Theodore L. Steck

Cross-linking the major proteins of the isolated erythrocyte membrane☆

Abstract Isolated human red blood cell membranes were reacted with a series of protein cross-linking reagents. The consequences were assessed by electrophoresis of the polypeptides on polyacrylamide gels containing sodium dodecyl sulfate. Each agent caused distinctive alterations in the gel pattern. Certain bands diminished and disappeared while new bands of two or more times their molecular weight appeared in a reciprocal fashion. Some components were distinctly unreactive. The preferential cross-linking reactions suggest that some of the membrane polypeptides may exist in specific oligomeric associations rather than in a random array.

Inside-Out Red Cell Membrane Vesicles: Preparation and Puirification

Plasma membranes purified from human red cells were Converted into small vesicleS by disruption in alkaline buffer of low ionic strength. Most of these vesicles were inside-out. The presence of divalent cations prevented this inversion. The inside-out vesicles were separatcd from right-side-out vesicles by centrifugration to equilibrium in dextran density gradients.

Probing red cell membrane cholesterol movement with cyclodextrin.

We probed the kinetics with which cholesterol moves across the human red cell bilayer and exits the membrane using methyl-beta-cyclodextrin as an acceptor. The fractional rate of cholesterol transfer (% s(-1)) was unprecedented, the half-time at 37 degrees C being ~1 s. The kinetics observed under typical conditions were independent of donor concentration and directly proportional to acceptor concentration. The rate of exit of membrane cholesterol fell hyperbolically to zero with increasing dilution. The energy of activation for cholesterol transfer was the same at high and low dilution; namely, 27-28 Kcal/mol. This behavior is not consistent with an exit pathway involving desorption followed by aqueous diffusion to acceptors nor with a simple one-step collision mechanism. Rather, it is that predicted for an activation-collision mechanism in which the reversible partial projection of cholesterol molecules out of the bilayer precedes their collisional capture by cyclodextrin. Because the entire membrane pool was transferred in a single first-order process under all conditions, we infer that the transbilayer diffusion (flip-flop) of cholesterol must have proceeded faster than its exit, i.e., with a half-time of <1 s at 37 degrees C.

Cholesterol movement in Niemann-Pick type C cells and in cells treated with amphiphiles.

Cholesterol accumulates to massive levels in cells from Niemann-Pick type C (NP-C) patients and in cells treated with class 2 amphiphiles that mimic NP-C disease. This behavior has been attributed to the failure of cholesterol released from ingested low density lipoproteins to exit the lysosomes. However, we now show that the rate of movement of cholesterol from lysosomes to plasma membranes in NP-C cells is at least as great as normal, as was also found previously for amphiphile-treated cells. Furthermore, the lysosomes in these cells filled with plasma membrane cholesterol in the absence of lipoproteins. In addition, we showed that the size of the endoplasmic reticulum cholesterol pool and the set point of the homeostatic sensor of cell cholesterol were approximately normal in NP-C cells. The plasma membrane cholesterol pools in both NP-C and amphiphile-treated cells were also normal. Furthermore, the build up of cholesterol in NP-C lysosomes was not a physiological response to cholesterol overload. Rather, it appeared that the accumulation in NP-C lysosomes results from an imbalance in the brisk flow of cholesterol among membrane compartments. In related experiments, we found that NP-C cells did not respond to class 2 amphiphiles (e.g.trifluoperazine, imipramine, and U18666A); these agents may therefore act directly on the NPC1 protein or on its pathway. Finally, we showed that the lysosomal cholesterol pool in NP-C cells was substantially and preferentially reduced by incubating cells with the oxysterols, 25-hydroxycholesterol and 7-ketocholesterol; these findings suggest a new pharmacological approach to the treatment of NP-C disease.

THE BINDING OF KIDNEY-BEAN PHYTOHEMAGGLUTININ BY EHRLICH ASCITES CARCINOMA.

Abstract 1. 1. The phytohemagglutinin of the red kidney bean agglutinates 3 types of erythrocytes, 4 reticuloendothelial cell types, and 3 mouse tumors with different avidity. Evidence is provided that these cell types have surface receptors in common which are responsible for their agglutination. 2. 2. The reaction of the phytohemagglutinin with Ehrlich ascites carcinoma cells was examined in detail. It was shown that the agglutination of this tumor is not a simple function of phytohemagglutinin binding, but depends upon the state of the cell surface. The affinity constant for the reaction of phytohemagglutinin with Ehrlich ascites carcinoma cells was found to be 1.8·10 7 at 1°. The binding affinity was maximal at 18° and diminished at higher temperatures. The 6.6·10 7 receptor sites per Ehrlich ascites carcinoma cell appeared to be located at the cell surface. 0.3% of the receptor sites had to be occupied before agglutination was observed. The velocity of phytohemagglutinin binding increased with temperature and concentration. The rate constant was observed to diminish as the reaction progressed; thus at 1°, it decreased from 2·10 3 to 0.5·10 3 l·moles −1 ·sec −1 during the first 30 min. 3. 3. The affinity constant for the reaction of phytomegglutinin with human erythrocytes was found to be 8.4·10 7 ·4% of the 5·10 5 binding sites per erythrocyte must be occupied for agglutination to be detectable. Erythrocytes treated with trypsin and neuraminidase showed increased agglutinability.

Preparation of Impermeable Inside-Out and Right-Side-Out Vesicles from Erythrocyte Membranes

Each biological membrane operates differentially on the two compartments it separates and is thus anisotropic in its function. It is reasonable to suppose that the molecular constituents of the two surfaces differ and that this asymmetry constitutes a principal feature of membrane organization. Since membranes are only a few macromolecules thick, it is clear that enumeration of the components at each surface would do much to define the structure as a whole.

CIRCULATION OF CHOLESTEROL BETWEEN LYSOSOMES AND THE PLASMA MEMBRANE

The cholesterol in the lysosomes of cultured human fibroblasts was determined to constitute ∼6% of the cell total. This pool was enlarged by as much as 10-fold in Niemann-Pick type C cells. Certain amphiphiles (e.g. U18666A, progesterone, and imipramine) caused lysosomal cholesterol to increase to similarly high levels at a rate of ∼0.8% of cell cholesterol/h. Lysosomal cholesterol accumulated even in the absence of exogenous lipoproteins. Furthermore, nearly all of the lysosomal cholesterol in both of the two perturbed systems was shown to be derived from the plasma membrane. Oxysterols known to alter cholesterol movement and homeostasis blocked lysosomal cholesterol accretion in amphiphile-treated cells, suggesting that this process is regulated physiologically. Treating cells with amphiphiles slightly reduced the efflux of cholesterol from lysosomes and slightly increased the influx from the plasma membrane, causing the lysosomal cholesterol compartment to double in size in ∼15 h. After more prolonged amphiphile treatments, a population of buoyant lysosomes appeared that exchanged cholesterol with the plasma membrane completely but slowly. Niemann-Pick type C lysosomes were similarly buoyant and sluggish. We conclude that cholesterol circulates bidirectionally between the plasma membrane and lysosomes. The massive accumulation of lysosomal cholesterol in the perturbed cells does not appear to reflect disabled lysosomal transport but rather the formation of lysosomes modified for lipid storage, i.e. lamellar bodies.

CHOLESTEROL HOMEOSTASIS AND THE ESCAPE TENDENCY (ACTIVITY) OF PLASMA MEMBRANE CHOLESTEROL

We review evidence that sterols can form stoichiometric complexes with certain bilayer phospholipids, and sphingomyelin in particular. These complexes appear to be the basis for the formation of condensed and ordered liquid phases, (micro)domains and/or rafts in both artificial and biological membranes. The sterol content of a membrane can exceed the complexing capacity of its phospholipids. The excess, uncomplexed membrane sterol molecules have a relatively high escape tendency, also referred to as fugacity or chemical activity (and, here, simply activity). Cholesterol is also activated when certain membrane intercalating amphipaths displace it from the phospholipid complexes. Active cholesterol projects from the bilayer and is therefore highly susceptible to attack by cholesterol oxidase. Similarly, active cholesterol rapidly exits the plasma membrane to extracellular acceptors such as cyclodextrin and high-density lipoproteins. For the same reason, the pool of cholesterol in the ER (endoplasmic reticulum) increases sharply when cell surface cholesterol is incremented above the physiological set-point; i.e., equivalence with the complexing phospholipids. As a result, the escape tendency of the excess cholesterol not only returns the plasma membrane bilayer to its set-point but also serves as a feedback signal to intracellular homeostatic elements to down-regulate cholesterol accretion.

Quantitation of the Pool of Cholesterol Associated with Acyl-CoA:Cholesterol Acyltransferase in Human Fibroblasts

The esterification of cholesterol in homogenates of human fibroblasts was explored as a means of estimating the size of the pool of cholesterol associated with the endoplasmic reticulum (ER)in vivo. The rationale was that the acyl-coenzyme A:cholesterol acyltransferase (ACAT) in homogenates should have access only to cholesterol associated with the (rough) ER membrane fragments in which it resides. Reacting whole homogenates to completion with an excess of [14C]oleoyl-CoA converted ∼0.1–2% of total cell-free cholesterol to [14C]cholesteryl esters. Control studies indicated that membranes not associated with ACAT did not contribute cholesterol to this reaction. The extent of in vitro cholesterol esterification varied with pretreatment of the cells. Exposing intact cells to serum lipoproteins, oxysterols, or sphingomyelinase increased cholesterol esterification in homogenates severalfold; exposing the cells to mevinolin or cholesterol oxidase had the opposite effect. The variation in cholesterol esterification did not correlate with either the total cellular cholesterol or the intrinsic activity of ACAT, neither of which was changed significantly by the pretreatments. Rather, the total amount of cholesterol esterified in homogenates paralleled the rate of cholesterol esterification in the corresponding intact cells. The pool of cholesterol esterified in vitro therefore appears to reflect that associated with the ER in vivo. Since several of the mechanisms keeping cell cholesterol under tight feedback control are themselves located in the ER, this pool might not only be regulated physiologically, but could, in turn, help to regulate homeostatic effector pathways.

Effectors of Rapid Homeostatic Responses of Endoplasmic Reticulum Cholesterol and 3-Hydroxy-3-methylglutaryl-CoA Reductase

The cholesterol content of the endoplasmic reticulum (ER) and the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) imbedded therein respond homeostatically within minutes to changes in the level of plasma membrane cholesterol. We have now examined the roles of sterol regulatory element-binding protein (SREBP)-dependent gene expression, side chain oxysterol biosynthesis, and cholesterol precursors in the short term regulation of ER cholesterol levels and HMGR activity. We found that SREBP-dependent gene expression is not required for the response to changes in cell cholesterol of either the pool of ER cholesterol or the rate of cholesterol esterification. It was also found that the acute proteolytic inactivation of HMGR triggered by cholesterol loading required the conversion of cholesterol to 27-hydroxycholesterol. High levels of exogenous 24,25-dihydrolanosterol drove the inactivation of HMGR; lanosterol did not. However, purging endogenous 24,25-dihydrolanosterol, lanosterol, and other biosynthetic sterol intermediates by treating cells with NB-598 did not greatly affect either the setting of their ER cholesterol pool or the inactivation of their HMGR. In summary, neither SREBP-regulated genes nor 27-hydroxycholesterol is involved in setting the ER cholesterol pool. On the other hand, 27-hydroxycholesterol, rather than cholesterol itself or biosynthetic precursors of cholesterol, stimulates the rapid inactivation of HMGR in response to high levels of cholesterol.