Showing papers in "Journal of Supramolecular Structure in 1976"
TL;DR: SDS polyacrylamide gel electrophoresis reveals that this cytoskeleton of chick embryo fibroblasts is made up of three main proteins, one of which is 42,000 daltons and co-migrates with muscle actin and the other two components are 52,000 and 230,000Daltons, which remain quantitatively associated with the cytos skeleton during the detergent extraction.
Abstract: Treatment of chick embryo fibroblasts with 0.5% Triton X-100 extracts most of the cell protein, leaving an organized part of the cell structure attached to the tissue culture dish. This "Triton cytoskeleton" consists largely of intermediate-sized filaments and bundles of microfilaments. SDS polyacrylamide gel electrophoresis reveals that this cytoskeleton is made up of three main proteins. One protein component is 42,000 daltons and co-migrates with muscle actin. The other two components are 52,000 and 230,000 daltons and remain quantitatively associated with the cytoskeleton during the detergent extraction. The possible identity of these three protein components and their organization into a supramolecular structure is discussed.
367 citations
TL;DR: FRAP measurements indicate that over 75% of the wheat germ agglutinin receptor (WGA-receptor) complexes on the surface of human embryo fibroblasts are mobile, and the mean diffusion coefficient of these complexes are higher than that obtained from earlier FRAP measurements of the mobility of concanavalin A-recept (Con A) complexes in a variety of cell types.
Abstract: The use of fluorescence recovery after photobleaching (FRAP) techniques to monitor the lateral mobility of plant lectin-receptor complexes on the surface of single, living mammalian cells is described in detail. FRAP measurements indicate that over 75% of the wheat germ agglutinin receptor (WGA-receptor) complexes on the surface of human embryo fibroblasts are mobile. These WGS-receptor complexes diffuse laterally (as opposed to flow) on the cell surface with a diffusion coefficient in the range of 2 X 10(-11) to 2 X 10(-10) cm2/sec. Both the percentage of mobile WGA-receptor complexes and the mean diffusion coefficient of these complexes are higher than that obtained from earlier FRAP measurements of the mobility of concanavalin A-receptor (Con A-receptor) complexes in a variety of cell types. The possible reasons for the differing mobilities of WGA and Con A receptors are discussed.
180 citations
TL;DR: It is concluded that in tissue culture cells tropomyosin binds preferentially to actin filaments involved in structural support to confer rigidity upon them as well as aid them in maintaining a stretched phenotype.
Abstract: In the assembly of actin filaments that takes place during the spreading of a polulation of human lung cells, after trypsin detachment off the substratum and replating, tropomyosin exhibits a considrable lag in its association with the newly forming filament bundles; it begins to associate with them during the later stages of cell spreading as the actin filament bundles normally seen in interphase cells begin to organize. This lag is evident in a number of cell types that are spreading onto a substratum; it does not appear to be due to a selective degradation of this molecule during rounding up of the cells, since tropmyosin associates with the actin filament bundles after this lag even under conditions where the protein synthetic activity of the cell is inhibited to more than 95% by cycloheximide. The preferential binding of tropomyosin to fully assembled filament bundles but not to newly formed bundles of actin filaments suggests therefore the existence of two classes of action filaments: those that bind tropomyosin and those that do not. This selective localization of tropomyosin and those that do not. This selective localization of tropomyosin on actin filaments was further pursued by examining the localization of this molecule in membrane ruffles. The immunofluorescent results indicate that ruffling is an actin-filament-dependent, microtubule-independent phenomenon. Tropomyosin is absent from membrane ruffles under a variety of circumstances where ruffling is expressed and, more generally, from any other cellular activity where actin filaments are expected to be in a dynamic state of reorganization or are required to be in a flexible configuraion. It is concluded that in tissue culture cells tropomyosin binds preferentially to actin filaments involved in structural support to confer rigidity upon them as well as aid them in maintaining a stretched phenotype. The absence of tropomyosin from certain motile phenomena where actin filaments are involved indicates that these classes of actin filaments are regulated by cytoplasmic mechanisms distinct from that by which tropomyosin (and troponin) mediates contractility in skeletal mulscle; it opens the possibility that different types of actin filaments enagaged in different cellular motile phenomenon in tissue culture cells may be regulated by a host of coexisting regulatory mechanisms, some as yet undetermined.
168 citations
TL;DR: New models of hormone action must be considered which integrate the progress in knowledge of both the complex mechanisms regulating hormone binding to their surface receptors, and the dynamic properties of the cell membrane.
Abstract: The binding of many polypeptide hormones to cell surface receptors does not appear to follow the law of mass action. While steady-state binding data are consistent in many cases with either heterogeneous populations of binding sites or interactions of the type known as negative cooperativity, study of the kinetics of dissociation of the type known as negative cooperativity, study of the kinetics of dissociation of the hormone receptor complex allows an unambiguous demonstration of cooperative interactions. Negative cooperativity, which seems to be wide-spread among hormone receptors, provides exquisite sensitivity of the cell at low hormone concentrations while buffering against acutely elevated hormone levels. The molecular mechanisms underlying the cooperativity are still largely unknown. Cooperativity may stem from a conformational transition in individual receptors or involve receptor aggregation in the fluid membrane (clustering) or more extensive membrane phenomena. Thus, new models of hormone action must be considered which integrate the progress in our knowledge of both the complex mechanisms regulating hormone binding to their surface receptors, and the dynamic properties of the cell membrane.
157 citations
TL;DR: The receptor-mediated binding steps in the transport process are most easily studied in the chicken because of the enormous amount of oocyte membrane available from a given oocyte and because up to 1 gm of protein is normally transported per day per oocyte.
Abstract: Proteins are selectively sequestered by a number of cell types. However, only in oocytes is the process sufficiently aggravated and specific to be readily studied. In these cells certain serum proteins are taken up in proportions different from those found in the serum. In vitro incubations of hormonally stimulated and synchronous mosquito oocytes show that the only protein capable of initiating the transport process is the female specific yolk protein. Heterologous proteins such as IgG, bovine serum albumin, cytochrome C, and ferritin are inactive. The female specific protein is a phosphoglycolipoprotein. It is synthesized in the fat body, a liver analog in the insect, and passed into the serum before being transported into the oocytes. Preliminary kinetic analysis shows the uptake process to be specific with an apparent Km of about 10(-7) M. Glycolytic inhibitors stop protein uptake. The receptor-mediated binding steps in the transport process are most easily studied in the chicken because of the enormous amount of oocyte membrane available from a given oocyte and because up to 1 gm of protein is normally transported per day per oocyte. IgG and the hen specific phosvitin lipovitellin are two of the physiologically important proteins that are transported intact into the chicken oocytes. The uptake appears selective as shown by studies with iodinated proteins. Ferritin conjugated to IgG is shown by electron microscopy to bind to isolated plasma membranes only where coated pits have formed, whereas ferritin alone is not seen localized on any membrane surface. These very specialized regions of the membrane are similar to micropinocytotic pits but, in addition, possess on their cytoplasmic side dense ridges that form the coat. Transport involves binding to the coated pits, the pinching off of the pits, and the subsequent movement of the coated vesicles in the cytoplasm.
154 citations
TL;DR: It is apparent that the proteins appear as a response to low intracellular iron rather than being controlled by the ton B gene, and may be located in the active transport system of the cytoplasmic membrane.
Abstract: The functional interaction of outer membrane proteins of E. coli can be studied using phage and colicin receptors which are essential components of penetration systems. The uptake of ferric iron in the form of the ferrichrome complex requires the ton A and ton B functions in the outer membrane of E. coli. The ton A gene product is the receptor protein for phage T5 and is required together with the ton B function by the phages T1 and o80 to infect cells and by colicin M and the antibiotic albomycin, a structural analogue of ferrichrome, to kill cells. The ton B function is necessary for the uptake of ferric iron complexed by citrate. Iron complexed by enterochelin is only transported in the presence of the ton B and feu functions. Cells which have lost the feu function are resistant to the colicins B, I or V while ton B mutants are resistant to all 3 colicins. The interaction of the ton A, ton B, and feu functions apparently permits quite different "substrates" to overcome the permeability barrier of the outer membrane. It was shown for ferrichrome dependent iron uptake that the complexing agent was not altered and could be used repeatedly. Only very low amounts of 3H-labeled ferrichrome were found in the cell. It is possible that the iron is mobilized in the membrane and that desferri-ferrichrome is released into the medium without having entered the cytoplasm. Growth on ferrichrome as the sole iron source was used to select revertants of T5 resistant ton A mutants. All revertants exhibited wild-type properties with the exception of partial revertants. In these 4 strains, as in the ton A mutants, the ton A protein was not detectable by SDS polyacrylamide gel electrophoreses of outer membranes. Albomycin resistant mutants were selected and shown to fall into 5 categories: 1) ton A; 2) ton B mutants; 3) mutants with no iron transport defects and normal ton A/ton B functions, which might be target site mutants; 4) mutants which were deficient in ferrichrome-mediated iron uptake but had normal ton A/ton B functions. We tentatively consider that the defect might be located in the active transport system of the cytoplasmic membrane; 5) a variety of mutants with the following general properties: most of them were resistant to colicin M, transported iron poorly, and, like ton B mutants, contained additional proteins in the outer membrane. The outer membrane protein patterns of wild-type and ton B mutant strains were compared by slab gel electrophoresis in an attempt to identify a ton B protein. It was observed that under most growth conditions, ton B mutants overproduced 3 proteins of molecular weights 74,000-83,000. In extracted, iron-deficient medium, both the wild-type and ton B mutant strains had similar large amounts of these proteins in their outer membranes. The appearance of these proteins was suppressed by excess iron in both wild-type and mutant. From this evidence it is apparent that the proteins appear as a response to low intracellular iron rather than being controlled by the ton B gene...
143 citations
TL;DR: This is a review of the evidence that the cytoplasmic contractile proteins function as a cytoskeletal system in the cytopskeletal matrix and shows that actin filaments can form a solid gel under conditions likely to exist in living cells.
Abstract: This is a review of the evidence that the cytoplasmic contractile proteins function as a cytoskeletal system in the cytoplasmic matrix. Biochemical experiments show that cycoplasmic actin filaments can form a solid gel under conditions likely to exist in living cells. The actin filaments are associated with other proteins which amy stabilize the gel and which are involved with motile force generation like myosin. Ultrastructural studies show that actin filaments are difficult to preserve, but that under stabilizing conditions networks of actin filaments are found throughtout the cytoplasmic matrix.
99 citations
TL;DR: A hypothesis is presented relating microtubule assembly and surface membrane modulation suggesting that calcium is the primary modulating signal in the presence of elevated levels of calcium.
Abstract: Indirect immunofluorescence analyses using antibodies directed against 6S tubulin have shown an elaborate cytoplasmic microtubule complex (CMTC) in nontransformed cells in culture. The CMTC is strikingly altered in cells that have been transformed spontaneously by viruses or by chemicals. Assembly of microtubules in vitro and in vivo is markedly inhibited in the presence of elevated levels of calcium. Alteration of the surface of normal cells by brief treatment with low concentrations of trypsin initiate a rapid breakdown of cytoplasmic microtubules. Finally, a hypothesis is presented relating microtubule assembly and surface membrane modulation suggesting that calcium is the primary modulating signal.
82 citations
TL;DR: The localization and organization of actin-like microfilaments in normal, SV-40 and adenovirus transformed cells are determined by the coordinated use of light optical, electron optical and biochemical techniques, and microfilament meshworks appear to be the predominant organizational form of cellular action.
Abstract: The localization and organization of actin-like microfilaments in normal, SV-40 and adenovirus transformed cells are determined by the coordinated use of light optical, electron optical and biochemical techniques. In adenovirus-type 5 transformed hamster embryo cells, microfilament meshworks appear to be the predominant organizational form of cellular action, while in normal hamster cells, microfilament bundles are prevalent. Differences between 3T3 and SV-40 transformed 3T3 cells are less apparent and may be related to the packing and intracellular distribution of microfilament bundles. Attempts at relating these ultrastructural changes in transformed cells to the images obtained following reaction with fluorescein-labelled myosin fragments and indirect immunofluorescence with smooth muscle myosin antibody are discussed. In several instances the fluorescence microscope images to not correspond to the ultrastructural observations. The results are discussed in terms of the possible relationships between alterations in cytoplasmic contractile elements and the abnormal behavior of transformed cells.
81 citations
TL;DR: Evidence indicates that SAM is the remnant of "footpad" vesicle by which the cell adheres to the substrate and that EGTA treatment weakens the subsurface cytoskeleton, allowing these footpad vesicles to be pinched off from the rest of the cell.
Abstract: The proteins and polysaccharides which are left adherent to the tissue culture substrate after EGTA-mediated removal of normal, virus-transformed, and revertant mouse cells (so-called SAM, or substrate-attached material), and which have been implicated in the cell-substrate adhesion process, have been characterized by SDS-PAGE and other types of analyses under various conditions of cell growth and attachment. The following components have been identified in SAM: 3 size classes of hyaluronate proteoglycans; glycoprotein Co (the LETS glycoprotein); protein Ca (a myosin-like protein); protein Cb(MW 85,000); protein C1 (MW 56,000, which is apparently not tubulin); protein C2 (actin); proteins C3-C5 (histones) which are artifactually bound to the substrate as a result of EGTA-mediated leaching from the cell; and proteins Cc, Cd, Ce, and Cf. The LETS glycoprotein (Co) and Cd appear in newly-synthesized SAM (which is probably enriched in "footpad" material--"footpads" being focal areas of subsurface membraneous contact with the substrate in greater relative quantities than in the SAM accumulated over a long period of time (which is probably enriched in "footprint" material--remnants of footpads left behind as cells move across the substrate). CO and Cd turn over very rapidly following short radiolabeling periods during chase analysis. The SAM's deposited during a wide variety of cellular attachment and growth conditions contained the same components in similar relative proportions. This may indicate well-controlled and coordinate deposition of a cell "surface" complex involving the hyaluronate proteoglycans, the LETS glycoprotein, actin-containing microfilaments with associated proteins, and a limited number of additional proteins in the substrate adhesion site. Evidence indicates that SAM is the remnant of "footpad" vesicles by which the cell adheres to the substrate and that EGTA treatment weakens the subsurface cytoskeleton, allowing these footpad vesicles to be pinched off from the rest of the cell. Three different models of cell-substrate adhesion are presented and discussed.
68 citations
TL;DR: Results from surface label experiments reveal that cells from drug-resistant Chinese hamster ovary clones possess a surface carbohydrate component of apparent molecular weight 165,000 which is absent from wild-type cells.
Abstract: Surface label experiments using the galactose oxidase-[3 H] -borohydride technique reveal that cells from drug-resistant Chinese hamster ovary clones possess a surface carbohydrate component of apparent molecular weight 165,000 which is absent from wild-type cells. The component may also be demonstrated by [14C] glucosamine incorporation but not by [3 H] leucine incorporation or by the lactoperoxidase surface labeling reaction.
TL;DR: The quantitative correlations strongly support the view that the receptor studied by ligand binding corresponds to the receptor study by pharmacological methods, and also applies to direct binding studies of [3H] acetylcholine.
Abstract: The binding of one irreversible and two reversible radioactive antagonists to muscarinic receptors in synaptosome preparations of rat cerebral cortex has been studied. The ligands all bind to the same receptor pool and directly and competitively yield self-consistent binding constants closely similar to those obtained by pharmacological methods on intact smooth muscle. The binding process for antagonists seems to be a simple mass action-determined process with a Hill slope of 1.0. The quantitative correlations strongly support the view that the receptor studied by ligand binding corresponds to the receptor studied by pharmacological methods. Inhibition of antagonist binding by most agonists shows a reduced Hill slope which also applies to direct binding studies of [3H] acetylcholine. Mechansims that might account for the behavior of agonists are discussed but do not conclusively point to any single mechanism.
TL;DR: Analysis of the experimental data suggests that a gradient which increases serine concentration by a factor of 2 in 10 mm is sufficient to double the average duration of a trajectory for a bacterium moving directly up the gradient, which suggests a microscopic model for the dependence of bacterial behavior on the serine gradient.
Abstract: The details of the chemotactic response of Salmonella typhimurium to gradients of L-serine have been examined in some detail. Two relatively macroscopic techniques have been employed to measure the bacterial response. These include measurements of the average velocity as the bacterial population moves toward attractants, and measurement of the upward-to-downward flux ratio, R, in the stable preformed attractant gradients. The dependence of the average velocity on gradient appears to be hyperbolic in nature, while the flux ratio depends linearly on the gradient. These data suggest a microscopic model for the dependence of bacterial behavior on the serine gradient. The model involves a linear dependence of the mean lifetime of a bacterial trajectory on the gradient for those bacteria moving toward higher attractant concentration. Those moving toward low concentrations of attractant do not change the mean duration of their trajectories, or the speed at which a given bacterium swims through the solution. This model generates the observed dependences of the average velocity and flux ratio on gradient. Interpretation of the experimental data suggests that a gradient which increases serine concentration by a factor of 2 in 10 mm is sufficient to double the average duration of a trajectory for a bacterium moving directly up the gradient. The concentration dependence of the chemotactic response to serine is more complicated. It suggests that more than one receptor of serine may be involved in determining chemotactic behavior to this attractant.
TL;DR: The detailed similarities between the disease of receptor-immunized animals "experimental autoimmune myasthenia gravis" (EAMG), and myast Hennessy gravis are compared and reactions of antisera from animals with EAMG with receptor from Electrophorus and Torpedo are studied.
Abstract: Immunochemical techniques for the study of acetylcholine receptors are described. Immunization of rabbits, rats, guinea pigs, and goats with acetylcholine receptor protein purified from Electrophorus electric organ tissue results in muscular weakness and death due to impaired neuromuscular transmission. Serum from immunized animals contains high concentrations of antibodies directed at receptors from the electric organ and low concentrations of antibodies directed at receptors from skeletal muscle. The detailed similarities between the disease of receptor-immunized animals "experimental autoimmune myasthenia gravis" (EAMG), and myasthenia gravis are compared. Reactions of antisera from animals with EAMG with receptor from Electrophorus and Torpedo are studied. Antireceptor antibodies in these antisera are directed predominantly and determinants other than the acetylcholine-binding site.
TL;DR: It is proposed that tertiary amine local anesthetics displace plasma membrane-bound Ca2+, resulting in disengagement of microfilament systems from the plasma membrane and increased cellular Ca2+ concentration to levels which disrupt microtubular organization.
Abstract: Tertiary amine local anesthetics (dibucaine, tetracaine, procaine, etc.) modify cell morphology, concanavalin A (Con A)-mediated agglutinability and redistribution of Con A receptors. Con A agglutination of untransformed mouse 3T3 cells was enhanced at low concentrations of local anesthetics, and the dynamics of fluorescent-Con A indicated that ligand-induced clustering was increased in the presence of the drugs. In contast, these drugs inhibited Con A-induced receptor capping on mouse spleen cells. These effects can be duplicated by combinations of vinblastine (or colchicine) and cytochalasin B suggesting that local anesthetics act on microtubule and microfilament assemblies which are involved in the trans-membrane control of cell surface receptor mobility and distribution. It is proposed that tertiary amine local anesthetics displace plasma membrane-bound Ca2+, resulting in disengagement of microfilament systems from the plasma membrane and increased cellular Ca2+ concentration to levels which disrupt microtubular organization. The possible involvement of cellular Ca2+ in cytoskeletal destruction by local anesthetics was investigated utilizing Ca2+-specific ionophores A23187 and X537A. In media containing Ca2+ and cytochalasin B these ionophores caused effects similar to tertiary amine local anesthetics.
TL;DR: It is shown that the addition of cholesterol to the dispersion broadens and decreases the magnitude of the transition, which is interpreted in terms of a change in the polarizability of the acyl chains of a lipid bilayer undergoes a thermal transition.
Abstract: The use of conjugated polyene fatty acids as probes of membrane structure is examined, alpha- and beta-parinaric acid (cis, trans, trans, cis- and all trans-9, 11, 13, 15-octadecatetraenoic acid) and synthetic lecithins containing an alpha-parinaric acid chai in position 2 have been prepared, and their absorption and fluorescence properties have been determined. Their absorption spectra are at sufficiently long wavelength to be unobscured by cellular chromophores such as nucleotides and aromatic amin acids. Parinaric acid absorption does, however, overlap tryptophan emission which allows fluorescence energy transfer. Potential uses of these fluorescent probes are presented with studies on mode systems with known physical properties. Dipalmitoyl phosphatidylcholine exhibits a sharp phase transition 1 degree wide at 42 degrees C, as monitored by the fluorescence intensit of parinaric acid. The magnitude of the transition is independent of probe concentration, but the width of the transition and hysteresis are dependent upon such factors as the probe concentration and whether or not sonication is used in sample preparation. Using both fluorescence and absorption properties of the probe, we show that the addition of cholesterol to the dispersion broadens and decreases the magnitude of the transition. These results are interpreted in terms of a change in the polarizability of the acyl chains of a lipid bilayer undergoes a thermal transition. Lipid-protein interactions are studied by the binding of alpha-parinaric acid to bovine serum albumin. Fluorescence enhancement, absorption spectral shifts, and quenching of tryptophan fluorescnece are observed when alpha-parinaric acid binds to bovine serum albumin. Calculations based on these measurements are consistent with two binding sites of KB approximately 10(8) (M-1) and three to four binding sites of KB approximately 10(6)-10(7) (M-1), similar to known values for the binding of other long-chain fatty acids. Biosynthetic incorporation of beta-parinaric acid into the E. coli fatty acid auxotroph 30E betaox has been accomplished and phase transitions in cells and isolated phospholipids are shown.
TL;DR: A comparison of the capsomere morphologies and protein compositions of the giant phage capsids showed that all T4D giants are identical but differ from T2L: the T4Ds has a complex (6 + 6 + 1)-type morphology, whereas the T2 L has a simple 6-type.
Abstract: A study has been made of the structure of the capsids of T4D giant phage produced from mutants in gene 23 and temperature-sensitive mutants in gene 24, and T4D and T2L giant phage formed by the addition of L-canavanine followed by an L-arginine chase in the growth medium.
All the giant phage capsids have been shown to be built according to the same geometrical architecture. This consists of a near-hexsagonal surface net, lattice constant 129.5 A, folded into a left-hand T = 13 prolate icosahedron elongated along one of its fivefold symmetry axes. Their only apparent difference from wild-type T-even phage capsids is their abnormally elongated tubular part.
A comparison of the capsomere morphologies and protein compositions of the giant phage capsids showed that all T4D giants are indentical but differ from T2L: The T4D capsomere has a complex (6+6+1)-type morphology, whereas the T2L has a simple 6-type. T2L phage, however, lack two capsid proteins, “soc” and “hoc”, present in T4D. The difference in capsomere morphology can therefore be related to the difference in the protein compositions of these two phage.
Possible differences between the initiation and means of length regulation of giant phage heads and the aberrant polyheads are discussed.
TL;DR: The linear organization of the contractile apparatus of the retinal cone cell makes this cell a promising model for morphological characterization of the disposition of actin and myosin filaments during contraction in a nonmuscle cell.
Abstract: Teleost retinal cones contract in light and elongate in darkness. This paper describes the disposition of microtubules and cytoplasmic filaments in cone cells of 2 species of fish (Haemulon sciurus and Lutjanus griseus). In Haemulon, the neck-like “myoid” region of the cone changes in length from 5 μ to 75 μ. Maximal observed rates of elongation and contraction are comparable to that of chromosome movement in mitosis (2–3 μ/min). Microtubules presumably participate in cone elongation, since numerous longitudinal microtubules are present in the myoid region, and colchicine blocks dark-induced elongation.
Myoid shortening, on the other hand, appears to be an active contractile process. Disruption of microtubules in dark-adapted cones does not produce myoid shortening in the absence of light, and light-induced myoid shortening is blocked by cytochalasin-B. Cone cells possess longitudinally-oriented thin filaments which bind myosin subfragment-1 to form arrowhead complexes typical of muscle actin. Myoid thin filaments are clearly observed in negatively stained preparations of isolated cones which have been disrupted with detergent after attachment to grids. These myoid filaments are not, however, generally preserved by conventional fixation, though bundles of thin filaments are preserved in other regions of the cell. Thus, actin filaments are poorly retained by fixation in precisely the region of the cone cell where contraction occurs. Cone cells also possess longitudinally-oriented thick filaments 130–160A in diameter. That these thick filaments may be myosin is suggested by the presence of side-arms with approximately 150 A periodicity. The linear organization of the contractile apparatus of the retinal cone cell makes this cell a promising model for morphological characterization of the disposition of actin and myosin filaments during contraction in a nonmuscle cell.
TL;DR: It is proposed that toxin action involves the initial formation of an inactive toxin-ganglioside complex which subsequently migrates and is somehow transformed into an active species which involves relocation within the two-dimensional structure of the membrane with direct perturbation of adenylate cyclase molecules (virtually irreversibly).
Abstract: The enterotoxin from Vibrio cholerae is a protein of 100,000 mol wt which stimulates adenylate cyclase activity ubiquitously The binding of biologically active 125I-labeled choleragen to cell membranes is of extraordinary affinity and specificity The binding may be restricted to membrane-bound ganglioside GM1 This ganglioside can be inserted into membranes from exogenous sources, and the increased toxin binding in such cells can be reflected by an increased sensitivity to the biological effects of the toxin Features of the toxin-activated adenylate cyclase, including conversion of the enzyne to a GTP-sensitive state, and the increased sensitivity of activation by hormones, suggest analogies between the basic mechanism of action of choleragen and the events following binding of hormones to their receptors The action of the toxin is probably not mediated through intermediary cytoplasmic events, suggesting that its effects are entirely due to processes involving the plasma membrane The kinetics of activation of adenylate cyclase in erythrocytes from various species as well as in rat adipocytes suggest a direct interaction between toxin and the cyclase enzyme which is difficult to reconcile with catalytic mechanisms of adenylate cyclase activation Direct evidence for this can be obtained from the comigration of toxin radioactivity with adenylate cyclase activity when toxin-activated membranes are dissolved in detergents and chromatographed on gel filtration columns Agarose derivatives containing the "active" subunit of the toxin can specifically absorb adenylate cyclase activity, and specific antibodies against the choleragen can be used for selective immunoprecipitation of adenylate cyclase activity from detergent-solubilized preparations of activated membranes It is proposed that toxin action involves the initial formation of an inactive toxin-ganglioside complex which subsequently migrates and is somehow transformed into an active species which involves relocation within the two-dimensional structure of the membrane with direct perturbation of adenylate cyclase molecules (virtually irreversibly) These studies suggest new insights into the normal mechanisms by which hormone receptors modify membrane functions
TL;DR: The process of cell wall biogenesis in Oocystis is compared to that in higher plants and microfibril development in relation to a coordinated multienzyme complex is discussed.
Abstract: Cell wall structure and biogenesis in the unicellular green alga, Oocystis apiculata, is described. The wall consists of an outer amorphous primary layer and an inner secondary layer of highly organized cellulosic microfibrils. The primary wall is deposited immediately after cytokinesis. Golgi-derived products contribute to this layer. Cortical microtubules underlie the plasma membrane immediately before and during primary wall formation. They function in maintaining the elliptical cell shape. Following primary wall synthesis, Golgi-derived materials accumulate on the cell surface to form the periplasmic layer. This layer functions in the deposition of coating and cross-linking substances which associate with cellulosic microfibrils of the incipient secondary wall. Secondary wall microfibrils are assembled in association with the plasma membrane. Freeze-etch preparations of untreated, living cells reveal linear terminal complexes in association with growing cellulosic microfibrils. These complexes are embedded in the EF fracture face of the plasma membrane. The newly synthesized microfibril lies in a groove of the outer leaflet of the plasma membrane. The groove is decorated on the EF fracture face by perpendicular structures termed "ridges". The ridges interlink with definitive rows of particles associated withe PF fracture face of the innter leaflet of the plasma membrane. These particles are termed "granule bands", and they function in the orientation of the newly synthesized microfibrils. Microfibril development in relation to a coordinated multienzyme complex is discussed. The process of cell wall biogenesis in Oocystis is compared to that in higher plants.
TL;DR: It is found that methyl xanthines inhibited the activation of glutathione reductase and that glutathion oxidizing agents act as mitotic inhibitors and reports that mixed disulfides of glutATHione and proteins can act as substrates for GR are discussed.
Abstract: It is shown that cyclic nucleotides can have a variety of effects on cell division, cell shape, cell adhesion, and cell movement, depending on the cells selected and the conditions under which they are used. For example, while CHO cells elongate under the influence of exogenous dibutyryl CAMP, Y-1 adrenal tumor cells round up and polyoma-transformed 3T3 cells show no change in shape. The totality of experience with cyclic nucleotides suggests that where they have been used by cells as control elements involving the four processes listed above, they are superimposed on basic cellular processes that progress in their absence--that is, they must be acting indirectly. In attempting to understand the inhibitory action of methyl xanthines on egg development, we were forced to abandon the idea that they acted through cyclic nucleotides. We found that methyl xanthines inhibited the activation of glutathione reductase and that glutathione oxidizing agents act as mitotic inhibitors. Further, we found that tubulin polymerizability, NAD-kinase activity, and a mitotic apparatus associated Ca+2-ATP-ase were all inhibited by oxidation of some of their sulfhydryls and were activated by reduction of the resulting disulfides. These results are discussed in terms of reported cycles and activations of glutathione reductase (GR) in cells and reports that mixed disulfides of glutathione and proteins can act as substrates for GR. Using the fact that a CAMP-dependent protein kinase has been reported to be activated by glutathione, we have suggested potential sites where sulfhydryl control processes and cyclic nucleotide control processes and cyclic nucleotide control processes may interact in certain restricted cases.
TL;DR: It is concluded that Balb/3T3 cells and C3H/10T1/2 cells are preneoplastic and give rise to spontaneously transformed clones when implanted in vivo attached to a solid substrate.
Abstract: The Balb/3T3 and C3H/10T1/2 lines, noted for their marked postconfluence inhibition of proliferation and anchorage dependence, and frequently studied as non-tumorigenic lines that are compared with tumorigenic sublines transformed with various agents, produced tumors within two to four months at low-cell dosage (3 X 10(4) cells) when implanted subcutaneously attached to 1 X 5 X 10 mm polycarbonate platelets. Platelets alone did not produce tumors. The cultured Balb/3T3 tumor cells showed loss of both postconfluence inhibition of proliferation and anchorage dependence. Tumors arising from attached Balb/3T3 cells in (BALB/c X C57B1/6)F1 hybrids were shown to be transplantable to BALB/c but not to C57B1/6 mice, proving that the tumors were derived from Balb/3T3 and not from host cells. The tumors exhibited unique transplantation rejection antigens that did not cross-react with each other. Scanning electronmicroscopy of Balb/3T3 cells and derived tumor cells on Teflon substrates (on which only the tumor cells and not the parent Balb/3T3 cells could grow) revealed that the two cell types were remarkably similar in appearance, except that the tumor cells were larger and showed many more microvilli that tended to concentrate over the nucleus. We conclude that Balb/3T3 cells and C3H/10T1/2 cells are preneoplastic and give rise to spontaneously transformed clones when implanted in vivo attached to a solid substrate.
TL;DR: The increased resolution obtained using a linear-gradient SDS polyacrylamide gel system now shows that both cAMP and 8-N3cAMP stimulate the phosphorylation by [gamma-32P]-ATP of the same red cell membrane proteins.
Abstract: An earlier report (1a) has shown the utility of 8-N3cAMP (8-azidoadenosine-3', 5'-cyclic monophosphate) as a photoaffinity probe for cAMP binding sites in human erythrocyte membranes. The increased resolution obtained using a linear-gradient SDS polyacrylamide gel system now shows that: 1) both cAMP and 8-N3cAMP stimulate the phosphorylation by [gamma-32P]-ATP of the same red cell membrane proteins; 2) the protein of approximately 48,000 molecular weight whose phosphorylation by [gamma-32P]-ATP is stimulated by cAMP and 8-N3cAMP migrates at a slower rate than the protein in the same molecular weight range which is heavily photolabeled with [32P]-8-N3cAMP;3) other cyclic nucleotide binding sites exist besides those initially reported; 4) the variation in the ratio of incorporation of [32P]-8-N3cAMP into the two highest affinity binding sites appears to be the result of a specific proteolysis of the larger protein.
TL;DR: The results indicate that loss of the transformation-sensitive surface proteins is due not to loss of synthesis but to lack of insertion of the protein in the neoplastic cell surface, which creates an impressive difference between the surface properties of normal and malignant cells.
Abstract: We have recently described a cell type-specific surface (SF) antigen that is deleted in chick fibroblasts transformed by Rous sarcoma virus. SF antigen is a major surface component and makes up about 0.5% of the total protein on normal cultured fibroblasts. The antigen is shed from normal cells and is present in circulation (serum, plasma), and in vivo, also, in tissue boundary membranes. The molecular equivalents of both cellular and serum SF antigen are distinct, large polypeptides, one of which (SF210, MW 210,000) is glycosylated and, on the cell surface, highly susceptible to proteases and accessible to surface iodination. Immunofluorescence and scanning electron microscopy have indicated that the antigen is located in fibrillar structures of the cell surface, membrane ridges, and processes.
Human SF antigen is present in human fibroblasts and in human serum. We have recently shown that human SF antigen is identical to what has been known as the “cold-insoluble globulin” and that it shows affinity toward fibrin and fibrinogen. Our results also indicate that loss of the transformation-sensitive surface proteins is due not to loss of synthesis but to lack of insertion of the protein in the neoplastic cell surface. Both normal and transformed cells produce the SF antigen, but the latter do not retain it in the cell surface.
The loss of SF antigen, a major cell surface component, from malignant cells creates an impressive difference between the surface properties of normal and malignant cells. The possible significance of SF antigen to the integrity of the normal membrane and its interaction to surrounding structures is discussed.
TL;DR: Con A-methacrylate microsphere conjugates prepared by a two-step glutaraldehyde reaction were used to label Con A-binding sites on bovine rod photoreceptor cells for visualization by scanning electron microscopy.
Abstract: Con A-methacrylate microsphere conjugates prepared by a two-step glutaraldehyde reaction were used to label Con A-binding sites on bovine rod photoreceptor cells for visualization by scanning electron microscopy. A dense distribution of markers was observed on the surface of the rod outer segment, the inner segment, and the synaptic region. Disk membranes also appear to be heavily labeled with the Con A-microsphere conjugates. The Con A inhibitor, alpha-methyl mannoside, inhibited the binding of the conjugate to the surface of these visual cells.
TL;DR: The specificity of lactoperoxidase-catalyzed iodination for the proteins of the hepatoma tissue culture cell plasma membrane was examined by histochemical, biochemical, and cell fractionation techniques.
Abstract: The specificity of lactoperoxidase-catalyzed iodination for the proteins of the hepatoma tissue culture cell plasma membrane was examined by histochemical, biochemical, and cell fractionation techniques. Light microscope autoradiography of sectioned cells shows the incorporated label to be localized primarily at the periphery of the cell. Most of this label can be released from the cell by trypsin but not by collagenase or hyaluronidase. The label is recovered from the cells as either monoiodotyrosine or diiodotyrosine after hydrolysis of cell extracts with a mixture of proteolytic enzymes. The label co-purifies during cell fractionation with an authentic liver cell plasma membrane marker enzyme, 5′-nucleotidase. Thus, the incorporated iodide is itself a valid marker for those membrane polypeptides having tyrosine residues accessible to the lactoperoxidase. The polypeptide complexity of the purified plasma membrane was examined by high resolution dodecyl sulfate-polyacrylamide gel electrophoresis. At least 50 polypeptides in the membrane are accessible to iodination. These polypeptides probably represent the bulk of the protein mass of the membrane and iodinating them does not affect cell viability, growth rate, or cell function. Labeling experiments with fucose and glucosamine show that at least nine of the iodinated peptides may be glycoproteins.
TL;DR: The sea urchin egg surface at fertilization has been examined with the scanning electron microscope to reveal the movements of the spermatozoon from the exterior, through the surface layers, and into the egg cytoplasm.
Abstract: The sea urchin egg surface at fertilization has been examined with the scanning electron microscope to reveal the movements of the spermatozoon from the exterior, through the surface layers, and into the egg cytoplasm. The layers that the spermatozoon encounter have been studied to determine their physical and chemical natures and their role in early development. By studying the outside of whole eggs and the inner face of surfaces isolated shortly after fertilization, it has been possible to compile data on the movements of the spermatozoon through the egg surface. The spermatozoon initially contacts the egg with the elongated acrosomal process. The vitelline sheet, the outermost layer of the egg, separates slightly next to the attached spermatozoon. As membrane fusion between the gametes occurs, the plasma membrane from the egg engulfs the spermhead, the cortical granules start to discharge their contents, and a spreading surface deformation, concommitant with a distortion of the fibrous cortex, is initiated. A cluster of elongate microville surround the perpendicularly fusing spermatozoon. These microvilli interidigitate as the spermatozoon is forced to lie upon the egg surface between the plasma membrane and the matrix of cortical fibers. The spermatozoon then rotates additionally to enter the egg cytoplasm with the posterior end first; it has rotated 180 degrees through the cell surface. Finally, it detaches into the egg cytoplasm, leaving a scar in the cortex through which it penetrated. The egg cortex, previously unobserved by electron microscopy, is revealed to be composed of 50-200 nm fibers. At fertilization they are uniformly organized but during later development this order is lost. The cortex is from 0.2-0.5 micronm thick and is a contractile structure. The role of the outer surface in releasing the cell from the metabolic constraints of the unfertilized egg is shown, and the apparent differences in the mobilities of the membranes derived from the sperm and from the egg are demonstrated. The relation of these layers to the movements of the spermatozoon, to the activation of the egg, to the block to polyspermy, and to each other are discussed.
TL;DR: Observations suggest that protein synthesis elongation factor Tu from E. coli and actin-like proteins could have evolved from a common ancestor, although more information will be necessary to prove or disprove this hypothesis.
Abstract: Protein synthesis elongation factor Tu from E. coli shares several physical, chemical, and functional properties with actin-like proteins. Limited tryptic degradation indicates that the two polypeptides have a similar molecular architecture. These observations suggest that they could have evolved from a common ancestor, although more information will be necessary to prove or disprove this hypothesis. A partial sequence, comprising 22 aminoacid residues from the aminoterminal end of the large tryptic fragment of elongation factor Tu is presented.
TL;DR: It is believed that isolated cortical granules may be an excellent model system for the biochemical study of exocytosis.
Abstract: Cortical granules are secretory vesicles bound to the inner surface of the plasma membrane of sea urchin eggs. Intact granules can be isolated by shearing away the cytoplasm of eggs which have been bonded to a protamine-coated surface. When Ca2+ is added to preparations of isolated granules the granules fuse with each other and release their contents. It is believed that isolated cortical granules may be an excellent model system for the biochemical study of exocytosis.
TL;DR: In a partially resistant lymphoma (BW5147/RCA3) that shows only a slight reduction in the total number of ricin-binding sites, ricin bound rapidly to the cell surface, but was endocytosed significantly less at low ricin doses compared to its parental line, indicating a possible difference in cell surface behavior.
Abstract: The binding, mobility, and mode of cell entry of the plant toxin ricin (or RCAII) were investigated on susceptible and partially resistant murine cell lines. When susceptible cells (SV40-transformed 3T3 fibroblast cells and BW5147 lymphoma cells) were examined, ricin bound rapidly, induced endocytosis, and entered the cell cytoplasm via broken endocytotic vesicles to inhibit cell protein synthesis, as found previously (1). Addition of lactose within 15 min after initial ricin binding prevented toxicity. After this time lactose addition no longer blocked the inhibition of protein synthesis. In a partially resistant lymphoma (BW5147/RCA3) that shows only a slight reduction in the total number of ricin-binding sites, ricin bound rapidly to the cell surface, but was endocytosed significantly less at low ricin doses compared to its parental line, indicating a possible difference in cell surface behavior. The exposed surface proteins on the BW5147 parental and BW5147/RCA3 resistnat lines were examined by 125I-labeling utilizing lactoperoxidase-catalyzed iodination. The radiolabeled components were solubilized and separated by slab electrophoresis in sodium dodecyl sulfate. Autoradiograms of the slab gels indicated that two surface components of approximately 80,000 and 35,000 mol wt were much less exposed or were missing on the resistant line.