Phosphorylation of component a of the human erythrocyte membrane in myotonic muscular dystrophy

The Journal of Membrane Biology - Endogenous membrane protein kinase activity in fresh erythrocyte ghosts is altered in myotonic muscular dystrophy. Phosphorylation of erythrocyte Component a,...     

Isolation of an abnormally phosphorylated erythrocyte membrane band 3 glycoprotein from patients with myotonic muscular dystrophy

Summary A fraction of erythrocyte Band 3 (M _r , 93,000) glycoprotein that demonstrates decreased autophosphorylation in membranes from myotonic muscular dystrophy patients is demonstrated. Sequential affinity chromatography of Triton X-100 solubilized erythrocyte membrane proteins separated three specifically retained glycoprotein fractions on a Ricin Communis I-Sepharose 4B column. One fraction contains a portion of the major sialoglycoprotein (apparentM _r , 78,000) and is specifically eluted from the column by 10mm NaCl and 100mm d-galactose (10/100). The two other glycoprotein fractions are eluted by 100mm NaCl, 10mm d-galactose (100/10) and 100mm NaCl, 100mm d-galactose (100/100). The composition of both fractions contains greater than 95% Band 3 (apparentM _r , 93,000) glycoprotein.The quantities of glycoprotein in each fraction obtained from erythrocytes of myotonic dystrophy patients did not differ from the quantities obtained from control erythrocytes. Following endogenous protein kinase incubations of ghosts with [-³²P]ATP, the specific [³²P] phosphorylation of the 10/100 and 100/10 fractions are identical. The 100/100 fraction, which makes up approximately 3% of the total erythrocyte membrane protein, demonstrates a different pattern for myotonic dystrophy patients; specific phosphorylation was reduced by 50% relative to activity in control experiments. These findings are consistent with previous experiments that demonstrated decreased autophosphorylation of the glycoprotein portion of Band 3 (Roses & Appel, 1975,J. Membrane Biol. 20: 51) and are consistent with the autosomal dominant mode of inheritance in this disease.     

Electron spin resonance studies of membrane proteins in erythrocytes in myotonic muscular dystrophy.

A goat antibody produced against bovine adrenal ferredoxin has been employed to establish immunochemically the involvement of adrenal ferredoxin in the cholesterol side-chain cleavage reaction catalyzed by mammalian adrenal mitochondria. When added to preparations of bovine adrenocortical mitochondria, this antibody was found to inhibit the conversion of cholesterol to pregnenolone and progesterone, the 11β-hydroxylation of deoxycorticosterone and the NADPH-dependent reduction of cytochrome c. These observations demonstrate that, similar to the NADPH-cytochrome c reductase and steroid 11β-hydroxylase reactions, adrenal ferredoxin is also required for the oxidative cleavage of the cholesterol side-chain catalyzed by bovine adrenocortical mitochondria.The goat antibody to bovine adrenal ferredoxin was also found to interact with the comparable iron-sulfur proteins present in mitochondria prepared from sheep, rat, mouse, cat, dog, guinea pig, rabbit, and human adrenals. The interaction of the antibody with these iron-sulfur proteins resulted in the inhibition of both the cholesterol side-chain cleavage and NADPH-cytochrome c reductase activities catalyzed by these adrenal mitochondria. The NADH-dependent reduction of cytochrome c catalyzed by mammalian adrenal mitochondria was not inhibited by the goat antibody to adrenal ferredoxin. These results demonstrate the immunochemical similarity existing among mammalian adrenal ferredoxins and their involvement in the adrenal cholesterol side-chain cleavage reaction.     

Substrate heterogeneity of component a of the human erythrocyte membrane

Component a of the erythrocyte membrane is a specific substrate for endogenous protein kinase activity and its phosphorylation is significantly decreased under assay conditions in myotonic muscular dystrophy (Roses, A. D., and Appel, S. H., J. Membr. Biol. 20:51–58 (1975)). We have demonstrated substrate heterogeneity of two fractions of component a separated by concanavalin A (Con-A) sepharose chromatography. The fraction of component a that is retarded by Con A and eluted with α-methyl-D-glucoside does not accept the transfer of phosphate from [γ-³²P] ATP as a substrate for endogenous protein kinase activity. The nonretarded fraction contains > 90% of the radioactive label. These experiments also confirm the carbohydrate heterogeneity of component a (Findley, J. B. C., J. Biol. Chem. 249:4398 (1974)).     

Erythrocyte membrane fluidity in chicken muscular dystrophy.

An increased rigidity of erythrocyte membranes in four-week old dystrophic chickens compared to closely related normal controls has been suggested using electron spin resonance. These findings suggest that similar to the case of human Duchenne muscular dystrophy, chicken muscular dystrophy may be associated with a generalized membrane defect.     

Phosphorylation of erythrocyte membrane liberates calcium

Washed and permeabilized human erythrocyte ghosts were found to discharge calcium on treatment with ATP. Concomitantly, there was a decrease in phosphatidylinositol (PI) and an increase in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2). These results support the hypothesis that an inositide shuttle, PI in equilibrium PIP in equilibrium PIP2, operates to maintain intracellular Ca2+ levels. The cation is thought to be sequestered in a cage formed by the head groups of two acidic phospholipid molecules, e.g., phosphatidylserine and phosphatidylinositol, with participation of both PO and fatty acid ester CO groups. These cages are stabilized by inter-headgroup hydrogen bonding. When the inositol group is phosphorylated in positions 4 and 5, inter-lipid hydrogen bonding is disrupted and the cage opens to release its Ca2+.     

Respiratory abdominal muscle recruitment and chest wall motion in myotonic muscular dystrophy.

Abdominal muscles are selectively active in normal subjects during stress and may increase the potential energy for inspiration by reducing the end-expiratory lung volume (EELV). We hypothesized that a similar process would occur in subjects with myotonic muscular dystrophy (MMD), but would be less effective, because of to their weakness and altered chest wall mechanics. Fine-wire electromyography (EMG) of the transversus abdominis (TA), internal oblique (IO), external oblique, and rectus abdominis was recorded in 10 MMD and 10 control subjects. EMG activity, respiratory inductive plethysmography, and gastric pressure were recorded during static pressure measurement and at increasing levels of inspiratory resistance breathing. EELV was reduced and chest wall motion was synchronous only in controls. Although the TA and IO were selectively recruited in both groups, EMG activity of the MMD group was twice that of controls at the same inspiratory pressure. In MMD subjects with mildly reduced forced vital capacity, significant differences can be seen in abdominal muscle recruitment, wall motion, work of breathing, and ventilatory parameters.     

Abnormal insulin receptor binding in cultured monocytes in myotonic muscular dystrophy

Even though one of the characteristic features of myotonic dystrophy (MyD) is the high level of circulating insulin, ¹²⁵I-insulin-binding data in MyD have been controversial. In the present study we utilized cultured monocytes to avoid problems of reproducibility and variability in age, and examined the affinity and binding characteristics of ¹²⁵I-insulin binding in MyD patients and controls. The B_max and mean affinity constant, K_a, were significantly lower, while the number of receptors per cell had increased in the patient group as compared to the controls. The data confirm our earlier findings that there is no primary defect in insulin receptors in MyD, and the disturbed insulin response may be due to an abnormality in the membrane environment. Since the insulin receptor is an integral membrane protein, abnormal plasma membrane lipid composition may lead to impaired lipid-protein interactions, and hence affect the binding characteristics in MyD.     

Electron spin resonance investigations of membrane proteins in erythrocytes in muscle diseases. Duchenne and myotonic muscular dystrophy and congenital myotonia.

Comparison of electron spin resonance spectra of spin labeled erythrocyte membranes from patients with the dystrophic conditions Duchenne and myotonic muscular dystrophy with those of normal controls suggests that alterations in membrane protein conformation and/or organization are present in these disease states. These protein alterations are not apparent in the non-dystrophic disease congenital myotonia. The results suggest a correlation between changes in the physical state of proteins in membranes with the presence of dystrophy. In addition, the present results from erythrocytes lend support for the concept of a generalized membrane defect in these diseases.     

Electron spin resonance investigations of membrane proteins in erythrocytes in muscle diseases. Duchenne and myotonic muscular dystrophy and congenital myotonia

Comparison of electron spin resonance spectra of spin labeled erythrocyte membranes from patients with the dystrophic conditions Duchenne and myotonic muscular dystrophy with those of normal controls suggests that alterations in membrane protein conformation and/or organization are present in these disease states. These protein alterations are not apparent in the nondystrophic disease congenital myotonia. The results suggest a correlation between changes in the physical state of protein in membranes with the presence of dystrophy. In addition, the present results from erythrocytes lend support for the concept of a generalized membrane defect in these diseases.     

Heritable variation in erythrocyte membranes of mice with muscular dystrophy

Several physical and chemical characteristics of erythrocyte membranes from dystrophic mice differ from those of controls. In this study it is postulated that there is a heritable antigenic characteristic in erythrocyte membranes of dystrophic mice that is associated with muscular dystrophy. Accordingly, antisera were prepared in goats against erythrocyte membranes from dystrophic mice. These antisera discriminated between membranes from control mice and membranes from three different strains of dystrophic mice. Preliminary data on carrier detection are consistent with the hypothesis that the antigen is manifest in membranes of carriers.     

Insulin resistance in myotonic dystrophy.

The aim of the present study was to obtain a comprehensive picture of the rate of insulin secretion and of tissue sensitivity to the endogenous hormone in myotonic dystrophy patients (MyD). The minimal model approach was utilized for the analysis of frequently sampled intravenous glucose tolerance test data (FSIGT). This method provided the characteristic parameters: SI, insulin sensitivity index; SG fractional glucose disappearance independent of dynamic insulin; n, fractional insulin clearance; phi 1 and phi 2 first and second phase insulin delivery sensitivities to glucose stimulation. In MyD patients SI was reduced (p less than 0.01) by 71% to 1.4 +/- 0.3 x 10(-4) min-1/(microU/ml), whereas in controls it was 4.85 +/- 0.77; SG was within the normal range: 0.044 +/- 0.012 min-1 in MyD patients and 0.036 +/- 0.017 min-1 in controls; phi 1 increased in MyD patients (7.4 +/- 1.3 min (microU/ml)/(mg/dl) versus 4.1 +/- 1.2 in controls); phi 2 increased in MyD patients (126 +/- 47 x 10(4) min-2/(microU/ml)/(mg/dl) versus 17 +/- 6 in controls; p less than 0.05). MyD patients showed a normal tolerance with the glucose disappearance constant, KG within the normal range: 2.75 versus 2.62% min-1 in controls. In MyD patients insulin resistance was associated with a higher than normal insulin delivery for both secretory phases, although the second phase was responsible for releasing a greater amount of hormone. In conclusion MyD patients try to compensate for overall insulin resistance by a more marked pancreatic response.