Thomas F. Uchytil

The stimulation of coupling factor 1 ATPase by tentoxin

Tentoxin at 10--1000 micrometer causes a marked species-selective stimulation of coupling factor 1 Ca2+-dependent ATPase activity (Ka 6.3 . 10(3) M-1). This effect decreases the Km for ATP to about 0.3 mM and increases V 2.75-fold. Above 1.6 micrometer tentoxin the rate of coupled electron transport was reduced to basal without uncoupling.

Tentoxin An uncompetitive inhibitor of lettuce chloroplast coupling factor 1

The interaction of tentoxin [cyclo-(-L-leucyl-N-methyl-(Z)-dehydrophenylalanyl-glycyl-N-methyl-L-alanyl-)] with solubilized lettuce chloroplast coupling factor 1 was characterized by direct binding studies, measurement of the time course of ATPase inhibition, and steady-state enzyme kinetics. Neither substrates, products or Ca2+ competed with the tentoxin binding site, nor did they induce any large change in tentoxin affinity. The inhibition of lettuce chloroplast coupling factor 1 ATPase was found to be the time dependent, and at equilibrium the affinities estimated by equilibrium ultrafiltration and enzyme inhibition were similar (1.8 . 10(8) M-1). The steady-state kinetics best fit an uncompetitive pattern suggesting that the inhibited steps follow an irreversible step occurring after ATP binding.

Recombination of Solanum brevidens chromosomes in the second backcross generation from a somatic hybrid with S. tuberosum

Solanum brevidens synteny groups were examined with 47 widely-distributed RFLP markers in 17 BC2 progeny from six fertile BC1 plants. The BC1 plants were derived from a single S. brevidens + S. tuberosum somatic hybrid backcrossed with S. tuberosum (potato). Probes which were linked in potato and tomato were also found to be syntenic along each of the 12 S. brevidens chromosomes. More than half of the S. brevidens synteny groups had lost one or more S. brevidens-specific RFLPs in the BC2, suggesting that recombination had occurred. For 8 of the 12 S. brevidens RFLP synteny groups, the frequency of recombinant chromosomes exceeded that of intact parental chromosomes. Using the RFLP data, 161 RAPD markers were tentatively located throughout the S. brevidens genome. Further analyses with 39 of these 161 RAPD markers generally showed that RAPD and RFLP results were comparable, but some inconsistencies were noted with 14 of the 39 RAPD markers. The extent of marker loss and the high frequency of synteny groups which were marked by a single S. brevidens-specific RFLP marker suggest that the S. brevidens chromosomes have some pairing affinity with potato chromosomes. This interaction should facilitate the transfer of novel disease-resistance traits into potato breeding lines. One plant was recovered with the chromosome number of S. tuberosum (2n=48) that carried a single S. brevidens RFLP marker, suggesting transfer of this S. brevidens marker into the genome of S. tuberosum.

Introgression and stabilization oferwinia tuber soft rot resistance into potato after somatic hybridization ofsolanum tuberosum ands. brevidens

Resistance to potato tuber soft rot caused byErwinia carotovora was transferred fromSolanum brevidens to the cultivated potato over the course of four backcross generations originating from a somatic hybrid. Soft rot reactions were determined via a tuber plug inoculation method developed during the course of these experiments. Soft rot resistance was highest in the somatic hybrid (only ca. 20% of tubers and plugs showed evidence of severe rotting) and lowest among progeny of control potato x potato crosses (ca. 80% of tuber plugs showed severe rot). Backcross generations involving somatic hybrids were intermediate in their reaction, and resistance stabilized to about 60% of tuber plugs showing severe rot in the BC2 through the BC4. Reciprocal crosses showed no difference in the inheritance of soft rot resistance, indicating that neitherS. brevidens norS. tuberosum donor cytoplasm had a significant effect on the expression of resistance. Crosses between BC3 siblings where noS. brevidens genetic markers were detected but resistance was segregating demonstrated a dosage effect for soft rot resistance. We conclude that introgression of soft rot resistance has occurred and that at least one locus responsible for resistance inS. brevidens now resides in theS. tuberosum genome.

The binding of tentoxin to a tryptic digest of chloroplast coupling factor 1

Abstract The binding of tentoxin to lettuce chloroplast coupling factor 1 and its inhibition of Ca +2 -dependent ATPase involves the α and β subunits which remain after trypsin treatment. The tentoxin-binding properties of the digest are not greatly altered from those previously reported for the untreated protein.

Chromatographic amino acid analysis of protein hydrolysed in polyacrylamide after removal of ammonia

SummaryA simple method for the removal of NH3 from amino acids is presented. The method is based on a cation-exchange resin from which amino acids are eluted with NH4OH. The eluent is then removed under reduced pressure. The method allows the ninhydrin-based detection of amino acids after hydrolysis of stained protein bands in polyacrylamide gels. This was previously not possible since NH3 produced by the hydrolysis of polyacrylamide interferes with the ninhydrin-detection of basic amino acids. The method should also be applicable to the detection of amino acids with o-phthalaldehyde.