Interactions between oil substrates and glucose on pure cultures of ruminal lipase-producing bacteria.

The hydrolysis of free fatty acids from lipids is a prerequisite for biohydrogenation, a process that effectively saturates free fatty acids. Anaerovibrio lipolyticus 5s and Butyrivibrio fibrisolvens have long been thought to be the major contributors to ruminal lipolysis; however, Propionibacterium avidum and acnes recently have been identified as contributing lipase activity in the rumen. In order to further characterize the lipase activity of these bacterial populations, each was grown with three different lipid substrates, olive oil, corn oil, and flaxseed oil (3 %). Because different finishing rations contain varying levels of glycogen (a source of free glucose) this study also documented the effects of glucose on lipolysis. P. avidum and A. lipolyticus 5s demonstrated the most rapid rates (P < 0.05) of lipolysis for cultures grown with olive oil and flaxseed oil, respectively. A. lipolyticus, B. fibrisolvens, and P. avidum more effectively hydrolyzed flaxseed oil than olive oil or corn oil, especially in the presence of 0.02 % glucose. Conversely, P. acnes hydrolyzed corn oil more readily than olive oil or flaxseed oil and glucose had no effect on lipolytic rate. Thus, these bacterial species demonstrated different specificities for oil substrates and different sensitivities to glucose.

Glycerol inhibition of ruminal lipolysis in vitro.

Supplemental glycerol inhibits rumen lipolysis, a prerequisite for rumen biohydrogenation, which is responsible for the saturation of dietary fatty acids consumed by ruminant animals. Feeding excess glycerol, however, adversely affects dry matter digestibility. To more clearly define the effect of supplemental glycerol on rumen lipolysis, mixed populations of ruminal bacteria were incubated with 6 or 20% glycerol (vol/vol). After 48-h anaerobic incubation of mixed culture rumen fluid, rates of free fatty acid production (nmol/mL per h) for the 6 and 20% glycerol-supplemented samples were decreased by 80 and 86%, respectively, compared with rates from nonsupplemented control cultures (12.4±1.0; mean ± SE). Conversely, assay of the prominent ruminal lipase-producing bacteria Anaerovibrio lipolyticus 5S, Butyrivibrio fibrisolvens 49, and Propionibacterium species avidum and acnes revealed no effect of 2 or 10% (vol/vol) added glycerol on lipolytic activity by these organisms. Supplementing glycerol at 6% on a vol/vol basis, equivalent to supplementing glycerol at approximately 8 to 15% of diet dry matter, effectively reduced lipolysis. However, the mechanism of glycerol inhibition of ruminal lipolysis remains to be demonstrated.

Side chain orientation in the selectivity filter of a voltage-gated Ca2+ channel.

Four glutamate residues (EEEE locus) are essential for ion selectivity in voltage-gated Ca(2+) channels, with ion-specific differences in binding to the locus providing the basis of selectivity. Whether side chain carboxylates or alternatively main chain carbonyls of these glutamates project into the pore to form the ion-binding locus has been uncertain. We have addressed this question by examining effects of sulfhydryl-modifying agents (methanethiosulfonates) on 20 cysteine-substituted mutant forms of an L-type Ca(2+) channel. Sulfhydryl modifiers partially blocked whole oocyte Ba(2+) currents carried by wild type channels, but this block was largely reversed with washout. In contrast, each of the four EEEE locus glutamate --> cysteine mutants (0 position) was persistently blocked by sulfhydryl modifiers, indicating covalent attachment of a modifying group to the side chain of the substituted cysteine. Cysteine substitutions at positions immediately adjacent to the EEEE locus glutamates (+/-1 positions) were also generally susceptible to sulfhydryl modification. Sulfhydryl modifiers had lesser effects on channels substituted one position further from the EEEE locus (+/-2 positions). These results indicate that the carboxylate-bearing side chains of the EEEE locus glutamates and their immediate neighbors project into the water-filled lumen of the pore to form an ion-binding locus. Thus the structure of the Ca(2+) channel selectivity filter differs substantially from that of ancestral K(+) channels.