropriate buffers for adenylate cyclase assays. The characteristics of the purified membrane fraction have been described in detail. ~ On electron microscopy, it is seen to be composed of vesicles and large sacs, some containing mitochondria. :No brush border elements are detectable in the preparation. It is enriched with respect to both adenylate cyclase and :Na,K-ATPase but depleted of brush border enzymes such as alkaline phosphatase and 5'-nucleotidase. Thus, this preparation of adenylate cyclase represents membranes not derived from the brush border; it is activated by parathyroid hormone, calcitonin, vasopressin, or isoproterenol. Where indicated, the kidneys can be dissected into gross anatomical regions prior to processing. This allows segre-
152
BIOSYNTHESIS OF CYCLIC NUCLEOTIDES
[19]
ADENYLATE CYCLASE ACTIVITY a
Addition Species Rat
Cow Man
None
PTH
SCT
AVP
2.6±0.0 3.8~0.2 2.3~0.1
15.2±2 8.6±0.5 12.1±0.3
24.1±0.3 3.4~0.2 2.9 ~0..1
25.7±0.1 9.3±0.2 2.6±0.1
, Enzyme was prepared as described from renal eorticomedullaryjunction of the indicated species and tested with maximally effective amounts of parathyroid hormone, salmon ealcitonin, or arginine vasopressin. In the bovine and human preparations, similar results were obtained using bovine or human calcitonins, respectively. Results are the mean ___SE of three determinations, expressed as nanomoles of cyclic AMP generated per milligram of protein in 30 minutes at 22°. The abbreviations used are PTH, bovine parathyroid hormone; SCT, salmon ealcitonin; AVP, arginine vasopressin. gation into regions specifically sensitive to particular hormones; the cortical adenylate cyelase is most responsive to parathyroid hormone, the medullary junction most responsive to vasopressin, and that from the eorticomedullary junction most responsive to calcitonin or vasopressin. 1,2 The same method has been used to prepare enzyme from kidneys of the rat, cow, and man, in which a similar distribution of parathyroid hormone and vasopressin-sensitive enzyme has been found. The bovine and human kidneys, however, contain much less calcitonin-sensitive enzyme (see the table). In contrast to the crude enzyme preparation, the stability of the purified (38% sucrose fraction) adenylate cyclase on storage has been variable whether the material has been stored in Tris buffer A, or Tris with 10% DMSO. Thus, it is best to prepare the membranes freshly for use in the adenylate cyclase system. On the other hand, the preparation seems stable (stored in aliquots as pellets) for radioligand studies. Specific binding activity for 12~I-labeled salmon calcitonin was not significantly altered after storage in liquid nitrogen for many months. Preparation of E n z y m e from N e o n a t a l B o n e
Materials Neonatal rats (0-24 hours old) Stainless steel mortar and pestle Electrically driven rotating Teflon pestle (Tri-R $63) Buffer C: 10 mM Tris, 20 mM Na2EDTA, pH 7.5 in 10% DMSO
[20]
VISUAL PHOTORECEPTOR MEMBRANES
153
Solutions are kept on ice throughout the procedure. Calvaria from 20-40 rat fetuses are dissected and suspended in buffe~ C at 4 °. They are then transferred to a stainless steel mortar (prechilled on dry ice) and powdered while frozen using a prechilled stainless steel pestle. The preparation is then suspended in 6 volumes of buffer C and homogenized for 30 seconds with an electrically driven rotating Teflon pestle. The homogenate is then filtered once through glass wool and centrifuged 15 minutes at 2200 g at 4 °. This pellet can then be resuspended in appropriate buffers for adenylate cyclase assays. The adenylate cyclase in this preparation is sensitive to parathyroid hormone and to calcitonin. The calvarial preparation contains calcitoninbinding receptors similar to those in the kidney. 2 Earlier experiments showed no effect of porcine caleitonin on adenylate cyclase in fetal calvaria2 Recent studies, however, using the buffer (C) containing DMSO show marked stimulation of the enzyme by the highly potent molecule salmon calcitonin. 4 In these latter studies, salmon calcitonin in concentrations as low as 1 ng/ml caused activation of the skeletal enzyme. Porcine calcitonin was also active, but at a higher dose range. L. R. Chase, S. A. Fedak, and G. D. Aurbach, Endocrinology 84, 761 (1969).
[20] P r e p a r a t i o n o f V e r t e b r a t e P h o t o r e c e p t o r M e m b r a n e s for Study of Adenylate Cyclase, Guanylate Cyclase, and Cyclic Nucleotide Phosphodiesterase
By J. J. KEIRNS, ~. MIKI, and M. W. BITENSKY
Principle Photoreceptor membranes (vertebrate rod outer segments) are purified by flotation on heavy sucrose. Instructions for Isolation 1,=
All procedures except deliberate light adaptation are carried out in the complete absence of visible light with the aid of infrared light sources (made from ordinary microscope or high intensity tungsten lamps fitted with a Corning CS No. 7-56 infrared filter) and infrared image converters M. W. Bitensky, R. E. Gorman, and W. H. Miller, Proc. Nat. Acad. Sci. U.S. 68, 561 (1971). 2 N. Miki, J. J. Keirns, F. R. Marcus, and M. W. Bitensky, Exp. Eye Re.s. 18 (1974) in press.