cDNA clones for mouse parotid proline-rich proteins. mRNA regulation by isoprenaline and the nucleotide sequence of proline-rich protein cDNA MP5.

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 (MP5) 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 PGNQQGPPPQGGPQQ(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-gene-specific oligonucleotides established that MP3, MP4 and MP5 PRP mRNA were all induced by isoprenaline.

Characterization of tetanus toxin binding to rat brain membranes. Evidence for a high-affinity proteinase-sensitive receptor.

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.

Capping of cholera toxin-ganglioside GM1 complexes on mouse lymphocytes is accompanied by co-capping of alpha-actinin.

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 degrees 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-Ig complexes, requires two antibodies, and is inhibited at high toxin concentrations. Cholera toxin-GM1, like surface-Ig 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 alpha-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 alpha-actinin, and 57% of the cells that had a cholera toxin-GM1 cap displayed a co-cap of alpha-actinin. Time course studies revealed similar kinetics of external ligand cap formation and the formation of alpha-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.

Evidence for negative cooperativity in human erythrocyte sugar transport.

1. When D-glucose exchange influx is measure over a wide range of concentrations then two affinity constants (2.27 and 26.0 mM) are evident. This is consistent with a transport model (the allosteric pore model) in which there is negative cooperativity between subunits of the transport protein. 2. The equations for the allosteric pore model interacting with two substrates (or a substrate and an inhibitor) have been derived and have been used to analyse data from exchange inhibition and for mixed infinite-trans uptake experiments. 3. The exchange inhibition of tracer 3-O-methyl-D-glucose, D-xylose and D-fructose uptake by D-glucose also shows evidence for negative cooperativity and for two inhibition constants which are approximately equal to the D-glucose equilibrium exchange affinity constants. 4. The uptake of D-glucose into infinite-trans D-glucose or 3-O-methyl-D-glucose gives Km values of 2.6 and 2.33 mM, respectively. The uptake of 3-O-methyl-D-glucose into infinite-trans D-glucose or 3-O-methyl-D-glucose gives Km values of 6.0 and 4.6 mM, respectively. V values are slightly higher when the internal sugar is 3-O-methyl-D-glucose. 5. In cells that are treated with fluorodinitrobenzene the apparent Ki value for D-glucose inhibition of tracer D-fructose uptake is lowered. It is proposed that this is due to a partially selective effect of FDNB on the internal subunit interface stability constant (the internal pore gate).