Mechanism of action of bovine testicular hyaluronidase. Mapping of the active site.

The reactions of purified, homogeneous bovine testicular hyaluronidase have been studied with radioactively labeled oligomers of hyalobiuronic acid, (GlcUA-GlcNAc)n, as substrates and acceptors. Transglycosylation occurs by transfer of a glycosyl residue with retention of configuration from a leaving group to an acceptor. On the basis of detailed examination of cleavage and transglycosylation patterns for the trimer; comparison of trimer, tetramer, and polymer as substrates; comparison of acceptors; equilibrium binding; and other data, it is proposed that the enzyme's active site consists of five subsites for hyalobiuronate residues. In the terminology of Schechter, I., and Berger, A. ((1966) Biochemistry 5, 3371), these are s2-s1-s' 2-s3, where the reducing terminus is to the right, and cleavage occurs between s1 and s' 1. It is proposed that subsite s'2 has a high affinity for a substrate residue, while s1 and s'1 have low substrate affinity, and s2 and s' 3 are intermediate in affinity. This proposal is seen to have mechanistic implications. The reactions of several substrates show similar bell-shaped pH dependences, with optima in the region of pH 5 to 5.5.     

Molecular mapping of Or, a gene inducing beta-carotene accumulation in cauliflower (Brassica oleracea L. var. botrytis).

The cauliflower (Brassica oleracea L. var. botrytis) Or gene is a semi-dominant, single-locus mutation that induces the accumulation of high levels of beta-carotene in various tissues of the plant, turning them orange. As part of a map-based cloning strategy, molecular mapping of the Or gene in the cauliflower genome was undertaken in a mapping population consisting of 195 F2 individuals. By using amplified fragment length polymorphism (AFLP) in conjunction with bulked segregant analysis, we identified 10 AFLP markers closely linked to the Or gene. Four of the most closely linked flanking markers were converted into restriction fragment length polymorphism (RFLP) markers. Mapping of these markers in the mapping population placed two of them at 0.5 cM from the Or locus on one side, while another marker flanked the Or gene at 1.6 cM on the other side. Three of these markers were also successfully converted into sequence-characterized amplified region (SCAR) markers. These PCR-based markers will be useful for a large-scale application in facilitating the positional cloning of the Or gene.     

Effects of low-chloride solutions on action potentials of sheep cadiac purkinje fibers

The rapid repolarization during phase 1 of the action potential of sheep cardiac purkinje fibers has been attributed to a time- and voltage-dependent chloride current. In part, this conclusion was based on experiments that showed a substantial slowing of phase 1 when larger, presumably impermeant, anions were substituted for chloride in tyrode’s solution. We have re- examined the electrical effects of low-chloride solutions. We recorded action potentials of sheep cardiac purkinje fibers in normal tyrode’s solution and in low-chloride solutions made by substituting sodium propionate, acetylglycinate, methylsulfate, or methanesulfonate for the NaCl of Tyrode’s solution. Total calcium was adjusted to keep calcium ion activity of test solutions equal to that of control solutions. Propionate gave qualitatively variable results in preliminary experiments; it was not tested further. Low-chloride solutions made with the other anions gave much more consistent results: phase 1 and the notch that often occurs between phases 1 and 2 were usually unaffected, and the action potential duration usually increased. The only apparent change in the resting potential was a transient 3-6 mV depolarization when low-chloride solution was first admitted to the chamber, and a symmetrical transient hyperpolarization when chloride was returned to normal. If a time- and voltage-dependent chloride current exists in sheep cardiac purkinje fibers, our results suggest that it plays little role in generating phase 1 of the action potential.