Abstract
The phosphorylation and dephosphorylation of proteins in neutrophils from normal children and patients with chronic granulomatous disease (CGD) were studied with two-dimensional gel electrophoresis and autoradiography, followed by densitometric scanning. In normal neutrophils the radio-activities of 11 spots among approximately 50 radioactive spots were changed by stimulation with phorbol 12-myristate 13-acetate (PMA) and 6 of the 11 spots were also changed by stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP) and NaF. The phosphorylation of only two spots (Mr=48000 and 62000) was inhibited by 2-deoxyglucose and N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7), which inhibits superoxide production, while it was not affected by dibutyryl cAMP, KCN and ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA), which do not affect superoxide production. The observation indicates that the Mr=48000 and 62000 proteins may be involved in the activation process of superoxide production. When the neutrophils of four male and two female CGD patients were examined, the changes in 11 spots on stimulation were similar to those of normal children, indicating that the (de)phosphorylation of the proteins which seems to be involved in the activation process is not affected in CGD neutrophils.
Similar content being viewed by others
Abbreviations
- CGD:
-
chronic granulomatous disease
- PMA:
-
phorbol 12-myristate 13-acetate
- FMLP:
-
N-formyl-methionylleucyl-phenylalanine
- W-7:
-
N-(6-aminohexyl)-5-chloro-1-naphthalane sulphonamide
- EGTA:
-
ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid
- dBcAMP:
-
dibutyryl cyclic AMP
References
Andrews PC, Babior BM (1983) Endogenous protein phosphorylation resting and activated human neutrophils. Blood 61:333–340
Andrews PC, Babior BM (1984) Phosphorylation of cytosolic proteins by resting and activated human neutrophils. Blood 64:883–890
Baehner RL, Nathan DG (1967) Leukocyte oxidase: Defective activity in chronic granulomatous disease. Science 155:835–836
Borregaard N, Johansen KS (1979) Cytochrome b and chronic granulomatous disease. Lancet I:1397–1398
Böyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation at Ig. Scand J Lab Invest 21 [Suppl 97]:77–89
Brautigan DL, Bornstein P, Gallis B (1981) Phosphoryrosyl-protein phosphatase: specific inhibition by Zn2+. J Biol Chem 256: 6519–6522
Cantrell SJ, Babitch JA, Torres S (1981) Protein-load effects on the pH gradient of isoelectric focusing on polyacrylamide gels. Anal Biochem 116:168–173
Fujita I, Irita K, Takeshige K, Minakami S (1984) Diacylglycerol, 1-oleoyl-2-acetyl-glycerol, stimulates superoxide-generation from human neutrophils. Biochem Biophys Res Commun 120:318–324
Gabig TG, Lefker BA (1984) Deficient fravoprotein component of the NADPH-dependent O -2 -generating oxidase in the neutrophils from three male patients with chronic granulomatous disease. J Clin Invest 73:701–705
Holmes B, Page AR, Good RA (1967) Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function. J Clin Invest 46:1422–1432
Holn DC, Lehner RI (1975) NADPH oxidase deficiency in X-linked chronic granulomatous disease. J Clin Invest 55:707–713
Irita K, Takeshige K, Minakami S (1984) Phosphorylation of myosin light chain in intact pig leukocytes stimulated by phorbol 12-myristate 13-acetate. Biochim Biophys Acta 803:21–28
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275
Matsumoto T, Takeshige K, Minakami S (1979) Inhibition of phagocytic metabolic changes of leukocytes by an intracellular calcium-antagonist 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate. Biochem Biophys Res Commun 88:974–979
McPhail LC, DeChatelet LR, Shirley PS, Wilfert C, Johnston RB Jr, McCall CE (1977) Deficiency of NADPH oxidase activity in chronic granulomatous disease. J Pediatr 90:213–217
Nakagawara A, Takeshige K, Minakami S (1974) Introduction of a phagocytosis-like metabolic pattern in polymorphonuclear leukocytes by cytocharasin E. Exp Cell Res 87:392–394
O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021
Okamura N, Ohashi S, Nagahisa N, Ishibashi S (1984) Changes in protein phosphorylation in guinea pig polymorphonuclear leukocytes by treatment with membrane-perturbing agents which stimulate superoxide anion production. Arch Biochem Biophys 228: 270–277
Robinson JM, Badwey JA, Karnovsky ML, Karnovsky MJ (1984) Superoxide release by neutrophils: synergistic effects of a phorbol ester and a calcium ionophore. Biochem Biophys Res Commun 122:734–739
Schneider C, Zanetti M, Romeo D (1981) Surface-reactive stimuli selectively increase protein phosphorylation in human neutrophils. FEBS Lett 127:4–8
Segal AW, Peters TJ (1976) Characterization of the enzyme defect in chronic granulomatous disease. Lancet I:1363–1365
Segal AW, Cross AR, Garcia RC, Borregaard N, Valerius NH, Soothill JF, Jone OTG (1983) Absence of cytochrome b-245 in chronic granulomatous disease. N Engl J Med 308:245–251
Silver PJ, Still JT (1982) Quantitation of myosin light chain phosphorylation in small tissue samples. J Biol Chem 257:6137–6144
Stewart AA, Ingebritsen TS, Manalan A, Klee CB, Cohen P (1982) Discovery of a Ca2+- and calmodulin-dependent protein phosphatase. FEBS Lett 137:80–84
Stewart AA, Ingebritsen YTS, Cohen P (1983) The protein phosphatases involved in cellular regulation. 5. Purification and properties of a Ca2+/calmodulin-dependent protein phosphatase (2B) from rabbit skeletal muscle. Eur J Biochem 132:289–295
Takeshige K, Minakami S (1981) Involvement of calmodulin in phagocytotic respiratory burst of leukocytes. Biochem Biophys Res Commun 99:484–490
Wakeyama H, Takeshige K, Minakami S (1982) Superoxide-forming NADPH oxidase preparation of pig polymorphonuclear leukocytes. Biochem J 205:593–601
Weber K, Osborn M (1969) The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ishii, E., Irita, K., Fujita, I. et al. Protein phosphorylation of neutrophils from normal children and patients with chronic granulomatous disease. Eur J Pediatr 145, 22–26 (1986). https://doi.org/10.1007/BF00441847
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00441847