Lipid-protein globules of avian egg yolk. Isolation and properties of globules stable in concentrated sodium chloride solution.

A new type of globular particle, the 'insoluble yolk globule', was isolated from the egg yolk of three avian species (hen, duck, and emu) by centrifugation or gel-filtration chromatography. These globules are stable in NaCl and urea solutions at concentrations that dissolve or disrupt other constituents of yolk, The isolated globules are about 1% of the dry yolk of hen's and duck's eggs but about 8% emu's-egg yolk. Most of these globules are less than 2 micrometer in diameter. Electron micrographs of sections show a preponderance of globules in the range 0.125-0.25 micrometer, each with a thick shell surrounding a feature-less anterior. Globules with the same appearance were seen in sections of unfractionated yolk. Two kinds of larger particles were also observed: (i) particles with a distinct outer membrane and a vesiculated interior; (ii) featureless spheres, possibly of lipid. The insoluble yolk globules comprise protein (8-11% by dry wt.), phospholipid (31-35% total lipid), triacylglycerols (49-53%), cholesterol (8%) and cholesteryl esters (2-3%); the variations being among species. The phospholipid is accessible to phospholipase C. The isolated protein is heterogeneous and resembles the apoprotein from the yolk low-density lipoprotein.     

Structural variation in bacterial glyoxalase I enzymes: investigation of the metalloenzyme glyoxalase I from Clostridium acetobutylicum

The glyoxalase system catalyzes the conversion of toxic, metabolically produced α-ketoaldehydes, such as methylglyoxal, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the glyoxalase system, glyoxalase I (GlxI), from yeast, protozoa, animals, humans, plants, and Gram-negative bacteria, have suggested two metal activation classes, Zn(2+) and non-Zn(2+) activation. Here, we report a biochemical and structural investigation of the GlxI from Clostridium acetobutylicum, which is the first GlxI enzyme from Gram-positive bacteria that has been fully characterized as to its three-dimensional structure and its detailed metal specificity. It is a Ni(2+)/Co(2+)-activated enzyme, in which the active site geometry forms an octahedral coordination with one metal atom, two water molecules, and four metal-binding ligands, although its inactive Zn(2+)-bound form possesses a trigonal bipyramidal geometry with only one water molecule liganded to the metal center. This enzyme also possesses a unique dimeric molecular structure. Unlike other small homodimeric GlxI where two active sites are located at the dimeric interface, the C. acetobutylicum dimeric GlxI enzyme also forms two active sites but each within single subunits. Interestingly, even though this enzyme possesses a different dimeric structure from previously studied GlxI, its metal activation characteristics are consistent with properties of other GlxI. These findings indicate that metal activation profiles in this class of enzyme hold true across diverse quaternary structure arrangements.     

Stimulation of 16-dehydroprogesterone and progesterone reductases of Eubacterium sp. strain 144 by hemin and hydrogen or pyruvate.

Suspensions of Eubacterium sp. strain 144, prepared from cells grown with 16-dehydroprogesterone, catalyzed the reduction of this steroid to 17-isoprogesterone at a very low rate. Modifications of the assay to optimize the pH (5.5) and increase the steroid solubility (10% [vol/vol] methanol) did not significantly enhance the reaction. However, growth of strain 144 in the presence of hemin was found to stimulate 16-dehydroprogesterone reductase during the initial 30 min of incubation, giving a biphasic time course. These biphasic kinetics could be eliminated by providing the cells with an exogenous electron donor. Strain 144 used either H2 or pyruvate for this purpose, and 17-isoprogesterone formation was nearly complete after 20 to 30 min of incubation. However, under these conditions, strain 144 further converted 17-isoprogesterone to products which lacked UV absorbance (254 nm). When progesterone was used as a substrate, it was found that strain 144 could reduce the C4-C5 double bond of this steroid by a progesterone reductase to give mostly 5 beta-pregnadione and some 5 alpha-pregnadione. Furthermore, the 3-keto group of 5 beta-pregnadione steroid was also reduced to a hydroxy function. The maximum activities of both 16-dehydroprogesterone and progesterone reductases in cell suspensions required the growth of strain 144 with hemin and 16-dehydroprogesterone and the presence of H2 or pyruvate.