Phosphatidic acid and polyphosphoinositide metabolism in rod outer segments. Differential role of soluble and peripheral proteins.

The phosphorylation of endogenous diacylglycerol (DAG) and phosphoinositides by [tau-32P]ATP was studied in bovine rod outer segments (ROS) selectively depleted of soluble or peripheral and soluble proteins by treatment with moderate (100 mM) or low (5 mM) ionic strength medium, respectively. DAG kinase activity was similar in bleached and non-bleached ROS extracted with 100 mM medium, and amounted to 70% of that observed in the corresponding non-extracted ROS. Phosphatidic acid (PtdH) labelling in ROS extracted in the dark with low ionic strength medium was markedly lower than in those extracted in light. Thus, even when a major proportion of DAG kinase was associated to the membrane, a soluble form also occurred. Most of the membrane-bound fraction behaved as a peripherally associated protein, its binding to the membrane being modified by light. Ir ROS extracted at moderate ionic strength the labelling of inositides was similar to that in non-extracted ROS. A marked enhancement in polyphosphoinositide labelling was observed in ROS extracted in the dark with low ionic strength. Alkaline treatment of ROS also produced inhibition of polyphosphoinositide phosphorylation. A peripheral form of a type C phospholipase, or a peripheral protein-mediated activation of a particulate form thereof, is suggested. Labelled polyphosphoinositides were more actively hydrolyzed in the light and in the dark plus GTP tau S than in the dark-incubated membranes. The results of phosphorylation experiments in membranes where differential extraction of the alpha subunit of transducin was carried out suggest that alpha and beta tau subunits may play opposite modulating roles in PtdH and polyphosphoinositide metabolism.     

Cytosol protein-independent translocation of isomeric spin-labelled radioactive lipids from isolated guinea pig liver microsomal to mitochondrial membranes

Intermembranous translocation of membrane-bound radioactive lipids covalently labelled with 5-, 12, and 16-doxyl stearic acid was studied. Guinea pig liver microsomal membranes containing known amounts of isomeric spin-labelled radioactive phosphatidic acid, phosphatidylcholine, and diglycerides were incubated with unlabelled mitochondria; reisolated mitochondria contained around 28-31% of microsomal labelled lipids above the microsomal contamination. The effect of adding crude or 'pH 5.1' 105 000 X g cytosol supernatant on the amount and composition of translocated labelled lipids was studied. While the translocation of labelled phosphatidylcholine was slightly stimulated by the addition of these cytosol supernatants, no significant increase of the amount of translocated labelled phosphatidic acic and diglycerides was observed by this addition. In view of these results, a probable mechanism for the cytosol protein-independent translocation of lipids between biological membranes is proposed.     

GTP and cytosol stimulate phosphoinositide hydrolysis in isolated platelet membranes

Hydrolysis of polyphosphoinositides by phospholipase C was examined in isolated membranes prepared from [32P]labelled platelets. In the presence of GTP gamma S, thrombin increased the release of inositol triphosphate and inositol biphosphate approximately 500%. GTP gamma S alone stimulated release 2 fold. Maximal activation of thrombin-induced phosphoinositide hydrolysis was observed at 10 uM GTP. Although addition of calcium had no effect, 2 mM EGTA completely inhibited inositolphosphate release. Addition of high speed supernatant to [32P]labelled membranes stimulated the release of inositolphosphates. This hydrolysis was further enhanced by the addition of GTP. These data demonstrate that the breakdown of polyphosphoinositides in isolated platelet membranes is dependent on GTP and stimulated by platelet cytosol.     

Metabolism of phosphoinositides in the rat erythrocyte membrane. A reappraisal of the effect of magnesium on the 32P incorporation into polyphosphoinositides

The metabolism of phosphoinositides was investigated in the red blood cell membrane of the rat by measuring 32P-incorporation into phospholipids after incubation of membranes with [gamma-32P]ATP in a medium containing magnesium. A new chromatographic procedure has been developed which facilitates the separation of triphosphoinositide, diphosphoinositide and phosphatidylinositol from the phospholipids present in lipid extracts of incubated 'ghost' under our experimental conditions only two phospholipids, diphosphoinositide and triphosphoinositide, were 32P-labelled. Furthermore, the results indicate that either di-or triphosphoinositide could be labelled preferentially, depending upon the magnesium concentration of the incubation medium. This clarifies some apparent discrepancies reported in the literature between the 32 P labelling of polyphosphoinositides observed in intact erythrocytes and that observed with 'ghost' membranes. In addition, the enzymatic pathways involved in the phosphoinositide metabolism are discussed.     

Potential activity of the external pathway of NADH oxidation in mitochondria

High rate of exogenic NADH oxidation (up to 200 mg-at. oxygen for 1 min per 1 mg of protein and higher) along the rothenone, antimycin-nonsensitive pathway is observed under interaction of mitoplasts with the external membrane and cytochrome c. In the medium with low ionic strength the interaction of external and internal membranes is not a sufficient condition for activating the external pathway of the NADH oxidation: the presence of exogenic cytochrome c is also necessary. With saturated cytochrome c concentrations the addition of outer membranes leads to further stimulation of the NADH oxidation. In the medium with high ionic strength external membranes stimulate oxidation of NADH when exogenic cytochrome c is absent; the subsequent addition of cytochrome c stimulates the NADH oxidation in this medium to a greater extent than in the medium with the low ionic strength. Under the nonlimited interaction of external and internal membranes and cytochrome c the potential activity of the outer pathway of NADH oxidation in the liver mytoplasts of hybernating gophers is lower than in the liver mytoplasts of rats.     

Parameters of activation of the membrane-bound O2- generating oxidase from bovine neutrophils in a cell-free system

Parameters governing the extent of activation of the O2- generating oxidase in a cell-free system derived from bovine neutrophils were examined. The reconstituted system consisted of the following: a particulate fraction enriched in plasma membrane and containing the oxidase, a soluble fraction containing cytosolic factor(s) required for oxidase a soluble fraction containing cytosolic factor(s) required for oxidase activation, a non hydrolyzable analog of GTP, and either arachidonic acid or sodium dodecyl sulfate. When the amount of arachidonic acid or sodium dodecyl sulfate was maintained at a fixed value with respect to the amount of membrane used, a sigmoidal response of oxidase activity to increasing amounts of cytosol added was observed. In contrast, when the concentration of arachidonic acid or sodium dodecyl sulfate was properly adjusted with respect to that of membrane and cytosol, the curve relating oxidase activity to cytosol was hyperbolic, pointing to a simple michaelian relationship for the dependence of oxidase activation on the activating factor(s) of cytosol. Another parameter affecting oxidase activation was the ionic strength of the reconstitution medium, the extent of activation being lower at high ionic strength.     

Binding of the PH and polybasic C-terminal domains of ARNO to phosphoinositides and to acidic lipids

The activity on ARF of the guanine nucleotide exchange factor ARNO depends on its membrane recruitment, induced by binding of its PH domain to phosphoinositides. A polycationic C-terminal extension to the PH domain might also contribute to its specific binding to phosphatidylinositol 4,5-bisphosphate [(4,5)PIP2] and to phosphatidylinositol 3,4,5-trisphosphate [(3,4,5)PIP3], and to ionic binding to other acidic lipids. We have analyzed in vitro the relative contributions to phospholipid binding of the PH domain and C-terminal extension by cosedimentation of "PH+C domain" and "nominal PH domain" protein constructs including or not including the polycationic C-terminus, with sucrose-loaded unilamellar vesicles made of equal proportions of the neutral lipids phosphatidylcholine and phosphatidylethanolamine, and supplemented or not with 30% acidic phosphatidylserine (PS) and 2% of various phosphoinositides. Binding was measured as a function of the vesicle concentration and of the medium ionic strength. Both proteins bound with higher affinity to (3,4,5)PIP3 than to (4,5)PIP2, the selectivity for (3,4,5)PIP3 being highest for the nominal PH domain. We observed also a clear selectivity of (3,4,5)PIP3 over (4,5)PIP2 for stimulating the activity of ARNO on ARF with vesicles containing 10% PS and 1% PIP2 or PIP3. Our data suggest that the PH domain provides the specific phosphoinositide binding site and some unspecific ionic interaction with acidic PS, whereas the polybasic C domain contributes to binding mainly by unspecific ionic interactions vith PS. Phosphorylation by protein kinase C of a serine in the C domain reduces the ionic affinity of the PH+C domain for PS, but does not affect the phosphoinositide specificity.     

Stimulation of phosphoinositide breakdown in rat pancreatic islets by glucose and carbamylcholine

In the presence of Li⁺, glucose, 2-ketoisocaproate and carbamylcholine induced the rapid formation of ³H-inositol phosphates in rat pancreatic islets prelabelled with ³H-inositol. The production of labelled inositol phosphates continued up to 20 min of incubation. Glibenclamide and ionophore A23187 had no significant effect on labelled inositol phosphate production. The effects of carbamylcholine and to a lesser extent, glucose were found to persist in the absence of added Ca²⁺, but both were strongly inhibited by excess EGTA. In general, the rise in ³H-inositol phosphate production was associated with a fall in lipid bound radioactivity, although the latter was found to occur more slowly, and was of a smaller magnitude than labelled inositol phosphate formation. The results suggest that nutrient secretagogues and cholinergic agonists stimulate hydrolysis of phosphoinositides in pancreatic islets by a phospholipase C mechanism. This effect is Ca²⁺-dependent, but probably not triggered by increased Ca²⁺ uptake into the islet.     

Isolation, characterization and metal-ion-binding properties of the alpha-subunit from S-100a protein.

The present experiments were undertaken to investigate the role of the phosphoinositides phosphatidylinositol 4-phosphate (PtdIns-4-P) and phosphatidylinositol 4,5-biphosphate (PtdIns-4,5-P2) in the alpha 1-adrenergic stimulation of respiration in isolated hamster brown adipocytes. Exposure of isolated brown adipocytes to the alpha-adrenergic-receptor agonist phenylephrine provoked a breakdown of 30-50% of the PtdIns-4-P and PtdIns-4,5-P2 after prelabelling of the cells with [32P]Pi. Coincident with the breakdown of phosphoinositides was an accumulation of labelled phosphatidic acid, which continued for the duration of the cell incubation. The time course of phosphoinositide breakdown was defined more precisely by pulse-chase experiments. Under these conditions, phenylephrine caused radioactivity in phosphatidylinositol, PtdIns-4-P and PtdIns-4,5-P2 to fall by more than 50% within 30 s and to remain at the depressed value for the duration of the incubation (10 min). This phospholipid response to alpha-adrenergic stimulation was blocked by exposure of the cells to phorbol 12-myristate 13-acetate (PMA); likewise phenylephrine stimulation of respiration was prevented by PMA. beta-Adrenergic stimulation of respiration and inhibition of respiration by 2-chloroadenosine and insulin were, however, unaffected by treatment with PMA. On the assumption that PMA is acting in these cells as an activator of protein kinase C, these results suggest the selective interruption of alpha-adrenergic actions in brown adipocytes by activated protein kinase C. These findings suggest that breakdown of phosphoinositides is an early event in alpha-adrenergic stimulation of brown adipocytes which may be important for the subsequent stimulation of respiration. The results from the pulse-chase studies also suggest, however, that phenylephrine-stimulated breakdown of inositol phospholipids is a short-lived event which does not appear to persist for the entire period of exposure to the alpha 1-adrenergic ligand.     

Subcellular site and mechanism of vasopressin-stimulated hydrolysis of phosphoinositides in rat hepatocytes

The intracellular site of vasopressin-induced phosphoinositide breakdown in rat hepatocytes was investigated. After 45 s of vasopressin treatment of hepatocytes prelabeled with 32Pi, the levels of 32P-labeled phosphatidylinositol 4-phosphate (PI-P) and phosphatidylinositol 4,5-bisphosphate (PI-P2) in the plasma membrane decreased by approximately 40%, then gradually returned to near control levels after 10 min of treatment. Only small changes in the levels of [32P] PI-P and [32P]PI-P2 were observed in the other subcellular fractions, and were attributed to contamination of these fractions by plasma membranes. The level of 32P-labeled phosphatidylinositol in the plasma membrane decreased by 15% after 45 s of vasopressin treatment and then increased above control levels at later times while 32P-labeled phosphatidic acid levels in the plasma membrane gradually increased to 2-fold greater than control after 5 min of treatment. Using 32P-labeled plasma membranes obtained from prelabeled hepatocytes, it was found that PI-P and PI-P2 were rapidly degraded by a calcium-dependent polyphosphoinositide-specific phosphodiesterase. The enzyme was activated by physiological concentrations (200 nM) of free calcium when assayed at low ionic strength, but the calcium requirement shifted to micromolar concentrations under isosmotic, intracellular-like, ionic conditions. Addition of vasopressin (200 nM) to the 32P-labeled plasma membranes stimulated the breakdown of 20% of the [32P]PI-P2 present in the plasma membranes in 1 min when assayed under isosmotic conditions in the presence of 2 nM MgCl2 and approximately 200 nM free calcium. This suggests that the phosphoinositide-specific phosphodiesterase is not active under normal cellular conditions, but is activated upon the addition of vasopressin to the intact cell.     

Synthesis of polyphosphoinositides in vertebrate photoreceptor membranes

Rod outer segments isolated from bovine retinas incorporated 32P into phospholipids after incubation with [gamma-32P]ATP in a Mg2+-containing medium. Only phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidate were labelled. The incorporation of label into lipids was detected as early as 20 s after the start of incubation and the products were stable for at least 10 min. The reactions were time, protein and ATP-concentration dependent. Entire rod outer segments showed higher diacylglycerol kinase and lower phosphatidylinositol and phosphatidylinositol 4-phosphate kinase activities than the disc membranes obtained from them. Exogenously added phosphatidylinositol (up to 1 mM) in the presence of Triton X-100 increased phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate labelling in rod outer segments (8- and 6-fold, respectively). Triton X-100 at a concentration of 0.4% stimulated phosphorylation of endogenous phosphoinositides. Diacylglycerol kinase activity was largely suppressed by the detergent, but this effect was partially reversed by addition of phosphatidylinositol. It is suggested that the rod outer segments contain phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase bound to disc membranes, as well as an active diacylglycerol kinase occurring either as a soluble or a peripherally bound protein in disc membranes.     

Adenosine-3':5'-monophosphate-dependent and plasma-membrane-associated protein kinase from bovine corpus luteum. Solubilization and properties of solubilized enzyme.

The solubilization of plasma membrane fractions FI and FII associated protein kinases has been attempted using monovalent salts of high ionic strength and various detergent treatments. Extraction of FI and FII plasma membranes with high ionic strength salt solutions did not release more than 20% of the protein kinase activity. Similarly, monovalent salts released little adenosine 3':5'-monophosphate (cyclic AMP) binding activity, but after extraction binding capacity of cyclic [3H]AMP to plasma membranes was increased about 150-200%. Triton X-100 was a better solubilizing agent that Lubrol WX or deoxycholate. In addition to solubilization, 0.1% Triton X-100 also stimulated the protein kinase activity 150-200%. The properties of Triton X-100 solubilized FI and FII and purified cytosol KII were characterized with respect to protein substrate specificity, effect of cyclic AMP, cyclic nucleotide specificity, effects of divalent metal ion and gonadotropins. Upon sucrose density gradient centrifugation, FI solubilized protein kinase and cyclic AMP binding activities co-sedimented with a sedimentation coefficient of 6.3 S. The FII solubilized protein kinase sedimented as two components with sedimentation coefficients of 7.7 S and 5.5 S. The cyclic AMP binding activity also sedimented as two components with sedimentation coefficient 6.7 S and 5.5 S. Cyclic AMP caused dissociation of solubilized protein kinase from FI into a single catalytic (4.8 S) and two cyclic AMP binding subunits (8.1 S and 6.7 S). FII solubilized enzyme was dissociated into one catalytic (4.8 S) and one cyclic AMP binding subunit (6.3 S). Fractionation of FI and FII solubilized enzymes on DEAE-cellulose column chromatography resolved them each into two peaks Ia, Ib and IIa, IIb, respectively. Peaks Ib and IIb were more sensitive to cyclic AMP STIMULATION THAN Ia and IIa peaks. From these studies it is concluded that the plasma-membrane associated and cytosol protein kinases have similar catalytic properties but differ in some of their physical properties.     

Norepinephrine causes alpha 1-adrenergic receptor-mediated decrease of phosphatidylinositol in isolated rat liver plasma membranes supplemented with cytosol

When purified rat liver plasma membranes were incubated with norepinephrine, rat liver cytosol, and Ca2+, the amount of membrane-bound phosphatidylinositol was reduced by up to 50%. The levels of other major membrane phospholipids underwent negligible change. The decrease in phosphatidylinositol levels was not observed if norepinephrine was omitted or cytosol was absent. The disappearance of phospholipid persisted when soluble Ca2+ was depleted by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The decrease was prevented by phentolamine, benextramine, and prazosin, but not by sotalol. The results show that norepinephrine elicits a specific alpha 1-adrenergic receptor-mediated breakdown of phosphatidylinositol in isolated plasma membranes which is dependent on the presence of cytosol.     

Darcy permeability of agarose-glycosaminoglycan gels analyzed using fiber-mixture and donnan models

Agarose-glycosaminoglycan (GAG) membranes were synthesized to provide a model system in which the factors controlling the Darcy (or hydraulic) permeability could be assessed in composite gels of biological relevance. The membranes contained a GAG (chondroitin sulfate) that was covalently bound to agarose via terminal amine groups, and the variables examined were GAG concentration and solution ionic strength. The addition of even small amounts of GAG (0.4 vol/vol %) resulted in a twofold reduction in the Darcy permeability of 3 vol/vol % agarose gels. Electrokinetic coupling, caused by the negative charge of the GAG, resulted in an additional twofold reduction in the open-circuit permeability when the ionic strength was decreased from 1 M to 0.01 M. A microstructural hydrodynamic model was developed, based on a mixture of neutral, coarse fibers (agarose fibrils), and fine, charged fibers (GAG chains). Heterogeneity within agarose gels was modeled by assuming that fiber-rich, spherical inclusions were distributed throughout a fiber-poor matrix. That model accurately predicted the Darcy permeability when the ionic strength was high enough to suppress the effects of charge, but underestimated the influence of ionic strength. A more macroscopic approach, based on Donnan equilibria, better captured the reductions in Darcy permeability caused by GAG charge.     

Feedback regulation of CTP:phosphocholine cytidylyltransferase translocation between cytosol and endoplasmic reticulum by phosphatidylcholine

The mechanism for the increased association of CTP:phosphocholine cytidylyltransferase (CT) with membranes of hepatocytes derived from choline-deficient, compared with choline-supplemented rats, has been investigated. The cells were maintained in culture for 4 h in a choline- and methionine-deficient medium. (Methionine is required for synthesis of phosphatidylcholine (PC) via methylation of phosphatidylethanolamine.) Afterward, the cells were incubated +/- choline for various times up to 4 h. In the presence, but not in the absence, of choline there was a translocation of CT activity from membranes to cytosol. During this time period there was no change in the amounts of unesterified fatty acids or diacylglycerol recovered from the hepatocytes. In addition, there was no evidence for a difference in the incorporation of 32P into CT or other cytosolic proteins isolated from hepatocytes +/- choline. In contrast, there was a highly significant correlation between the concentration of PC in the membranes and the increased activity of CT in the cytosol (R = 0.98) and the decreased activity in the membranes (R = 0.93). The concentration of PC could alternatively be altered by incubation of the choline-deficient hepatocytes with methionine or lyso-PC. With either of these supplementations highly significant correlation coefficients were observed between the concentration of PC in membranes and decreased activity of CT in membranes or increased activity in cytosol. The concentration of PC was reduced in the endoplasmic reticulum, but not the Golgi membranes, isolated from choline-deficient compared with choline-supplemented livers. The data suggest that the amount of PC in the endoplasmic reticulum feedback regulates the amount of CT associated with this membrane.     

Subcellular localization and membrane topology of sphingosine-1-phosphate lyase in rat liver

Sphingosine-1-phosphate lyase is responsible for the ultimate step in sphingolipid breakdown, converting phosphorylated long chain bases into ethanolamine phosphate and a fatty aldehyde. Using tritiated dihydrosphingosine-1-phosphate, prepared enzymatically from [4,5-3H]dihydrosphingosylphosphocholine, we have reinvestigated the subcellular distribution of this enzyme in rat liver. Upon cell fractionation by differential centrifugation, the enzyme showed a microsomal distribution. Further separation of the microsomal fraction by sucrose gradient centrifugation confirmed an association with the endoplasmic reticulum. By means of constrained nonlinear regression, no evidence for a significant association with mitochondrial membranes, as reported previously (Stoffel, W., LeKim, D., and Sticht, G. (1969) Hoppe Seyler's Z. Physiol. Chem. 350, 1233-1241), nor with other cell compartments was found. The lyase activity, which appeared to be sensitive to different detergents, but not to Triton X-100, was not latent. It could be solubilized with Triton X-100, but not by high ionic strength, indicating that it is an integral membrane protein whose catalytic site is most probably exposed to the cytosol. Treatment of intact microsomal vesicles with trypsin or thermolysin inactivated the lyase activity, confirming that its catalytic site(s) or other domains essential for activity face the cytosol.     

Insulin degradation by adipose tissue. Studies at several levels of cellular organization.

A systematic study of the degradation of physiological concentrations of 125I-labelled insulin was performed in intact fat-pads, isolated adipocytes and subcellular fractions of isolated adipocytes. The findings indicate that insulin is rapidly degraded to low-molecular-weight peptides and/or amino acids by the intact tissue and isolated cells. Of the total insulin-degradation products present after incubation with an intact fat-pad, 94% is recovered in the medium, indicating that these products are not retained by the cells or tissue. The plasma membranes do not degrade insulin significantly in the absence of reduced glutathione, and over 99% of the cellular degradative capacity is found in the postmicrosomal supernatant (cytosol). The cytosol degrades insulin to several labelled fragments that are intermediate in size between insulin and insulin A chain, as well as to the low-molecular-weight tissue degradation products. Inclusion of plasma membranes with cytosol accelerates the cleavage of the intermediate fragments to the size of the small products seen with the intact tissue. However, plasma membranes do not increase the initial step in the degradation of insulin when incubated with cytosol, suggesting that the insulin receptor is not involved with the direct cleavage of insulin. This study supports the hypothesis that the bulk of insulin degradation occurs in the adipocyte cytosol, where intermediate-sized fragments are generated and rapidly cleaved to smaller products by the plasma membrane and quickly released into the surrounding medium.     

Enhancement by Potassium of Carbachol-Stimulated Inositol Phospholipid Breakdown in Rat Cerebral Cortical Miniprisms: Comparison with Other Depolarising Agents

Increasing the [K+] in the assay medium from 5.7 to 17.8 mM produces a large enhancement of the inositol phospholipid breakdown response to the muscarinic agonist carbachol in rat cerebral cortical miniprisms, with minor effects on basal inositol phospholipid breakdown. This effect is also found with Rb+. The enhancement by a raised [K+] is not accompanied by a change in the composition of the labelled polyphosphoinositides. The carbachol-stimulated inositol phospholipid breakdown at 17.8 and 42.7 mM K+ was antagonised by veratrine (5-80 microM), 4-aminopyridine (5 mM), and tetraethylammonium (20 mM). These compounds, however, also inhibited the binding of [3H]quinuclidinyl benzilate to cortical membranes. BRL 34915 (0.2-20 microM) was without significant effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+.Mg2+ (10 mM) considerably reduced the carbachol-stimulated inositol phospholipid breakdown at 17.8, but not 42.7, mM K+. Inositol phospholipid breakdown was also stimulated, albeit to a small extent, by L-glutamate (100-3,000 microM) and quisqualate (1-100 microM), with the stimulation being additive to that produced by carbachol at both 5.7 and 17.8 mM K+. N-Methyl-D-aspartate (10-1,000 microM in Mg2+-free medium) had no significant effect on basal inositol phospholipid breakdown and had little or no effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+. It is concluded that it may not be correct to ascribe wholly the enhancement by K+ of carbachol-stimulated inositol phospholipid breakdown to the tissue-depolarising actions of this ion and that other actions of K+ may be involved.     

The effects of ionic strength on the protein conformation and the fluidity of porcine intestinal brush border membranes. Fluorometric studies using N-[7-dimethylamino-4-methylcoumarinyl]maleimide and pyrene

The effects of ionic strength on the conformation around the SH groups of the proteins and the lipid fluidity of porcine intestinal brush border membranes were studied using two fluorescent dyes, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM) and pyrene. The extent of DACM labeling to the SH groups of the membrane proteins was accelerated depending on the KCl concentrations in medium. A quenching study of DACM-labeled membranes with acrylamide showed that the proximity of the quencher to the fluorescence-labeled SH groups in the membrane proteins is increased with increasing ionic strength of medium. An implication of the conformational changes around SH groups in the membrane proteins with increase of ionic strength was also obtained from the stimulation of guanidine effect on the fluorescence parameters of DACM-labeled membranes by addition of KCl. On the other hand, the results of the quenching study with KI, excimer fluorescence, and polarization measurements of pyrene-labeled membranes suggested an increase of membrane fluidity on addition of KCl to medium. The temperature dependence of polarization of the complex strongly suggested that the rotational freedom of pyrene molecules embedded into the lipid layers of the membranes is increased by addition of KCl. In fact, the harmonic means of the rotational relaxation times of pyrene molecules in the membranes with and without 100 mM KCl were estimated to be about 2900 and 9000 ns at 25 degrees C, respectively. Based on these results, the salt-induced alterations of the conformation in the vicinity of the bound dyes of the membrane proteins and of the membrane fluidity are discussed.     

Intracellular protein degradation in serum-deprived human fibroblasts.

IMR90 human fibroblasts were labelled by incubation of cells for 48 h in medium containing 10% serum and [3H]leucine. The labelled protein was degraded at a rate of 1%/h during a subsequent incubation in medium with 10% serum. Incubation in medium without serum caused a transient enhancement of the degradation of endogenous protein, which was also found in cells labelled in medium without serum. The degradation of micro-injected haemoglobin was enhanced by serum deprivation in a non-transient manner. These results suggest that enhanced degradation in serum-free medium occurs only for a subpopulation of cell proteins and that it appears transient because the major part of the pool of susceptible endogenous proteins is being degraded during the first 20-30 h in serum-free unlabelled medium. Protein turnover in various cell compartments was measured by a double-labelling technique. Most of the enhanced degradation in serum-deprived cultures (73-83%) was due to breakdown of cytosolic proteins. The enhanced degradation of cytosolic proteins seemed to affect several proteins irrespective of their molecular mass or metabolic stability.