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The disposition of newly synthesized sterols in cultured human fibroblasts has been examined in this study. We began by demonstrating that cholesterol mass and exogenously added [3H]cholesterol both are markers for the plasma membrane, perhaps better than 5‘-nucleotidase. Cells were incubated with radioactive acetate to label their endogenous sterols biosynthetically, treated with cholesterol oxidase to convert plasma membrane cholesterol to cholestenone, and then homogenized and spun to equilibrium on sucrose gradients. The density gradient profiles of the various organelles were monitored using these markers: plasma membrane, radioactive cholestenone; smooth endoplasmic reticulum, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase); and Golgi apparatus, galactosyltransferase. The buoyant density profiles of radioactive intracellular cholesterol and lanosterol both had a peak at 1.12 g/cm3, similar to 5‘-nucleotidase and galactosyltransferase but not to HMG-CoA reductase. This result suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. Digitonin treatment shifted the profiles of both plasma membrane and intracellular cholesterol to higher densities. Pretreatment of intact cells with cholesterol oxidase abolished the digitonin shift of plasma membranes but not the intracellular cholesterol, indicating that these two membrane pools are not entirely physically associated. Because intracellular cholesterol was shifted more than any of the organelle markers, it must reside in a separate membrane. Since digitonin selectively shifts the density of membranes rich in cholesterol, we infer that newly synthesized cholesterol accumulates in such membranes prior to its delivery to the plasma membrane. Taken together, these results suggest that cholesterol may be concentrated for delivery to the plasma membrane by being synthesized from a sterol precursor such as lanosterol in a discrete but undefined intracellular membrane.

We have determined the amino acid sequence of the N alpha-terminal portion of band 3, the anion transport protein of the human erythrocyte membrane. The material analyzed was a 201-residue, 23,053-Da fragment cleaved from the cytoplasmic end of band 3 by S-cyanylation. The sequence had these notable features. 1) The N alpha-terminal region was extraordinarily acidic, second only to a segment of similar size from the sigma factor of Escherichia coli RNA polymerase. The first 33 residues contained 6 aspartic acid and 12 glutamic acid residues, no basic residue, and a blocked N alpha-amino group. 2) The first 11 residues of the protein had a striking resemblance to the following 11 residues. 3) In contrast to the acidic N alpha-terminal third, the COOH-terminal two-thirds of the 23,053-Da fragment had a predominantly basic character. The highly acidic character of the N alpha-terminal portion of band 3 accounts for the capacity of this part of the protein to bind glycolytic enzymes in a highly electrostatic fashion, presumably through interaction with their cationic substrate-binding sites.

Our previous study has shown that the decorated tubules (collectively known as the decorated spongiome) of the contractile vacuole complex (CVC) in Paramecium are the site of fluid segregation, as the binding of microinjected monoclonal antibody (mAb) DS-1 to the tubules reduced the CVC's fluid output. In this study, we showed by immunogold labeling on cryosections that the antigenic sites for mAb DS-1 were located on the 15 nm 'pegs' protruding from the cytosolic surface of the decorated tubules. In immunofluorescence studies, both polyclonal antibodies against the subunits of the V-ATPase of Dictyostelium discoideum and against the 57 kDa B-subunit of the V-ATPase of chromaffin granules gave identical labeling patterns to that produced by mAb DS-1. On cryosections, all three antigens were located most consistently near or on the pegs of the decorated tubules. These data support the notion that the pegs on the membrane of the decorated tubules represent the V1 complex of a proton pump. Concanamycin B, a potent inhibitor of V-ATPase activity and of acidification of lysosomes and endosomes, strongly and reversibly inhibited fluid output from the CVC but had minimal effect on the integrity of the decorated spongiome as observed by immunofluorescence. Such inhibition suggests that a V-ATPase is intimately involved in fluid segregation. Exposing Paramecium to 12 degrees C or 1 degrees C for 30 minutes resulted in the dissociation of the decorated tubules from the smooth spongiome that borders the collecting canals; thus the DS-1-reactive A4 antigen, the 75 kDa and 66 kDa antigens were all found dispersed in the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycolytic enzymes have been observed to associate in vitrowith membranes and cytoplasmic filaments in a variety of systems, but their distribution in vivois contested. We have therefore examined the distribution of glyceraldehyde-3-phosphate dehydrogenase (G3PD) in the intact human erythrocyte using indirect immunofluorescence and affinity-purified rabbit antibodies to G3PD. Antibody specificity was demonstrated by immunoblotting as well as immunofluorescence experiments with ghosts specifically depleted of and reconstituted with G3PD. Anti-G3PD immunolabeling experiments utilized both fixed whole cells and fixed cell suspensions infused with 2.3 Msucrose, frozen and thick-sectioned. In all experiments a two-stepfixation protocol was employed which ensured that cytoplasmic hemoglobin was retained when cells were subjected to Triton X-100 permeabilization, the antigenicity of G3PD was preserved, and antibody penetration was complete. We used mixtures of biotinylated affinity-purified antibodies to G3PD and dichlorotria-zinylaminofluorescein-labeled, affinity-purified antibodies to hemoglobin, followed by rhodamine-streptavidin, in double-label experiments.

Phosphofructokinase is known to associate with the human erythrocyte membrane both in vitro and in vivo. Such association activates the enzyme in vitro by relieving the allosteric inhibition imposed by ATP (Karadsheh, N.S., and Uyeda, K. (1977) J. Biol. Chem. 252, 7418-7420). We now demonstrate that ADP, ATP, and NADH, all of which are known to bind to the enzyme's adenine nucleotide activation site, are particularly potent in eluting the enzyme from the membrane. In addition, both inside-out red cell membrane vesicles and a 23-kDa fragment containing the amino terminus of the membrane protein, band 3, cause a slow, partial, and reversible inactivation of phosphofructokinase. The dependence of the residual phosphofructokinase activity on phosphofructokinase concentration demonstrates that inactivation occurs through the dissociation of active tetramers to inactive dimers. Dimers of phosphofructokinase associate with the membrane more avidly than tetramers. The kinetics of phosphofructokinase inactivation are consistent with the dissociation of tetramers in solution followed by the binding of dimers to the membrane. There is no indication of an association equilibrium between tetramers and dimers of phosphofructokinase bound to the membrane. Taken together, these results suggest that the amino-terminal segment of band 3 binds to the adenine nucleotide activation site, which is thought to be located in a cleft between the dimeric subunits of phosphofructokinase. As a result, band 3 not only rapidly activates the phosphofructokinase tetramer but also slowly inactivates the enzyme by preferentially binding its dissociated subunits.

The complete sequence of a retrotransposon from Dictyostelium discoideum , named skipper , was obtained from cDNA and genomic clones. The sequence of a nearly full-length skipper cDNA was similar to that of three other partially sequenced cDNAs. The corresponding retrotransposon is represented in approximately 15-20 copies and is abundantly transcribed. Skipper contains three open reading frames (ORFs) with an unusual sequence organization, aspects of which resemble certain mammalian retroviruses. ORFs 1 and 3 correspond to gag and pol genes; the second ORF, pro, corresponding to protease, was separated from gag by a single stop codon followed shortly thereafter by a potential pseudoknot. ORF3 (pol) was separated from pro by a +1 frameshift. ORFs 2 and 3 overlapped by 32 bp. The computed amino acid sequences of the skipper ORFs contain regions resembling retrotransposon polyprotein domains, including a nucleic acid binding protein, aspartyl protease, reverse transcriptase and integrase. Skipper is the first example of a retrotransposon with a separate pro gene. Skipper is also novel in that it appears to use stop codon suppression rather than frameshifting to modulate pro expression. Finally, skipper and its components may provide useful tools for the genetic characterization of Dictyostelium.

We have examined fragments of the filamentous network underlying the human erythrocyte membrane by high-resolution electron microscopy. Networks were released from ghosts by extraction with Triton X-100, freed of extraneous proteins in 1.5 M NaCl, and collected by centrifugation onto a sucrose cushion. These preparations contained primarily protein bands 1 + 2 (spectrin), band 4.1 and band 5 (actin). The networks were partially disassembled by incubation at 37 degrees C in 2 mM NaPi (pH 7), which caused the preferential dissociation of spectrin tetramers to dimers. The fragments so generated were fractionated by gel filtration chromatography and visualized by negative staining with uranyl acetate on fenestrated carbon films. Unit complexes, which sedimented at approximately 40S, contained linear filaments approximately 7-8 nm diam from which several slender and convoluted filaments projected. The linear filaments had a mean length of 52 +/- 17 nm and a serrated profile reminiscent of F-actin. They could be decorated in an arrowhead pattern with S1 fragments of muscle heavy meromyosin which, incidentally, displaced the convoluted filaments. Furthermore, the linear filaments nucleated the polymerization of rabbit muscle G-actin, predominantly but not exclusively from the fast-growing ends. On this basis, we have identified the linear filaments as F-actin; we infer that the convoluted filaments are spectrin. Spectrin molecules were usually attached to actin filaments in clusters that showed a preference for the ends of the F-actin. We also observed free globules up to 15 nm diam, usually associated with three spectrin molecules, which also nucleated actin polymerization; these may be simple junctional complexes of spectrin, actin, and band 4.1. In larger ensembles, spectrin tetramers linked actin filaments and/or globules into irregular arrays. Intact networks were an elaboration of the basic pattern manifested by the fragments. Thus, we have provided ultrastructural evidence that the submembrane skeleton is organized, as widely inferred from less direct information, into short actin filaments linked by multiple tetramers of spectrin clustered at sites of association with band 4.1.

The literature suggests that cholesterol and sphingomyelin might be essentially confined to plasma membranes in mammalian cells; however, this premise has thus far escaped a direct test. We explored the issue in three ways. First, we fractionated whole homogenates of cultured human fibroblasts by equilibrium sucrose density gradient centrifugation. We found that the profiles of cholesterol and sphingomyelin were indistinguishable from those of two plasma membrane markers, 5′ nucleotidase and [3H]galactose, which was conjugated to the surface of intact cells from an exogenous donor by galactosyltransferase.

The interaction of the amphipath chlorpromazine (CPZ) with the human erythrocyte membrane was evaluated. The partition coefficient of CPZ between the membrane bilayer and the aqueous compartment, measured spectrophotometrically, ranged between 1 and 3 X 10(3). An independent estimate, 4.6 X 10(3), was obtained by a novel method which avoided the measurement of binding and determined instead the variation of the hemolytic potency of the amphipath with the ratio of buffer volume to membrane volume. The maximal uptake of CPZ exceeded 2 X 10(9) molecules/red cell, corresponding to a volume greater than that of the bilayer itself. Such heavily loaded membranes were increased in thickness more than 2-fold, suggesting the formation of a CPZ-rich zone at the center of the bilayer. Ghosts loaded with massive levels of CPZ condensed approximately 20-fold in surface area and increased proportionately in thickness, suggesting the formation of a novel CPZ-lipid solution. CPZ caused hemolysis by a colloid-osmotic mechanism. By measuring the simultaneous uptake of mannitol and sucrose, we determined that CPZ induced holes of constant size but variable number. If circular, the holes would have had a diameter of approximately 14 A. The time-averaged number of holes ranged from 0.09 per cell (signifying intermittency) to 16. Freeze-fracture electron microscopy of CPZ-treated red cells revealed multiple round patches of nearly particle-free bilayer up to 0.3 micron in diameter with crowding of the intramembrane particles into the surrounding membrane. We interpret these images to signify lateral phase separation within the CPZ-treated bilayer. Hemolysis could, therefore, result from the intermittent opening of weak seams at phase boundaries; these could then be fluctuating slits approximately 14 A in width and of variable length, rather than simple circular holes.

The secretion of 3H-cyclic adenosine 3',5'-monophosphate (cAMP) by prelabeled and suitably differentiated Dictyostelium discoideum amoebae was elicited in a perfusion apparatus by 10(-10) to 10(-5) M [14C]cAMP stimuli of defined magnitude and duration. Exogenous stimuli evoked an immediate increase in the rate of [3H]cAMP secretion which accelerated continuously to reach a peak of up to 100 times the unstimulated rate after 2--3 min of stimulation. Withdrawal of the stimulus at any time during the response led to a rapid decline to basal levels. Furthermore, a spontaneous decline in secretion rate was observed during prolonged cAMP stimulation, with a return to basal levels after 3--8 min of stimulation. After the initial secretory event, cells did not respond further to the continued presence of external [14C]cAMP unless (a) it was interrupted by a brief recovery period or (b) the level of the stimulus was increased sufficiently. Since the second increment could follow the first at any time, continuous secretion of [3H]cAMP could be sustained for up to 30 min by progressively increasing the stimulus between 10(-10) and 10(-5) M cAMP. The total magnitude of spontaneously terminated responses depended on the size of the increment in applied cAMP, larger stimuli evoking both a more rapid acceleration and a slower deceleration in [3H]cAMP secretion rate. The integrated response to a given increment in stimulus level was apparently independent of its "shape" - i.e., the duration, magnitude, and number of sub-steps in the increment. These data support two mechanistic inferences: that amoebae respond in proportion to relative increases in extracellular cAMP concentration, but adapt to the concentration of cAMP itself. The data further suggest that the initiation and termination of the response are mediated by cellular component(s) beyond cAMP-occupied receptors.

We have characterized the interaction of bovine pancreatic deoxyribonuclease I (DNase I) with the filamentous (F-)actin of red cell membrane skeletons stabilized with phalloidin. The hydrolysis of [3H]DNA was used to assay DNase I. We found that DNase I bound to a homogenous class of approximately equal to 2.4 X 10(4) sites/skeleton with an association rate constant of approximately 1 X 10(6) M-1 S-1 and a KD of 1.9 X 10(-9) M at 20 degrees C. Phalloidin lowered the dissociation constant by approximately 1 order of magnitude. The DNase I which sedimented with the skeletons was catalytically inactive but could be reactivated by dissociation from the actin. Actin and DNA bound to DNase I in a mutually exclusive fashion without formation of a ternary complex. Phalloidin-treated red cell F-actin resembled rabbit muscle G-actin in all respects tested. Since the DNase I binding capacity of the skeletons corresponded to the number of actin protofilaments previously estimated by other methods, it seemed likely that the enzyme binding site was confined to one end of the filament. We confirmed this premise by showing that elongating the red cell filaments with rabbit muscle actin monomers did not appreciably add to their capacity to bind or inhibit DNase I. Saturation of skeletons with cytochalasin D or gelsolin, avid ligands for the barbed end of actin filaments, did not reduce their binding of DNase I. Furthermore, neither cytochalasin D nor DNase I alone blocked all of the sites for addition of monomeric pyrene-labeled rabbit muscle G-actin to phalloidin-treated skeletons; however, a combination of the two agents did so. In the presence of phalloidin, the polymerization of 300 nM pyrenyl actin on nuclei constructed from 5 nM gelsolin and 25 nM rabbit muscle G-actin was completely inhibited by 35 nM DNase I but not by 35 nM cytochalasin D. We conclude that DNase I associates uniquely with and caps the pointed (slow-growing or negative) end of F-actin. These results imply that the amino-terminal, DNase I-binding domain of the actin protomer is oriented toward the pointed end and is buried along the length of the actin filament.

We report the results of experiments which show that cholesterol transfer between membranes cannot proceed by aqueous diffusion, as widely held, but must involve a more complex mechanism. (a) The rate of transfer of [3H]cholesterol from red blood cells was found to vary inversely with the size of the acceptor particle (ghosts, vesicles of ghosts, liposomes, and plasma lipoproteins). (b) The transfer of [3H]cholesterol from red blood cells to ghosts was accelerated by the presence of plasma, even though the plasma competed with the ghosts as an acceptor. (c) The rate of transfer of [3H]cholesterol from red blood cells to ghosts decreased to zero with increasing dilution but was not simply second-order. (d) The cholesterol in retinal rod disc membranes is not at equilibrium with plasma lipoproteins in that disc cholesterol increased when the homogenates were incubated in vitro with plasma. (e) The kinetics of cholesterol transfer cannot be limited by unstirred layer effects since the transfer of lysolecithin in the same system was faster than that of cholesterol by 3 orders of magnitude. The simplest model compatible with all the data suggests a two-step pathway involving a first-order followed by a second-order process. The first step could be a unimolecular activation event, perhaps the movement of the sterol in the donor particle to a more exposed (hydrated) position. In the second step, the activated sterol would be transferred during transient collisions between donor and acceptor particles. When collision is not rate-limiting, the overall process would appear to be simply first-order, hence kinetically indistinguishable from the aqueous diffusion mechanism. The activation-collision model thus not only rationalizes our data but is also consistent with the simpler kinetics previously reported for the transfer of both membrane phospholipids and sterols.

Filamentous skeletons were liberated from isolated human erythrocyte membranes in Triton X-100, spread on fenestrated carbon films, negatively stained, and viewed intact and unfixed in the transmission electron microscope. Two forms of the skeleton were examined: (a) basic skeletons, stripped of accessory proteins with 1.5 M NaCl so that they contain predominantly polypeptide bands 1, 2, 4.1, and 5; and (b) unstripped skeletons, which also bore accessory proteins such as ankyrin and band 3 and small plaques of residual lipid. Freshly prepared skeletons were highly condensed. Incubation at low ionic strength and in the presence of dithiothreitol for an hour or more caused an expansion of the skeletons, which greatly increased the visibility of their elements. The expansion may reflect the opening of spectrin from a compact to an elongated disposition. Expanded skeletons appeared to be organized as networks of short actin filaments joined by multiple (5-8) spectrin tetramers. In unstripped preparations, globular masses were observed near the centers of the spectrin filaments, probably corresponding to complexes of ankyrin with band 3 oligomers. Some of these globules linked pairs of spectrin filaments. Skeletons prepared with a minimum of perturbation had thickened actin protofilaments, presumably reflecting the presence of accessory proteins. The length of these actin filaments was highly uniform, averaging 33 +/- 5 nm. This is the length of nonmuscle tropomyosin. Since there is almost enough tropomyosin present to saturate the F-actin, our data support the hypothesis that tropomyosin may determine the length of actin protofilaments in the red cell membrane.

Band 3, the anion transport protein of the human erythrocyte membrane, is known to be phosphorylated in ghosts at tyrosine 8. The band 3 tyrosine kinase is now shown to be associated with the Triton X-100 insoluble membrane skeleton but not with spectrin or actin. The kinase was reversibly dissociated from membranes and skeletons at elevated ionic strength (50% at mu = 0.15). The binding capacity of the membranes exceeded their native complement of the kinase by at least 60-fold. Prior removal of all peripheral proteins from the cytoplasmic surface of inside-out vesicles did not diminish the rebinding of the kinase, whereas prior removal of band 3 and other accessory proteins from skeletons abolished the rebinding of the kinase. An excess of glyceraldehyde-3-P dehydrogenase, which binds to band 3 in the region of the phosphate acceptor tyrosine 8, both inhibited the phosphorylation of band 3 and released the kinase into solution. Soluble 40/45-kDa chymotryptic fragments from the cytoplasmic pole of band 3 were phosphorylated at least as well as membranous band 3 and caused the release of the kinase from Triton-extracted skeletons. Membrane skeletons lacked most of the membrane band 3, but retained most of the kinase. Nevertheless, the band 3 population solubilized by Triton X-100 from prelabeled ghosts was as well phosphorylated as the population of band 3 retained by the skeletons. Furthermore, the fraction of band 3 not associated with the skeletons following Triton X-100 extraction was a good substrate for the solubilized kinase. We conclude that this tyrosine kinase is reversibly bound to the membrane through electrostatic interactions with the polyacidic sequence surrounding the phosphate accepting tyrosine 8 on band 3. The kinase appears to be preferentially linked to those band 3 molecules associated with the membrane skeleton, but it impartially phosphorylates band 3 species free in the bilayer as well as band 3 fragments in solution. The resemblance of its plasma membrane binding behavior to that of tyrosine kinases of certain viruses causing oncogenic transformation is discussed.

Nucleoids isolated from Escherichia coli strains carrying temperature-sensitive gyrA or gyrB mutations were examined by sedimentation in ethidium bromide-containing sucrose density gradients. A shift to restrictive temperature resulted in nucleoid DNA relaxation in all of the mutant strains. Three of these mutants exhibited reversible nucleoid relaxation: when cultures incubated at restrictive temperature were cooled to 0 degree C over a 4- to 5-min period, supercoiling returned to levels observed with cells grown at permissive temperature. Incubation of these three mutants at restrictive temperature also caused nucleoid sedimentation rates to increase by about 50%.

The binding of phosphofructokinase and aldolase to the membrane of the intact human erythrocyte was assessed by the rapid hemolysis/filtration method of Kliman and Steck (Kliman, H. J., and Steck, T. L. (1980) J. Biol. Chem. 255, 6314-6321). We found that about 50% of the phosphofructokinase was membrane-bound in fresh red cells prior to hemolysis. Binding was not significantly altered by deoxygenation. Approximately 40% of aldolase was membrane-associated in fresh red cells. In outdated, blood-banked red cells, aldolase was 73% membrane-bound while, following metabolic repletion, 40% of the enzyme was membrane-associated. These results support the hypothesis that certain glycolytic enzymes in the red cell are membrane-bound in a rapidly reversible and metabolically sensitive fashion.

We have examined the ameba Dictyostelium discoideum for evidence of a discrete, prelysosomal, acidic receiving compartment in endocytosis. We observed in the cytoplasm abundant round vacuoles with diameters up to 2 microns that concentrated acridine orange by a process inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). They were therefore taken to be acidic. The vacuoles were observed to fuse nearly quantitatively with primary phagosomes over 30 min and thereby to confer upon them the ability to accumulate acridine orange. The entry into lysosomes of phagocytic cargo occurred later. In the absence of phagocytosis, almost all of the acidic vacuoles rapidly accumulated fluorescent markers that had either been covalently coupled to the cell surface or fed as the soluble dextran conjugate. Therefore, these vacuoles also lie on the pathway of pinocytosis. A prominent subcellular ATPase activity inhibited by 25 microM NBD-Cl co-distributed on sucrose equilibrium density gradients with vacuoles capable of concentrating acridine orange in vitro. The peak was broad and more buoyant than that bearing lysosomal acid hydrolases, which contained only a minor amount of this ATPase. Also migrating in the buoyant peak were internalized plasma membrane markers; e.g., 3H-galactose had been covalently coupled to the surface of intact cells and allowed to enter pinosomes. We conclude that in D. discoideum an extensive prelysosomal vacuolar compartment provides the proton pumps that acidify both phagosomes and pinosomes.

We report a case of repeated fetal death at 31 gestational weeks associated with mild non-proteinuric pre-eclampsia and intrauterine growth retardation. After double intradermal immunisation with paternal leukocytes, a third pregnancy proceeded uneventfully until it ended at 38 weeks. Maternal anti-paternal blocking antibody activity was assessed by the erythrocyte antibody inhibition (EAI) test. Serologic testing revealed that the couple did not share HLA class I antigens. The mechanisms underlying the likely benefit from immunotherapy are discussed.

The kinetics of transfer of [3H]cholesterol between human erythrocytes and plasma at 37 degrees C in physiological buffer had these features. 1) Cholesterol transfer was strikingly similar in both directions. 2) Transfer progressed to isotopic equilibrium in a monotonic, apparently first order fashion, except for a minor rapid component (approximately 15%) observed in the transfer of cholesterol from cells to plasma. 3) The mechanism of transfer was not via transient collisions in that the rate of the reaction was quite insensitive to the concentration of reactants over a wide range. 4) The mechanism of transfer did not involve specific, stable complex formation in that there was little difference in the behavior of erythrocytes and inside-out plasma membrane vesicles derived therefrom or between plasma and sonicated liposomes as acceptors. Furthermore, transfer was not affected by vigorous proteolysis of either the cells or the plasma. 5) The kinetics of transfer were fully compatible with diffusion of cholesterol through the aqueous compartment. This was shown by fitting our data to a rigorous model for diffusion equilibrium between three compartments. 6) The partition coefficient of [3H]cholesterol between red cells and buffer was shown to be 10(7). 7) The rate constants for cholesterol release from both red cells and plasma were approximately 1 X 10(-4) s-1 (t 1/2 approximately 2 h). The rate constant for cholesterol uptake into red cells was approximately 1 X 10(3) s-1 (t 1/2 approximately 1 ms). 8) The similarity of the corresponding kinetic constants among red cells, plasma, and liposomes suggests that phospholipids in a variety of physical forms are equivalent solvents for cholesterol. We conclude that despite its extremely low solubility in water, cholesterol moves between lipid compartments by aqueous diffusion.