Bile salt deconjugation and cholesterol removal from media by Lactobacillus casei.

Lactobacillus casei N19 and E5 and Lactobacillus acidophilus L1 and ATCC 43121 were compared for their ability to deconjugate bile salts and remove cholesterol from MRS broth during growth at pH 6.0 and during growth without pH control. Samples grown without pH control dropped to pH 4.2 to 4.5 during 20 h of incubation, depending on the culture. The plate counts indicated that populations in all cultures were near their maximum numbers after 16 h of growth. The amount of cholesterol removed from the broth was similar for both strains of L. acidophilus grown with and without pH control. However, the strains of L. casei differed significantly in the amount of cholesterol removed during growth with or without pH control. Both cultures of L. casei that were grown at pH 6.0 removed very little cholesterol from the broth, but cells grown without pH control removed up to 60 micrograms of cholesterol/ml. All cultures of both species deconjugated 60 to 90% of the bile salts. Lactobacillus acidophilus L1 was the only culture to demonstrate differences between the two pH treatments in the amount of bile salts deconjugated; however, there was no difference in the amounts of cholesterol removed. These results indicate that most of the cholesterol removal from broth by L. acidophilus was due to assimilation, perhaps by the incorporation of cholesterol into the cellular membrane. Lactobacillus casei most likely removes cholesterol from broth by means of the destabilization of cholesterol micelles and the coprecipitation of the cholesterol with the deconjugated bile salts at pH less than 6.0.

Cloning and sequencing of a cDNA for firefly luciferase from Photuris pennsylvanica.

The first cDNA from the Photurinae subfamily of the Lampyridae encoding a firefly luciferase from lantern mRNA of Photuris pennsylvanica has been cloned, sequenced, the amino-acid sequence predicted and the sequence reported to GenBank. The cDNA was about 1.8 kb in length with the largest open reading frame coding for a 545-residue protein. The 5' noncoding region is 61 bp long and the 3' noncoding region is 135 bp in length. There is a 24-nucleotide poly(A) tail. When the amino-acid residues are aligned, P. pennsylvanica contains 154 (about 28% of the total residues) that are conserved in all 16 of the deduced luciferase sequences that are presently available. In this P. pennsylvanica luciferase, the amino acids at 276 of the positions are the same at corresponding positions of at least one of the other enzymes. There are two amino-acid differences between this luciferase and the unpublished sequence obtained by Dr. Keith Wood for a putative larval Photuris firefly luciferase cloned from a Maryland firefly. Signature amino-acid sequences and domains found in the deduced sequence are for adenylate kinase, the putative AMP-binding domain, luciferin 4-monooxygenase, 4-coumarate CoA ligase, long-chain fatty acid CoA ligase, 2-acylglycerophosphoethanolamine acyltransferase, the microbody-directing sequence, peptide-synthesizing complexes, and acyladenylate-synthesizing enzymes.

Use of firefly luciferase for ATP measurement: other nucleotides enhance turnover.

Firefly luciferase utilizes only ATP and a few closely related nucleotides as substrates for the formation of luciferyl adenylate which is an intermediate in the bioluminescent reaction sequence that oxidizes firefly luciferin. The enzyme shows two different time courses of light production depending on ATP concentration used: a flash with high concentrations of ATP (> 8 microM) or a fairly constant production of light with lower concentrations of ATP (< 1 microM). Many nucleotides, nucleotide-containing substances and other compounds, when added either prior to or 1 min after the addition of ATP, change the time course of light production. When added before ATP, these compounds yield a reaction mixture in which light production is fairly constant (at the level characteristic of the flash observed with that ATP concentration). When the compounds are added after ATP addition, light production is markedly stimulated and the higher rate of light production is maintained for several minutes. There is an increase in quanta of light produced per luciferase dimer from 1 to 5/min with the addition of any of several nucleotide analogues. These results are consistent with a stimulated release of the inhibitory product oxyluciferin, allowing turnover of the enzyme. This enzyme turnover permits more light output at high ATP concentrations, thus enhancing the sensitivity of enzyme determination.

Does the sulfhydryl or the adenine moiety of CoA enhance firefly luciferase activity?

Light production by firefly luciferase is limited by product release resulting in flash kinetics. Several compounds (CoA, PPi, and nucleotides) transform the flash-form of light production into continuous light production. The sulfhydryl group of CoA is required; however, since nucleotides are also active, at least two mechanisms (sites) must exist.

Comparisons of freshly isolated strains of Lactobacillus acidophilus of human intestinal origin for ability to assimilate cholesterol during growth.

Fecal isolates of Lactobacillus acidophilus were obtained from human volunteers and tested for bile tolerance, the ability to deconjugate bile salts, and the ability to assimilate (take up) cholesterol during growth. One hundred and twenty-three of the 304 isolates of lactobacilli obtained were identified as L. acidophilus. In most cases, isolates of L. acidophilus from the same volunteer varied significantly in the amount of cholesterol assimilated, bile salt deconjugated, and bile tolerance. The two cultures from each of nine volunteers that assimilated the most cholesterol were compared as a group to select the most active cultures. Lactobacillus acidophilus ATCC 43121 (an isolate from the intestines of a pig, which in an earlier study aided significantly in controlling serum cholesterol in pigs) was included in this comparison. Significant variation in the ability to assimilate cholesterol was observed among these isolates from different volunteers. Eight of 17 isolates assimilated numerically but not significantly more cholesterol than L. acidophilus ATCC 43121, and 4 isolates assimilated significantly less. Bile tolerance and bile salt deconjugation also varied significantly among the selected isolates. Six of the selected isolates were quantitatively but not significantly better able to deconjugate bile salts than L. acidophilus ATCC 43121, but none was significantly more bile tolerant. Based on characteristics tested, isolates B7, D3, L1, 016, and 017 have the most potential of those included in this study for use as dietary adjuncts to lower human serum cholesterol.