scholarly journals Protein kinases potentially capable of catalyzing the phosphorylation of p47-phox in normal neutrophils and neutrophils of patients with chronic granulomatous disease

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 940-947
Author(s):  
J Ding ◽  
JA Badwey ◽  
RW Erickson ◽  
KJ Balazovich ◽  
JT Curnutte
Keyword(s):  
Chronic Granulomatous Disease ◽  
Protein Kinases ◽  
Granulomatous Disease ◽  
Amino Acid Residues ◽  
Peptide Substrate ◽  
Uncharacterized Protein ◽  
Kinase C ◽  
Multiple Protein ◽  
Novel Protein ◽  
Stimulation Of

A procedure for uncovering novel protein kinases was used to search for enzymes in neutrophils that may catalyze the phosphorylation of the 47- Kd subunit of the NADPH oxidase system (p47-phox). This component of the oxidase can undergo phosphorylation on multiple sites. The method is based on the ability of renatured kinases to recognize exogenous substrates fixed in gels. We report that neutrophils contain several uncharacterized protein kinases that catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297 through 331 of p47-phox. Some of these enzymes are strongly activated on stimulation of the cells with phorbol 12-myristate 13-acetate (PMA). The results indicate that the phosphorylation of p47-phox in neutrophils may be more complicated than previously appreciated and may involve multiple protein kinases. In addition, we have examined both the renaturable protein kinases and the properties of protein kinase C (PKC) in neutrophils from patients with chronic granulomatous disease (CGD) who are deficient in cytochrome b558. Previous studies have shown that these cells exhibit incomplete phosphorylation of p47-phox on stimulation. In this study, we were unable to detect any alterations in the renaturable protein kinases or PKC in CGD neutrophils that could explain these defects in the phosphorylation of p47-phox.

scholarly journals Protein kinases potentially capable of catalyzing the phosphorylation of p47-phox in normal neutrophils and neutrophils of patients with chronic granulomatous disease

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 940-947 ◽  
Author(s):  
J Ding ◽  
JA Badwey ◽  
RW Erickson ◽  
KJ Balazovich ◽  
JT Curnutte
Keyword(s):  
Chronic Granulomatous Disease ◽  
Protein Kinases ◽  
Granulomatous Disease ◽  
Amino Acid Residues ◽  
Peptide Substrate ◽  
Uncharacterized Protein ◽  
Kinase C ◽  
Multiple Protein ◽  
Novel Protein ◽  
Stimulation Of

Abstract A procedure for uncovering novel protein kinases was used to search for enzymes in neutrophils that may catalyze the phosphorylation of the 47- Kd subunit of the NADPH oxidase system (p47-phox). This component of the oxidase can undergo phosphorylation on multiple sites. The method is based on the ability of renatured kinases to recognize exogenous substrates fixed in gels. We report that neutrophils contain several uncharacterized protein kinases that catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297 through 331 of p47-phox. Some of these enzymes are strongly activated on stimulation of the cells with phorbol 12-myristate 13-acetate (PMA). The results indicate that the phosphorylation of p47-phox in neutrophils may be more complicated than previously appreciated and may involve multiple protein kinases. In addition, we have examined both the renaturable protein kinases and the properties of protein kinase C (PKC) in neutrophils from patients with chronic granulomatous disease (CGD) who are deficient in cytochrome b558. Previous studies have shown that these cells exhibit incomplete phosphorylation of p47-phox on stimulation. In this study, we were unable to detect any alterations in the renaturable protein kinases or PKC in CGD neutrophils that could explain these defects in the phosphorylation of p47-phox.

scholarly journals Detection of carriers of the autosomal form of chronic granulomatous disease

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 505-507 ◽  
Author(s):  
AJ Verhoeven ◽  
ML van Schaik ◽  
D Roos ◽  
RS Weening
Keyword(s):  
Chronic Granulomatous Disease ◽  
Respiratory Burst ◽  
Granulomatous Disease ◽  
Mosaic Pattern ◽  
Simple Method ◽  
Nbt Test ◽  
Activated Neutrophils ◽  
Slide Test ◽  
Microscopic Slide ◽  
Stimulation Of

The NADPH:O2 oxidoreductase catalyzing the respiratory burst in activated phagocytes from healthy individuals is not operative in phagocytes from patients with chronic granulomatous disease (CGD). In a microscopic slide test using the dye nitroblue tetrazolium (NBT), carriers of X-linked CGD can be recognized by a mosaic pattern of NBT- positive and NBT-negative cells, governed by the expression of an unaffected or an affected X chromosome, respectively. Until now, it has not been possible to detect carriers of the autosomal form of CGD (other than by family studies) because all cells of these carriers stain positive in the NBT test. We have investigated whether neutrophils from carriers of autosomal CGD can be recognized by measurement of the rate of oxygen uptake upon stimulation of the cells. It was found that with the phorbol ester PMA as a stimulus, the respiratory burst is significantly lower in autosomal CGD carriers. With serum-treated zymosan as a stimulus, no difference between controls and carriers was observed. The addition of f-Met-Leu-Phe (1 microM) to PMA-activated neutrophils of control donors caused a transient increase in oxygen consumption of about 40%. Under these conditions, an increase of more than 100% was observed in neutrophils from carriers of autosomal CGD. These findings provide a simple method for the detection of carriers of the autosomal form of CGD.

scholarly journals Protein phosphorylation in neutrophils from patients with p67-phox- deficient chronic granulomatous disease

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4404-4410 ◽  
Author(s):  
PG Heyworth ◽  
J Ding ◽  
RW Erickson ◽  
DJ Lu ◽  
JT Curnutte ◽  
...  
Keyword(s):  
Chronic Granulomatous Disease ◽  
Protein Phosphorylation ◽  
Inorganic Phosphate ◽  
Granulomatous Disease ◽  
Cell Stimulation ◽  
Phagocyte Oxidase ◽  
Stimulation Of

Neutrophils are known to contain a major 67-kD protein that undergoes enhanced phosphorylation and translocation to the membrane during cell stimulation. Recent studies have assumed that this 67-kD phosphoprotein is the 67-kD subunit of the phagocyte oxidase (p67-phox). We compare here the protein phosphorylation patterns in lysates of normal neutrophils and neutrophils from patients with chronic granulomatous disease (CGD) that are completely deficient in p67-phox. The phosphoproteins were labeled by incubation of the cells with radioactive inorganic phosphate (32Pi) or by the addition of [gamma- 32P]ATP to electropermeabilized neutrophils. With either method, stimulation of the normal or CGD cells always resulted in an enhanced incorporation of 32p into two proteins in the 67-kD area. The extent of phosphorylation of these two proteins was very similar in the normal and CGD cells when permeabilized neutrophils loaded with [gamma - 32P]ATP were compared. Moreover, no overall differences in the protein phosphorylation patterns were observed between the normal and CGD cells. Our data indicate that the major 67-kD phosphoproteins observed in stimulated neutrophils are clearly different from p67-phox.

scholarly journals NADPH-binding component of the respiratory burst oxidase system: studies using neutrophil membranes from patients with chronic granulomatous disease lacking the beta-subunit of cytochrome b558.

1994 ◽  
Vol 179 (1) ◽  
pp. 291-297 ◽  
Author(s):  
S Tsunawaki ◽  
H Mizunari ◽  
H Namiki ◽  
T Kuratsuji
Keyword(s):  
Chronic Granulomatous Disease ◽  
Respiratory Burst ◽  
Beta Subunit ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Oxidase System ◽  
Cytochrome B558 ◽  
Respiratory Burst Oxidase ◽  
Stimulation Of ◽  
Binding Component

The NADPH-binding site of the respiratory burst oxidase system of neutrophils has been proposed to be either at a cytosolic component or at the beta-subunit of cytochrome b558. In this study, affinity labeling of resting and stimulated membranes, the latter having been assembled by all of the oxidase components from both membrane and cytosol, was carried out using [32P]NADPH dialdehyde (oNADPH). Stimulation of human neutrophils with PMA greatly increased O2(-)-generating activity and caused considerable translocation of the cytosolic components p47phox and p67phox. Nevertheless, PMA stimulation did not produce a labeled band which included positions at 47, 67, and approximately 32 kD. The most intense band reflected a molecular mass of 84 kD regardless of the state of activation, but a labeled band was never found near the beta-subunit (91 kD) of cytochrome b558. This 84-kD protein was further confirmed in neutrophils of 14 patients with gp91phox-deficient X-linked chronic granulomatous disease. These results indicate that the NADPH-binding component is not recruited from the cytosol, and also, that a membranous redox component besides cytochrome b558 must be involved in the NADPH oxidase system.

ANP stimulates phospholipase C in cultured RIMCT cells: roles of protein kinases and G protein

1991 ◽  
Vol 260 (4) ◽  
pp. F590-F595 ◽  
Author(s):  
T. Berl ◽  
J. Mansour ◽  
I. Teitelbaum
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinases ◽  
G Protein ◽  
Pertussis Toxin ◽  
Cyclic Nucleotide ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Dependent Protein ◽  
Stimulation Of

We examined the possibility that, in addition to stimulation of guanylate cyclase (GC), atrial natriuretic peptide (ANP) also activates phospholipase C (PLC) in cultured rat inner medullary collecting tubule (RIMCT) cells. ANP (10(-12)M) causes marked release of inositol trisphosphate (IP3) at a concentration that does not stimulate GC. Concentrations of ANP that stimulate GC (greater than or equal to 10(-10) M) result in attenuated IP3 release. Similarly, exogenous dibutyryl guanosine 3',5'-cyclic monophosphate (10(-6) M) markedly inhibits the response to 10(-10) M ANP. Inhibition of cyclic nucleotide-dependent protein kinase by H 8, but not inhibition of protein kinase C by H 7, restores the response to 10(-8) and 10(-6) M ANP. Therefore, activation of cyclic nucleotide-dependent protein kinase inhibits ANP-stimulated PLC activity. Activation of protein kinase C by phorbol 12-myristate-13-acetate (PMA) decreases ANP-stimulated IP3 production. Pretreatment with H 7, but not H 8, prevents inhibition by PMA. To explore a potential role for G proteins, we examined the effect of guanine nucleotide analogues on ANP-stimulated IP3 production in saponin-permeabilized cells. ANP-stimulated IP3 production is enhanced by GTP gamma S and is inhibited by GDP beta S. Similarly, preincubation with pertussis toxin prevents ANP-stimulated IP3 release. We conclude that ANP stimulates PLC in RIMCT cells via a pertussis toxin-sensitive G protein. Stimulation of PLC is inhibited on activation of either cyclic nucleotide or Ca2+-phospholipid dependent protein kinases.

scholarly journals Detection of carriers of the autosomal form of chronic granulomatous disease

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 505-507 ◽  
Author(s):  
AJ Verhoeven ◽  
ML van Schaik ◽  
D Roos ◽  
RS Weening
Keyword(s):  
Chronic Granulomatous Disease ◽  
Respiratory Burst ◽  
Granulomatous Disease ◽  
Mosaic Pattern ◽  
Simple Method ◽  
Nbt Test ◽  
Activated Neutrophils ◽  
Slide Test ◽  
Microscopic Slide ◽  
Stimulation Of

Abstract The NADPH:O2 oxidoreductase catalyzing the respiratory burst in activated phagocytes from healthy individuals is not operative in phagocytes from patients with chronic granulomatous disease (CGD). In a microscopic slide test using the dye nitroblue tetrazolium (NBT), carriers of X-linked CGD can be recognized by a mosaic pattern of NBT- positive and NBT-negative cells, governed by the expression of an unaffected or an affected X chromosome, respectively. Until now, it has not been possible to detect carriers of the autosomal form of CGD (other than by family studies) because all cells of these carriers stain positive in the NBT test. We have investigated whether neutrophils from carriers of autosomal CGD can be recognized by measurement of the rate of oxygen uptake upon stimulation of the cells. It was found that with the phorbol ester PMA as a stimulus, the respiratory burst is significantly lower in autosomal CGD carriers. With serum-treated zymosan as a stimulus, no difference between controls and carriers was observed. The addition of f-Met-Leu-Phe (1 microM) to PMA-activated neutrophils of control donors caused a transient increase in oxygen consumption of about 40%. Under these conditions, an increase of more than 100% was observed in neutrophils from carriers of autosomal CGD. These findings provide a simple method for the detection of carriers of the autosomal form of CGD.

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