Comparison of the characteristics of the immobilized and solubilized glyceraldehyde phosphate dehydrogenase of human erythrocyte membranes

Schrier, Stanley L.; Junga, Irene; Johnson, Muriel

doi:10.1016/0024-3205(75)90528-7

Cited by 3 publications

(2 citation statements)

References 5 publications

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“…After a 1 min preincubation in the absence or presence of Triton X-100 (0.2% v/v), the reaction (20-22 °C) was followed spectrophotometrically (Ultraspec III, Pharmacia LBK, Biochrom Ltd., Cambridge, U.K.) at 412 and 340 nm for the acetylcholinesterase and glyceraldehyde-3-phosphate dehydrogenase assay, respectively. The pH optimum of glyceraldehyde-3-phosphate dehydrogenase activity is about 8.4 (Schrier et al, 1975), but the activity was determined at pH 7.4-7.6 to obtain comparable conditions in the assays of sidedness and transport.…”

Section: Chemicalsmentioning

confidence: 99%

Cyclic AMP Stimulates the Cyclic GMP Egression Pump in Human Erythrocytes: Effects of Probenecid, Verapamil, Progesterone, Theophylline, IBMX, Forskolin, and Cyclic AMP on Cyclic GMP Uptake and Association to Inside-Out Vesicles

et al. 1998

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The knowledge about the structure and function of the protein families responsible for cGMP synthesis and metabolic conversion has grown vastly the last years, whereas little is known about proteins that account for the cellular export of cGMP. In the present study, we have employed a model with inside-out vesicles prepared from human erythrocytes to characterize modulation and regulation of cellular cGMP extrusion. The active transport was saturable (Km of 2.4 +/- 0.2 microM, mean +/- SEM, n = 3) and coupled to ATP hydrolysis since no accumulation was detected in the presence of ATP-gamma-S and AMP-PNP. The observation that 100 microM of cAMP caused a minimal inhibition (14.4 +/- 0.3%) of active cGMP transport showed that the extrusion system for cGMP was not shared with cAMP, but a competitive interaction occurred for the ATP-independent association to the inside out vesicles. In contrast, the lowest, but physiological relevant cAMP concentrations (0.1-5 microM) stimulated the active cGMP transport with 30-35%, an observation that suggests cAMP as an allosteric regulator of the cGMP transporter. Several well-known modulators of other energy-requiring membrane transport systems caused a competitive and concentration-dependent inhibition, including verapamil (Ki = 13.0 +/- 2.4 microM), forskolin (Ki = 13.5 +/- 1.4 microM) and probenecid (Ki = 27.0 +/- 1.3 microM). Progesterone, which was the most potent inhibitor (Ki = 2.2 +/- 0.3 microM), interacted with the active cGMP transport in a noncompetitive manner. The highest concentration (100 microM) of IBMX and theophylline reduced the active cGMP uptake with 29.5 +/- 1.9% and 21.6 +/- 2.1%, respectively. None of these substances interfered with the association of cGMP to the vesicles in absence of ATP. The present results show that human erythrocytes possess a cell membrane cGMP transporter which is coupled to an ATPase. Its activity is regulated by cAMP in an apparent allosteric manner and inhibited by substances previously known to interact with other membrane transport systems.

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Section: Chemicalsmentioning

confidence: 99%

Cyclic AMP Stimulates the Cyclic GMP Egression Pump in Human Erythrocytes: Effects of Probenecid, Verapamil, Progesterone, Theophylline, IBMX, Forskolin, and Cyclic AMP on Cyclic GMP Uptake and Association to Inside-Out Vesicles

et al. 1998

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“…lactate dehydrogenase and pyruvate kinase) under various conditions (28) indicating that this enzyme (and aldolase) constitute or is located exterior to a diffusion barrier (presumably the inner leaflet). These interpretations are corroborated by the demonstration that the enzymatic properties of GAPDH are not significantly different in the membrane-associated and sohbilized states (91).…”

Section: Molecular Composition Of the Two Leaflets Of The Human Erythmentioning

confidence: 72%

The Plasma Membrane Consists of Two Polar–Nonpolar–Polar Leaflets

Cervén

1977

Upsala Journal of Medical Sciences

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The implications of a double polar-nonpolar-polar leaflet construction of the plasma membrane are investigated. Experimental data from transmission electron microscopic and enzymologic characterization of plasma membranes are advantageously interpreted in these terms compared to interpretation in terms of lipid bilayer. X-ray diffraction and electron spin resonance studies do not differentiate between the present and previous models for the structure of plasma membranes but electron spin resonance data that fail to indicate a statistical distribution of spin labels also fail to support the fluid mosaic model for cell membranes. Results from experiments involving vectorial digestion and labelling of plasma membranes as well as freeze fracture electron microscopic data are compatible with the present model. The molecular composition of the human erythrocyte membrane is investigated whereby the band 111 protein and glycophorin are suggested to be the structural proteins of the outer leaflet and the spectrins those of the inner leaflet.

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