Transport function and subcellular distribution of purified human erythrocyte glucose transporter reconstituted into rat adipocytes.

In order to delineate the insulin-independent (constitutive) and insulin-dependent regulations of the plasma membrane glucose transporter concentrations in rat adipocytes, we introduced purified human erythrocyte GLUT-1 (HEGT) into rat adipocytes by poly(ethylene glycol)-induced vesicle-cell fusion and its transport function and subcellular distribution in the host cell were measured. HEGT in adipocytes catalysed 3-O-methylglucose equilibrium exchange with a turnover number that is indistinguishable from that of the basal adipocyte transporters. However, insulin did not stimulate significantly the HEGT function in adipocytes where it stimulated the native transporter function by 7-8-fold. The steady state distribution and the transmembrane orientation assays revealed that more than 85% of the HEGT that were inserted in the physiological, cytoplasmic side-in orientation at the adipocytes plasma membrane were moved into low-density microsomes (LDM), while 90% of the HEGT that were inserted in the wrong, cytoplasmic side-out orientation were retained in the plasma membrane. Furthermore, more than 70% of the LDM-associated HEGT were found in a small subset of LDM that also contained 80% of the LDM-associated GLUT-4, the insulin-regulatable, native adipocyte glucose transporter. However, insulin did not cause redistribution of HEGT from LDM to the plasma membrane under the condition where it recruited GLUT-4 from LDM to increase the plasma membrane GLUT-4 content 4-5-fold. These results demonstrate that the erythrocyte GLUT-1 introduced in adipocytes transports glucose with an intrinsic activity similar to that of the adipocyte GLUT-1 and/or GLUT-4, and enters the constitutive GLUT-4 translocation pathway of the host cell provided it is in physiological transmembrane orientation, but fails to enter the insulin-dependent GLUT-4 recruitment pathway. We suggested that the adipocyte plasma membrane glucose transporter concentration is constitutively kept low by a mechanism where a cell-specific constituent interacts with a cytoplasmic domain common to GLUT-1 and GLUT-4, while the insulin-dependent recruitment requires a cytoplasmic domain specific to GLUT-4.

The platelet cytoskeleton: evidence for its structure from interactions with ZnCl2.

Isolation of platelet membranes and cytoskeletons in the presence of ZnCl2 as a stabilization agent, suggests that the 255 kd mol. wt. actin-binding-protein (rather than the 100 kd alpha-actinin-like constituent) may be involved in linkage of cellular cytoskeletal constituents to 105- and 120-kd membrane glycoproteins (presumed to be GP IIb and IIIa). Lack of detection in these preparations of a constituent that binds RBC alpha-spectrin antibody plus the presence of significant quantities of actin, further suggests that the principal membrane skeletal element is actin with perhaps smaller quantities of a 240-kd component, identified by others as the protein talin. Electron micrographs of platelets that have adhered to, partially spread on a substrate in the presence of ZnCl2, and have been jet washed with buffer, suggest the presence in these cells of both a membrane skeleton and a transcellular cytomatrix. The relationships between the membrane, membrane skeleton, transcellular cytomatrix and platelet granules are depicted in a proposed model (Fig. 9).

The platelet reticular network.

Human platelets have been observed by scanning electron microscopy after spreading on glass substrates followed by brief incubation in 1 mM ZnCl2 and subsequent shearing with a jet of buffer. The surfaces of many cells in such preparations are noted to consist of a reticular meshwork similar in appearance to the membrane skeleton of erythrocytes. In some preparations the reticular network is partially or almost entirely disrupted revealing an internal trabecular cytoskeleton and in the central region of the spread cell, granules associated with the cytoskeletal matrix. These results confirm previous observations obtained in TEM and HVEM studies of platelets and in addition provide information concerning the relative disposition of the filament systems in such spread cells. Preliminary results from sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of platelets and isolated platelet membranes suggest a basis for the effect of ZnCl2 on various platelet cytoskeletal and membrane components.

Effect of calcium on the morphology of human platelets spread on glass substrates.

The morphology of human platelets spread on glass substrates is sensitive to the presence of calcium. In the absence of Ca2+, cells spread from buffered salt solution develop radially oriented filopodia and subsequently a broad hyalomere surrounding the central region of the cell from which granules are frequently exocytosed. In the presence of Ca2+ cell rounding and apparent withdrawal from the substrate occurs. Scanning (SEM) and transmission (TEM) electron microscopy of cells rounded in the presence of Ca2+ show fibrous elements connecting the cells to the substratum as well as adherent to the substrate in the vicinity of the rounded cells. Interference reflection microscope (IRM) images of these cells are heterogeneous: some contain small discrete darker regions suggesting the presence of focal specializations at the ventral cell surface. In contrast IRM images of cells spread in the absence of Ca2+ indicate predominantly broad areas of unspecialized contact with the substrate in agreement with TEM observations. These results suggest that Ca2+ may enhance platelet-substrate adhesion by initially promoting the formation of focal specializations which become more pronounced as cell rounding occurs possibly due to Ca2+ activation of an actomyosin-based contractile mechanism.

Cytochalasin B binding by human platelets.

Intact human platelets bind cytochalasin B (CB) with a capacity of 100-120 p mols CB/mg protein or approximately 7 x 10(4) molecules/cell and dissociation constants (KD) ranging from 2 x 10(-8) to 10(-6) M. Up to 85% of this saturable binding is displaced by 10(-5) M cytochalasin E (CE). This CE-sensitive binding also appears heterogeneous with KD similar to those of the overall binding. The CE-insensitive binding, however, appears as a single component with KD approximately equal to 4 x 10(-7) M. The sedimentable constituents from frozen, thawed, and washed cells also bind CB with KD ranging from 2.4 x 10(-8) to 1.5 x 10(-6) M and a total capacity of approximately 39 p mols/mg protein which accounts for only 4% of the ligand binding to the intact cell. The major portion (60-80%) of this CB binding is displaceable by 500 mM D-glucose and has a KD of 1.5 x 10(-6) M, while only 10-15% is CE-sensitive with a KD of 2.4 x 10(-8) M. It is concluded that 95% of the saturable CB binding in platelets is associated with the cytosol of which 80-85% is sensitive to CE and that only 3% of the cellular binding is glucose sensitive, membrane-associated binding. If the CE-sensitive binding associated with the cytosol is entirely to actin, the stoichiometry of this binding is approximately one CB to 30 actin monomers, which is greater by an order of magnitude than that for CB binding to muscle actin.

Erythrosomes: large proteoliposomes derived from crosslinked human erythrocyte cytoskeletons and exogenous lipid.

Large (3-micrometers diameter) mechanically stable proteoliposomes (erythrosomes) were prepared in good yield by coating crosslinked erythrocyte cytoskeletons with phosphatidylcholine. The erythrosomes consist of the polypeptides designated band 1, 2, 3, 4.1 + 4.2, and 5 (less than 4% of the endogenous lipid) and enough added lipid to form a bilayer coating the surface. Electron microscopy shows only the large proteoliposomes in sealed preparations. The trapping of bovine serum albumin, mannitol, sucrose, glucose, cytosine arabinoside, and sodium in the erythrosomes was demonstrated, yielding an apparent volume of up to 100 liters/mol of phospholipid. This preparation possesses an effective diffusion barrier to glucose, sucrose, and sodium ion with half-equilibration times of 34, 29, and 170 hr, respectively. The results of the present study suggest that erythrosomes may be useful for membrane transport protein reconstitution and encapsulation systems.

Effect of divalent cations on the proteolysis of vertebrate and invertebrate muscle myosin.

Analysis by SDS-polyacrylamide gel electrophoresis of the products of alpha-chymotryptic digestion of lobster abdominal muscle myosin has shown that the presence or absence of Ca2+ during proteolysis does not have a major influence on the site of cleavage of the parent molecule, as it does for proteolysis of vertebrate muscle myosin. By following H+ liberation on the pH-stat during proteolysis, it has been found that the divalent cation only suppresses digestion of lobster myosin by about 15% as compared with proteolysis in the absence of Ca2+. Additionally, the subfragment-1-like species made by alpha-chymotryptic digestion of lobster myosin at low ionic strength is much more susceptible to degradation by trypsin than is vertebrate muscle myosin sub-fragment-1, in accordance with the relative susceptibilities of the parent molecules to similar degradation.

Association of creatine phosphokinase with the cytoskeleton of cultured mammalian cells.

Using an antibody specific for creatine phosphokinase (CPK), we have discovered an association between that enzyme and the cytoskeleton. Immunofluorescence observations show that CPK is associated with intermediate filaments in PTK cells and BALB/3T3 cells. The CPK distribution also follows intermediate filaments when cells are treated with colchicine.

Comparative studies on the structure and aggregative properties of the myosin molecule. II. THe substructure of the lobster myosin molecule.

Previous results (Siemankowski, R.F. and Zobel, C.R. (1976) J. Mechanochem. Cell Motility 3, 171--184) demonstrated that, relative to myosin from rabbit skeletal muscle, myosin from lobster abdominal muscle has four times as many sites susceptible to tryptic fragmentation at the fast rate. The present studies show that limited tryptic digestion of lobster myosin results in the rapid production of three species of rod fragments, all of which are insoluble at low ionic strength; a subfragment-1-like species; and, in addition, the release of large amounts of small peptides (35%, w/w). From estimates of the yields of the tryptic fragments, it is found that although 1 mol equiv. of rod-fragment is produced per mol of myosin digested, only 1 mol equiv. of a subfragment-1-like species is found, suggesting that the lobster myosin subfragment-1-moiety is much more trypsin-labile than the analogous region of rabbit myosin. By two-dimensional electrophoretic analysis, it was found that two of the rod species comprise a large number of unique peptides, collectively, after denaturation in 9 M urea. Similar analysis demonstrates that the subfragment-1-like species contains a small (Mr 25 000), very basic peptide, and that during digestion with trypsin the larger light chain (Mr 20 000) is converted entirely into a more acidic light chain fragment (Mr 18 500). The smaller light chain (Mr 16 000) is resistant to tryptic proteolysis.

Proteolytic fragments from the lobster myosin molecule.

The fragments produced by proteolysis of lobster abdominal muscle myosin with trypsin, alpha-chymotrypsin and papain have been investigated by sodium dodecyl sulfate (SDS) gel electrophoresis. Essentially monodisperse populations of long rods are produced by alpha-chymotryptic and papain digestion of rabbit myosin but corresponding digestion of lobster myosin yields multicomponent species. Similarly the low ionic strength insoluble fraction from tryptic digestion of lobster myosin is polydisperse in contrast to essentially monodisperse light meromyosin from rabbit myosin. Comparative tryptic digestion of rabbit and lobster myosin papain long rods shows that the latter have five susceptible cleavage sites in the subfragment-2 region while rabbit long rods have only one: both long rods appear to have three cleavage sites in the light meromyosin region. The fragments produced by tryptic digestion of rabbit myosin papain long rods have been tentatively identified by comparison with fragments isolated from papain digests of rabbit heavy meromyosin and tryptic digests of rabbit light meromyosin. The results suggest differences in sensitivity to enzymic proteolysis between the subfragment-2 regions in rabbit and lobster myosin as well as relative differences in proteolytic sensitivity between the subfragment-2 and light meromyosin region within the individual molecules. Partial explanation of the observation is proposed on the basis of differences in heavy chain compositions.

Comparative studies on the structure and aggregative properties of the myosin molecule. I. The structure of the lobster myosin molecule.

Myosin purified from the abdominal flexor muscle of the lobster, Homarus americanus, has a number average length of 1559 +/- 218 A, a rod like tail 1335 A long and a globular head 225 X 45 A as determined from electron microscopic observations on platinum shadowed preparations. The mass of the molecule was determined to be ca. 486,000 daltons from high speed equilibrium centrifugation studies at neutral and alkaline pH, and by SDS-acrylamide gel electrophoresis. Both sedimentation equilibrium centrifuge studies at alkaline pH and SDS-acrylamide gel electrophoresis experiments, indicate that the molecule contains a heavy chain core (two polypeptide chains weighing ca. 210,000 daltons each) and ca. four light chains of two weight classes (ca. 16,000 and 20,000 daltons). The amino acid composition of the myosin was determined. The specific activities of the Mg2+ -activated, K+/EDTA-activated, and Ca2+ -activated ATPases of the myosin were determined. Kinetic analysis of the digestion of lobster myosin with trypsin suggests that lobster myosin contains three classes of lysine and arginine residues; slowly split (k = 2.07 +/- 0.31 X 10(-2) moles/min2), rapidly split (k = 11.0 +/- 1.83 X 10(-2) moles/min2) and trypsin insensitive. There are 187 +/- 22 slowly split residues, 280 +/- 35 rapidly split residues, and 144 +/- 41 trypsin insensitive bonds per molecule. Comparison of these molecular parameters with those for the vertebrate skeletal muscle myosin indicates that the two myosins are similar in terms of mass, shape and overall polypeptide chain composition but may be considerably different in terms of local polypeptide chain conformation or composition.

Comparative studies on the structure and aggregative properties of the myosin molecule. III. The in vitro aggregative properties of the lobster myosin molecule.

The solubility of rabbit skeletal and lobster abdominal muscle myosin has been studied in monovalent salt solutions as a function of pH (over the range 4.75 to 8.5) and ionic strength (50-500 mM). Rabbit skeletal muscle myosin was found to precipitate over a narrower pH range than the lobster abdominal muscle myosin but at equivalent pH values and ionic strengths the former exhibited greater solubility. Comparison of the solubility of rabbit myosin, per se with that of light meromyosin and lobster myosin with its equivalent proteolytically produced fragment (fraction B1) showed that both rod fragments were more soluble than their parent molecules. Under conditions of low solubility (low ionic strength and pH) the quantitiy of protein in solution remained essentially constant with increasing total protein, thus suggesting that the aggregation phenomenon is of a phase transition type. Examination of the aggregates by electron microscopy revealed that rabbit myosin formed classical, elongate, spindle-shaped filaments similar to those previously observed by others. In contrast lobster myosin only formed short, dumbbell-shaped filaments 0.2-0.3 mum long. Consideration of the pH ranges over which aggregation occurred suggests that protonation of histidine residues may be involved in rabbit myosin filament formation while for lobster myosin, aggregation may involve protonation of epsilon-amino or guanidino groups. The possible relationship between the distribution of these groups along the rod portion of the myosin molecule and the formation of elongate filaments has been explored.

Effect of solution composition and proteolysis on the conformation of axonemal components.

The effect of solution composition and enzymic proteolysis on axonemes prepared from the sperm of sea urchins, Tripneustes gratilla, has been investigated. Aliquots of axonemes, prepared by treatment of sperm with Triton X-100 and differential centrifugation, were transferred to solutions of different composition with and without intervening tryptic proteolysis, and the particle conformations observed by dark-field and electron microscopy. In most solutions particles in partially digested preparations underwent conformational transformations to coiled or helix-like forms. Proteolysis was accompanied by an increase in the ATPase activity of the digest: by centrifuging down the insoluble digestion products it was shown that digestion resulted in the appearance of ATPase activity in the soluble phase with a concomitant decrease in ATPase activity in the pellet fraction. Gel electrophoresis showed this corresponded to the appearance of dynein in the supernatant and a decrease in dynein associated with the insoluble fraction. Supernatant dynein had a greater specific ATPase activity than dynein extracted from axonemes. Observations on specimens prepared for electron microscopy by thin sectioning allowed a rough correlation to be made between the dark-field observations, chemical analyses, and morphological alterations attendant with the proteolysis and solution conditions. It is concluded that in the intact axoneme the doublet tubules are under considerable tension and that proteolytic destruction of physical restraining elements allows spontaneous conformational alterations of the digestion products. In addition, proteolysis increases the specific ATPase activity of dynein and removes a portion of it from the axonemal structure.