Summary
Ultrastructural localization of adenylate cyclase (AC) activity was investigated in suspensions of unfixed isolated rat thymocytes using a medium containing 0.6 mM 5′-adenylylimidodiphosphate (AMP-PNP) as a substrate, 10 mM MgSO4 as an activator, 5 mM theophylline as an inhibitor of 3′,5′-AMP-phosphodiesterase and 2 mM lead nitrate as a capturing agent. AC activity was demonstrated in plasma membrane, perinuclear space, endoplasmic reticulum, Golgi complex, centriole microtubules and mitochondria. AC was activated with 10−4 M adrenalin in the presence of 5′-guanylylimido-diphosphate (GMP-PNP) as well as with 10−2 M NaF. In the cells incubated in a medium devoid of theophylline and containing 5′-AMP instead of AMP-PNP, 5′-nucleotidase activity was observed in the same cell structures as AC activity. Hydrolysis of 5′-AMP in the nucleus was much stronger than that of AMP-PNP. 10 mM NaF markedly inhibited hydrolysis of 5′-AMP in all cell structures. No staining was observed with 2 mM β-glycerophosphate as a substrate. Incubation of unfixed thymocytes in media containing AMP-PNP, 5′-AMP or p-nitrophenyl phosphate, but not β-glycerophosphate, induced both in the nucleus and in the cytoplasm in some cells an appearance of a transitory reticular formation consisting of about 30 nm thick strands which could penetrate the nuclear envelope and plasma membrane and form connections with adjacent cells. The transitory reticular formation seems to belong to the cytoskeleton and to be involved in cell aggregation.
Similar content being viewed by others
References
Buchwalow IB (1973) Ultrastructural localization of enzyme systems in the cell nucleus. Uspekhi Sovrem Biol 76:382–394 (In Russian)
Buchwalow IB, Unger E, Schulze W, Schön R, Raikhin NT (1975) Hemmung und Stimulierung der ATPase-Aktivität und die nichtenzymatische Hydrolyse von ATP in der elektronenmikroskopischen Histochemie. Histochemie 11:1–11
Buchwalow IB, Unger E, Raikhlin NT, Mashkowtsev JuW (1977) Electron-histochemical investigation of the enzyme activity of nuclear ribonucleoproteins. Acta Histochem Cytochem 10:360–370
Buchwalow IB, Kopiov OV, Schulze W (1979) Endocellular reticulum — a new cell organelle. Proceedings of the XI-th All-Union Conference on Electron Microscopy, Tallin-Moscow, Vol 3, pp 144–145 (In Russian)
Atmar VJ, Westland JA, Garcia G, Kuehn GD (1976) Adenylate cyclase in Physarum polycephalum: Inhibition of a nuclear enzyme by polyamines. Biochem Biophys Res Commun 68:561–568
Cheng H, Farquhar MC (1976a) Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. 1. Biochemical determination. J Cell Biol 70:660–670
Cheng H, Farquhar MC (1976b) Presence of adenylate cyclase activity in Golgi and other fractions of rat liver. II. Cytochemical localization within Golgi and ER membranes. J Cell Biol 70:671–684
Dedman JR, Brinkley BR, Means AR (1979) Regulation of microfilaments and microtubules by calcium and cyclic AMP. Adv Cycl Nucl Res 11:131–174
Entman ML, Levey GS, Epstein SE (1969) Demonstration of adenyl cyclase activity in canine sacroplasmic reticulum. Biochem Biophys Res Commun 35:728–733
Liao S, Lin AH, Tymoszko JL (1971) Adenyl cyclase of cell nuclei isolated from rat ventral prostate. Biochim Biophys Acta 230:535–538
Lyubskii SL, Buchwalow IB, Raikhlin NT (1979) Ultrastructural localization of ATPase and 5′-nucleotidase activity in chinese hamster mitotic cells. Acta Histochem Cytochem 12:1–6
McKeel DW, Jarett L (1974) The enrichment of adenylate cyclase in the plasma membrane and Golgi subcellular fractions of porcine adenohypophysis. J Cell Biol 62:231–236
Monneron A, D'Alayer J (1980) Subcellular localization of adenylate cyclase in thymocytes. Biochim Biophys Acta 629:50–60
Rabinowitz ML, Deslles L, Meisler J, Lorand L (1965) Distribution of adenyl cyclase activity in rabbit muscle fractions. Biochim Biophys Acta 97:29–36
Ray TK, Forte JC: Adenyl cyclase of oxyntic cells. Its association with different cellular membranes. Biochim Biophys Acta 363:320–329
Seraydarian K, Mommaerts FHM (1965) Density gradient separation of sarcotubular vesicles and other particulate constituents of rabbit muscle. J Cell Biol 26:641–656
Soifer D, Hechter O (1971) Adenyl cyclase activity in rat liver nuclei. Biochim Biophys Acta 230:539–542
Solntseva TI, Belousova AK (1977) Content of cAMP and its metabolism at various stages of hepatoma 22a growth. Bull Exp Biol Med 10:472–474 (In Russian)
Sulakhe PV, Dhalla NS (1973) Adenylate cyclase of heart sacrotubular membranes. Biochim Biophys Acta 293:379–396
Tu JC, Malhotra KS (1973) Histochemical localization of adenylate cyclase in the fungus Phycomyces blakesleeanus. J Histochem Cytochem 21:1041–1046
Unger E, Halbhuber K-J, Buchwalow IB, Schütz H (1978) Phosphationenaufnahme durch Biologische Materialien, Phosphationendiffusion und elektronenoptischer Phosphohydrolase-Nachweis. Acta Histochem 62:86–94
Wagner CR, Bitensky MW (1974) Adenylate cyclase. In: Hayat MA (ed) Electron microscopy of enzymes. Principles and methods vol 2. Van Nostrand, New York, pp 110–131
Wedner HJ, Hoffer BJ, Bloom FE, Parker CM (1973) Catecholamine stimulation of cyclic AMP in the nucleus of the human peripheral lymphocytes. Fed Proc 32:744–750
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Buchwalow, I.B., Kopiov, O.V. & Schulze, W. Ultracytochemical localization of adenylate cyclase activity in rat thymocytes. Histochemistry 72, 625–634 (1981). https://doi.org/10.1007/BF00493279
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00493279