We used cholera toxin, which binds exclusively and with a high affinity to the ganglioside GM1, as a probe to investigate the distribution of this glycolipid on the surface of mouse lymphocytes. When lymphocytes are incubated with cholera toxin (or its B subunit) and then sequentially with horse anti-toxin and FITC-swine anti-horse Ig at 37°C, the cholera toxin-ganglioside GM1 complex is redistributed to a cap at one pole of the cell. The capping of cholera toxin-GM1 complexes is slower than the capping of surface-lg complexes, requires two antibodies, and is inhibited at high toxin concentrations. Cholera toxin-GM, like surfaceIg capping, is an energy-dependent process and is inhibited by sodium azide, low temperatures, or cytochalasin B, but is unaffected by demecolcine. An affinity-purified antibody against ~-actinin was used to examine the distribution of this cytoskeletal component during the capping process. 88% of the cells that had a surface Ig cap displayed a co-cap of c~-actinin, and 57% of the cells that had a cholera toxin-GM~ cap displayed a co-cap of c~-actinin. Time course studies revealed similar kinetics of external ligand cap formation and the formation of ~-actinin co-caps. We conclude that capping of a cell-surface glycolipid is associated with a reorganization of the underlying cytoskeleton. The implications of such an association are discussed in the context of current models of the mechanism of capping.

Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialogangliosides were effective inhibitors of toxin binding (IC50 10 nM) and trisialogangliosides inserted into membranes lacking a toxin receptor were able to bind toxin with high affinity (KD 2.6 nM). The results are consistent with previous studies and the hypothesis that di- and trisialogangliosides act as the primary receptor for tetanus toxin under these conditions. In contrast, when toxin binding was assayed in Krebs-Ringer buffer, pH 7.4, binding was greatly reduced, was non-saturable and competition binding studies showed evidence for a small number of high-affinity sites (KD 0.42 nM, Bmax. 0.90 pmol/mg) and a larger number of low-affinity sites (KD 146 nM, Bmax. 179 pmol/mg). Treatment of membranes with proteinases, heat, and neuraminidase markedly reduced binding. Trisialogangliosides were poor inhibitors of toxin binding (IC50 11.0 microM), and trisialogangliosides inserted into membranes bound toxin with low affinity. The results suggest that in physiological buffers tetanus toxin binds with high affinity to a protein receptor, and that gangliosides represent only a low-affinity site.

cDNA clones for mRNA sequences regulated by isoprenaline in mouse parotid glands were identified by differential colony hybridisation and all hybridised to a diagnostic proline-rich protein (PRP) oligonucleotide. They were divided into two cross-hybridisation groups, A and B, which were shown by hybrid-selected translations to encode acidic PRP and basic PRP, respectively.The A-type subgroup consisted of sequences homologous to the previously identified mouse PRP genes MP2 and MP3. The B-type subgroup comprised clones for the previously identified cDNA pUMP125 (MP4) as well as other PRP sequences. Six of the B-type clones contained a novel PRP cDNA (MPS) and these were sequenced. The composite MP5 cDNA was 897 nucleotides long and contained an open reading frame capable of encoding a 260-residue-long salivary PRP precursor (30% Pro, 19% Gln and 18% Gly), containing nine variant repeat units of consensus PGNQQGP-PPQGGPQQ(GPP)R(PPQ). MP5 was 80% identical to the sequence of MP4 and had a high degree of similarity (60%) at its 3'-untranslated region to rat salivary glutamate/glutamine-rich protein (GRP) cDNA. Two MP5 clones contained a 273-bp intron-like insertion in the 3' untranslated region, being derived, therefore, from incompletely spliced MP5 transcripts.Northern blotting showed that, although PRP mRNA species were induced by isoprenaline, a B-type PRP mRNA was present in normal parotid glands. RNA dot-blots probed with PRP-genespecific oligonucleotides established that MP3, MP4 and MP5 PRP mRNA were all induced by isoprenaline.Multiple salivary proline-rich proteins (PRP) [l] are induced by dietary plant polyphenols in rats and mice [2, 31. PRP bind polyphenols and protect rats against the toxic effects of polyphenols [4]. The induction of salivary PRP can be mimicked by treatment of rodents with the P-adrenergic agonist isoprenaline [2, 31.The multiple PRP species are encoded by a multigene family and in humans both differential RNA splicing and proteolytic cleavages generate more than 20 PRP from only six genes [5].In mice, induction of PRP by isoprenaline is due, at least in part, to increased transcription of PRP genes in parotid acinar cells [6]. Isoprenaline activates P-adrenoceptors and indirect evidence suggests that CAMP is the intracellular messenger [7], although the precise details of how induction occurs are still undetermined.Evidence from in vitro translations shows that in Balb/C mice there are six PRP mRNA species which are induced by isoprenaline, two of which encode acidic or A-type PRP and four of which encode basic or B-type PRP [8]. There are also at least two distinct constitutive PRP [9].

No abstract

No abstract