31P nuclear magnetic resonance study of enzymatically phosphorylated glycophorin A

Glycophorin A was phosphorylated using protein kinases and the new protein was investigated using³¹P NMR spectroscopy. Most of these ～30 moles of phosphate were found to be attached to Ser and Thr. Some of these phosphate residues appear to be affected by the carbohydrate residues present. The phosphorylated protein appears to be in a severe state of aggregation, with the degree of aggregationpH-dependent.     

Chondroitin sulphate proteoglycan in the substratum adhesion sites of Balb/c 3T3 cells. Fractionation on various ion-exchange and affinity columns.

Proteoglycans on the cell surface play critical roles in the adhesion of fibroblasts to a fibronectin-containing extracellular matrix, including the model mouse cell line Balb/c 3T3. In order to evaluate the biochemistry of these processes, long-term [35S]sulphate-labelled proteoglycans were extracted quantitatively from the adhesion sites of 3T3 cells, after their EGTA-mediated detachment from the substratum, by using an extractant containing 1% octyl glucoside, 1 M-NaCl and 0.5 M-guanidinium chloride (GdnHCl) in buffer with many proteinase inhibitors. Greater than 90% of the material was identified as a large chondroitin sulphate proteoglycan (Kav. = 0.4 on a Sepharose CL2B column), and the remainder was identified as a smaller heparan sulphate proteoglycan; only small amounts of free chains of glycosaminoglycan were observed in these sites. These extracts were fractionated on DEAE-Sepharose columns under two different sets of elution conditions: with acetate buffer (termed DEAE-I) or with acetate buffer supplemented with 8 M-urea (termed DEAE-II). Under DEAE-I conditions about one-half of the material was eluted as a single peak and the remainder required 4 M-GdnHCl in order to recover it from the column; in contrast, greater than 90% of the material was eluted as a single peak from DEAE-II columns. Comparison of the elution of [35S]sulphate-labelled proteoglycan with that of 3H-labelled proteins from these two columns, as well as mixing experiments, indicated that the GdnHCl-sensitive proteoglycans were trapped at the top of columns, partially as a consequence of their association with proteins in these adhesion-site extracts. Affinity chromatography of these proteoglycans on columns of either immobilized platelet factor 4 or immobilized plasma fibronectin revealed that most of the chondroitin sulphate proteoglycan and the heparan sulphate proteoglycan bound to platelet factor 4 but that only the heparan sulphate proteoglycan bound to fibronectin, providing a ready means of separating the two proteoglycan classes. Affinity chromatography on octyl-Sepharose columns to test for hydrophobic domains in their core proteins demonstrated that a high proportion of the heparan sulphate proteoglycan but none of the chondroitin sulphate proteoglycan bound to the hydrophobic matrix. These results are discussed in light of the possible functional importance of the chondroitin sulphate proteoglycan in the detachment of cells from extracellular matrix and in light of previous affinity fractionations of proteoglycans from the substratum-adhesion sites of simian-virus-40-transformed 3T3 cells.     

Cell surface heparan sulfate mediates some adhesive responses to glycosaminoglycan-binding matrices, including fibronectin

Proteins with affinities for specific glycosaminoglycans (GAC's) were used as probes for testing the potential of cell surface GAG's to mediate cell adhesive responses to extracellular matrices (ECM). Plasma fibronectin (FN) and proteins that bind hyaluronate (cartilage proteo-glycan core and link proteins) or heparan sulfate (platelet factor 4 [PF4]) were adsorbed to inert substrata to evaluate attachment and spreading of several 3T3 cell lines. Cells failed to attach to hyaluronate-binding substrata. The rates of attachment on PF4 were identical to those on FN; however, PF4 stimulated formation of broad convex lamellae but not tapered cell processes fibers during the spreading response. PF4-mediated responses were blocked by treating the PF4-adsorbed substratum with heparin (but not chondroitin sulfate), or alternatively the cells with Flavobacter heparinum heparinase (but not chondroitinase ABC). Heparinase treatment did not inhibit cell attachment to FN but did inhibit spreading. Cells spread on PF4 or FN contained similar Ca2+-independent cell-substratum adhesions, as revealed by EGTA-mediated retraction of their substratum-bound processes. Microtubular networks reorganized in cells on PF4 but failed to extend into the broadly spread lamellae, where fine microfilament bundles had developed. Stress fibers, common on FN, failed to develop on PF4. These experiments indicate that (a) heparan sulfate proteoglycans are critical mediators of cell adhesion and heparan sulfate-dependent adhesion via PF4 is comparable in some, but not all, ways to FN-mediated adhesion, (b) the uncharacterized and heparan sulfate-independent "cell surface" receptor for FN permits some but not all aspects of adhesion, and (c) physiologically compatible and complete adhesion of fibroblasts requires binding of extracellular matrix FN to both the unidentified "cell surface" receptor and heparan sulfate proteoglycans.     

Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

Clonal segregation of multiple and overlapping matrix adhesive responses in dorsal root neuronal derivative cells

Clones of F11 hybrid (neuroblastoma X dorsal root neuron) cells have been tested for adherence and neurite outgrowth on three different substrata on which the parental cells display some competence--plasma fibronectin (pFN) with its multiple receptors, cholera toxin subunit B(CTB) as a model ganglioside GM1-binding substratum, and platelet factor-4 (PF4) as a model proteoglycan-binding substratum. This paradigm tests for independently segregating and overlapping mechanisms of neuritogenesis via transmembrane processes in pluripotent hybrid cells based on random loss of chromosomes contributed by the parent neural cells. For the nine clones tested, attachment was significantly lower on CTB but much higher on PF4 for all clones when compared to their attachment on pFN. Supplementation of cells with GM1 stimulated attachment of only two clones (on all three substrata). Neurite outgrowth was observed in a substratum-specific pattern and varied from 0 to greater than 60% on pFN; on CTB and PF4 substrata, the patterns were similar to each other but differed markedly from the pattern on pFN. In contrast, PF4- and CTB-directed neurites differed morphologically from each other while sharing some characteristics with neurites on pFN. Supplementation with GM1 or GT1b, but not GD1a, was inhibitory for neurite outgrowth in certain clones. Cycloheximide pretreatment distinguished several classes of clones based on inhibition of neuritogenesis. While most clones on pFN were unaffected, all clones on CTB and PF4 displayed significant and comparable degrees of inhibition, suggesting the sharing of some protein intermediate(s) on these substrata. Exposure to cycloheximide only during the active period of neuritogenesis generated higher percentages and longer neurites for all clones, indicating a widely-used negative regulation mechanism. Based on substratum type and cycloheximide protocols, these data have resolved six or more different transmembrane signalling processes for generating different classes of neurites. Some mechanisms have been segregated into individual clones while others overlap in other clones, providing cell systems for biochemical and molecular biological dissection of these processes.     

Intrasynaptosomal Sequestration of [3H]Amphetamine and [3H]Methylenedioxyamphetamine: Characterization Suggests the Presence of a Factor Responsible for Maintaining Sequestration

We examined the incorporation of [3H]methylenedioxyamphetamine ([3H]MDA) and [3H]amphetamine into rat brain synaptosomes. Saturation studies, using increasing concentrations of nonradioactive ligand, revealed that [3H]-MDA interacted with two saturable sites that were sensitive to boiling of the tissue. Eadee-Scatchard plots of [3H]MDA saturation data were curvilinear; nonlinear curve-fitting analysis of these data suggested the presence of high- and low-affinity [3H]MDA sites of association: KD high = 295 nM, Bmax high = 32 pmol/mg of protein; KD low = 45 microM, Bmax low = 5.2 nmol/mg of protein. Association of [3H]MDA to the low-affinity site was dependent on the presence of isotonic sucrose in the incubation medium. The high capacities of these sites argue against a bimolecular interaction of [3H]MDA with monovalent protein binding sites. [3H]MDA incorporation was reduced under conditions that disrupt the integrity of plasma membranes, such as sonication, incubation in hypotonic media, and incubation in the presence of the detergent digitonin. These data indicate that [3H]MDA incorporation into synaptosomes may represent an internalization and sequestration phenomenon. [3H]MDA incorporation was also reduced by preincubation of the synaptosomal preparation at 37 degrees C or in hypotonic buffer at 4 degrees C, a result suggesting that this sequestration is maintained by an intrasynaptosomal component that is lost under the preincubation conditions described above. [3H]MDA incorporation was pH dependent (maximal at pH 7.5) and temperature sensitive (maximal incorporation occurred at 21 degrees C and was substantially reduced at 37 degrees C). [3H]Amphetamine was also incorporated into synaptosomes, and this incorporation was sensitive to the same physical manipulations of the tissue preparation as [3H]MDA incorporation. The synaptosomal sequestration of both [3H]MDA and [3H]amphetamine was inhibited by permeant cations, such as sodium and potassium, a result suggesting that the proposed intrasynaptosomal component that maintains the sequestration is anionic. Preliminary pharmacological profiles of [3H]MDA and [3H]amphetamine sequestration were identical. The rank order of inhibitor potencies for the incorporation of both ligands was desipramine greater than amphetamine greater than MDA greater than methylphenidate. This order of potency does not correspond to the lipophilicity of the test drugs. The synaptosomal incorporation and sequestration of [3H]MDA, [3H]methylenedioxymethamphetamine, and [3H]amphetamine described in the present report may be important in the molecular mechanism of action of monoamine release induced by the amphetamines.     

Deep-level diagnostic value of the rDNA-ITS region

The similarity of certain reported angiosperm rDNA internal transcribed spacer (ITS) region sequences to those of green algae prompted our analysis of the deep-level phylogenetic signal in the highly conserved but short 5.8S and hypervariable ITS2 sequences. We found that 5.8S sequences yield phylogenetic trees similar to but less well supported than those generated by a ca. 10-fold longer alignment from rDNA-18S sequences, as well as independent evidence. We attribute this result to our finding that, compared to 18S, the 5.8S has a higher proportion of sites subject to vary and greater among-site substitution rate homogeneity. We also determined that our phylogenetic results are not likely affected by intramolecular compensatory mutation to maintain RNA secondary structure nor by evident systematic biases in base composition. Despite historical homology, there appears to be no ITS2 primary sequence similarity shared sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that groups, however, share sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that ITS region sequences can diagnose organismal origins and phylogenetic relationships at many phylogenetic levels and provide a useful paradigm for molecular evolutionary study.      

Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.