Dieter Maretzki

A reappraisal of the binding of cytosolic enzymes to erythrocyte membranes

Several cytosolic proteins have been shown to be associated with hypotonic erythrocyte ghosts via electrostatic interactions with the anion transport band 3 protein. This article considers the problems of demonstrating binding under physiological conditions and reviews the evidence for the relevance of enzyme binding to the membrane for the regulation of glycolysis. The hypotheses for the existence of topological and sequential multienzyme complexes of the glycolytic enzymes in erythrocytes are also discussed.

[17] Assays for activation of opsin by all-trans-retinal

The data collected with the techniques discussed in this chapter suggest significant differences between the active conformation(s) of the opsin/atr complex, which are reversibly formed in the dark, and the active conformation (R*) of the meta-II photoproduct. First, there is good evidence for noncovalent opsin/atr complexes with considerable activity (although covalent binding of atr is found in mutant opsins. Even more intriguing, all-trans-retinal in an amount that saturates the activity of the opsin/atr complex toward Gt does not measurably inhibit the access of 11-cis-retinal to the light-sensitive binding site during regeneration (Fig. 2C). On the other hand, forced protonation at or near Glu-134 appears to be an integral mechanism for both the meta-II and the opsin-like activities (Fig. 4). Thus, it is not inconceivable that these two activities of the receptor arise from two fundamentally different conformations, one meta-II-like and one opsin-like. They would be similar with respect to the Gt (or RK) protein-protein interaction but different in their mode of retinal-protein interaction.

The membrane association and dissociation of human glyceraldehyde-3-phosphate dehydrogenase under various conditions of hemolysis Immunochemical evidence for the lack of binding under cellular conditions

Several authors reported an association of the GAPD to the cell membrane of red cells [l--5]. The varying extent of this association suggested to us a dependence on conditions of hemolysis and recorded the question of its artefactional nature. Therefore we investigated the membrane association and dissociation of the GAPD by means of immunochemical and other techniques under various conditions of hemolysis.

Interaction of pulmonary surfactant protein A with phospholipid liposomes: a kinetic study on head group and fatty acid specificity

Recent work on surfactant protein A (SP-A) has shown that Ca(2+) induces an active conformation, SP-A, which binds rapidly to liposomes and mediates their aggregation. Employing sensitive real time assays, we have now studied the lipid binding characteristics of the SP-A liposome interaction. From the final equilibrium level of the resonant mirror binding signal, an apparent dissociation constant of ca. K(d)=5 microM is obtained for the complex between SP-A and dipalmitoylphosphatidylcholine (DPPC) liposomes. At nanomolar SP-A concentrations, this complex is formed with a subsecond (0.3 s) reaction time, as measured by light-scattering signals evoked by photolysis of caged Ca(2+). With palmitoyloleoylphosphatidylcholine (POPC), the complex formation proceeds at half the rate, compared to DPPC, leading to a lower final equilibrium level of SP-A lipid interaction. Distearoylphosphatidylcholine (DSPC) shows a stronger interaction than DPPC. Regarding the phospholipid headgroups, phosphatidylinositol (PI) and sphingomyelin (SM) interact comparable to DPPC, while less interaction is seen with phosphatidylethanolamine (PE) or with phosphatidylglycerol (PG). Thus both headgroup and fatty acid composition determine SP-A phospholipid interaction. However, the protein does not exhibit high specificity for either the polar or the apolar moiety of phospholipids.

Activation and desensitization of glycolysis by stimulation of adenylate cyclase in rat reticulocytes

Isoproterenol or forskolin induce a 10–15‐fold increase in concentration of cyclic AMP in rat reticulocytes as compared with the basal level of 2.3 ± 0.3 μM. Glycolysis is stimulated by both compounds transiently more than 2‐fold with a peak after 7.5 min followed by an exponential decline. The glycolytic rate in the presence of 1O μM isoproterenol or 10 μM forskolin did not return to basal levels within 60 min of incubation, but was depressed by as much as 50% under the influence of 100 μM forskolin. This phenomenon is designated as metabolic desensitization. The stimulation of glycolysis is probably due to activation of phosphofructokinase as well as to stimulation of Na+,K+‐ATPase. The diminished glycolytic flux during the period of metabolic desensitization is accompanied by a decline of glucose 6‐phosphate and in the presence of high concentrations of forskolin also by a decrease in glucose 1,6‐bisphosphate. A lower rate of influx of glucose is postulated.

Dissociated and Associated Forms of NAD(P)-dependent Glyceraldehyde 3-phosphate Dehydrogenase after Dark-Light Transitions in Bean Leaves

The different states of plant NAD(P)-dependent glyceraldehyde 3-phosphate dehydrogenase EC 1.2.1.13) was investigated by sucrose density gradient centrifugation of crude extracts from the primary leaves of bean plants (Phaseolus vulgarisL.). Two molecular forms of NAD(P)-GADP were found: An associated form (MW 325 · 103 ± 20 · 103) and a dissociated form (MW 165 · 103 ± 35 · 103) which are mutually transformed by effectors. In the course of the greening process as well as during plant ontogenesis a shift of activity occurs toward the dissociated GAPD form. This fact indicates a dissociation process induced by effectors which are produced in the light reactions of photosynthesis. Reversibility of the association/dissociation process was proved by the addition of some metabolites at concentrations estimated for in vivo conditions in the light or in darkness. NADP(H) and ATP added to crude extracts and during the sedimentation increase the activity of the dissociated GAPD form and suppress the associated GAPD in a different extent. NAD favours the formation of the less active associated form. Complete dissociation induced by NADPH could be observed in recentrifugation experiments with the separated enzyme forms. In vivo only NADPH seems to act as a dissociating metabolite by causing a strong activation of NAD(P)-GAPD at concentrations present in the chloroplast. Simultaneous presence of NAD (and ATP) during sedimentation of crude extracts or in recentrifugation experiments strongly limits the effectiveness of NADP(H) for dissociation and maintain the associated enzyme form. The presence of metabolite combinations simulating light or darkness during sedimentation of crude leaf extracts from etiolated plants caused no reversible interconversion of the molecular forms but activation of the dissociated NAD(P)-GAPD.

ERYTHROCYTE BAND 3 PROTEIN STRONGLY INTERACTS WITH PHOSPHOINOSITIDES

85% of the phosphorus coisolated with band 3 protein during separation of the intrinsic proteins of the human erythrocyte membrane by zonal electrophoresis in high concentrations of acetic acid was found to be derived from phosphoinositides, mainly phosphatidylinositol 4,5‐bisphosphate. When native band 3 protein and pyrene‐labelled phospholipids were present in micelles of the nonionic detergent nonaethyleneglycol lauryl ether, strong resonance energy transfer was observed between the tryptophan residues and phosphatidylinositol 4,5‐bisphosphate and, to a smaller degree, phosphatidylinositol 4‐phosphate. We conclude that band 3 protein strongly interacts with phosphoinositides, in particular with phosphatidylinositol 4,5‐bisphosphate.

New fluorescent probes for visual proteins. Part II. 5-(Oxo)penta-2,4-dienyl-p-(N,N-dimethylamino)benzoateFor part 1, see ref. 1.

A new dual-fluorescent compound, 5-(oxo)penta-2,4-dienyl-p-(N,N-dimethylamino)benzoate (1), a derivative of dimethylaminobenzoic acid, has been synthesised and studied photophysically. This compound continues the series of potential fluorescent probes for visual and proton-pumping opsin proteins. The photophysical behaviour of this molecule, including charge-transfer interaction in the ground state and dual-fluorescence emission, is similar to that of the previously studied analogue cis-3-(oxo)propenyl-p-(N,N-dimethylamino)benzoate (cis-2). The presence of several theoretically calculated conformers of compound 2 was suggested to be responsible for the observed strongly red-shifted absorption and excitation wavelength dependence. These photophysical anomalies were also observed for molecule 1, though the models put forward to explain them in the cases of 1 and 2 are rather different. Based on theoretical calculations and experimental results, we propose that some of the stable conformers might be connected with either a charge-transfer complex or mesomeric interactions in the ground state. Upon changing the electronic nature of the oxo-pentadienyl acceptor moiety, e.g. protonation, chemical or biochemical reaction, the charge-transfer absorption disappears, which leads to a dramatic increase in the fluorescence quantum yield.

Metabolism of Phosphatidylinositol in Reticulocytes — Isoproterenol Affects the Turnover of Polyphosphoinositides

Pulse-chase experiments with rabbit reticulocytes and erythrocytes prelabelled with inorganic 32P-phosphate revealed a high turnover of the phosphomonoester groups of polyphosphoinositides in reticulocytes which declines strongly during maturation. Reticulocytes incorporated H-inositol into phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4, 5-bisphosphate (PIP2) in the proportion of 17: 1: 1.8, respectively. A slow release of 3H-labelled inositol phosphates was found in reticulocytes, indicating a very low phosphodiesterase activity (Phospholipase C) in the intact cells. Determination of the 32P/3H-ratio in double-labelled phosphatidylinositides in reticulocytes demonstrated an about 3000-fold turnover of the phosphodiester groups as compared with that of the phosphgdiester group. (-)-isoproterenol stimulates the incorporation of 32P-phosphate into the polyphosphoinositides in reticulocytes but did not influence the release of 3H-inositol phosphates. A rapid response to (-)-iso-proterenol (0.1 uM) with an increase of up to 60% was found in the 32P-labelling of PIP2. The effect was antagonized by alprenolol. The stimulation of the polyphosphoinositide phosphate turnover might be mediated by an elevated concentration of cyclic AMP in reticulocytes.