Proteolysis of apoprotein B during the transfer of very low density lipoprotein from hens' blood to egg yolk

We report an example of the enzymic cleavage of an apoprotein B (apoB), the main apoprotein in the very low density lipoprotein (VLDL) of laying hens' blood, in a normal biological process, the formation of egg yolk. Plasma VLDL was labeled in vivo with 3H-amino acids, isolated by centrifuging, and injected into another laying hen. Yolk VLDL was isolated and its apoproteins were separated. ApoB was not detected in this lipoprotein. Most of the label originally in apoB was distributed among four smaller yolk apoproteins, apovitellenins III to VI, which are a large proportion of the apoproteins of VLDL in yolk. This distribution of 3H suggested that 80% of apoB was cleaved at three places. One yolk apoprotein, apovitellenin II, was not labeled, indicating that it did not originate from an apoprotein in plasma VLDL. The site for cleavage of apoB in the ovarian tissue has not been determined, but cleavage may occur during receptor-mediated endocytosis. The pattern of cleavage of apoB during transfer to yolk was not imitated by some known proteolytic enzymes.     

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.     

Comparison of the nucleotide sequence of cloned DNA coding for an apolipoprotein (apoVLDL-II) from avian blood and the amino acid sequence of an egg-yolk protein (apovitellenin I): equivalence of the two sequences

We have compared the amino acid sequences of two low-molecular-weight avian apoproteins: apoVLDL-II from very low-density lipoproteins of hen plasma and apovitellenin I from hen egg yolk. The sequence of White Leghorn apoVLDL-II was derived from the nucleotide sequence of cloned apoVLDL-II DNA (Chan et al., 1980). The sequenator was used to determine the amino acid sequence of apovitellinin I from two breeds of hen (White Leghorn and Australorp). The sequences from the two breeds were not only identical, but they also completely matched the predicted sequence derived from the apoVLDL-II DNA sequence. The identity reported here establishes that this protein is transported intact from the blood to the egg yolk.     

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.     

Structure determination and refinement of bovine lens leucine aminopeptidase and its complex with bestatin.

The three-dimensional structure of bovine lens leucine aminopeptidase (EC 3.4.11.1) complexed with bestatin, a slow-binding inhibitor, has been solved to 3.0 A resolution by the multiple isomorphous replacement method with phase combination and density modification. In addition, this structure and the structure of the isomorphous native enzyme have been refined at 2.25 and 2.32 A resolution, respectively, with crystallographic R-factors of 0.180 and 0.159, respectively. The current structural model for the enzyme includes the two zinc ions and 481 of the 487 amino acid residues comprising the asymmetric unit. The enzyme is physiologically active as a hexamer, which has 32 symmetry, and is triangular in shape with a triangle edge length of 115 A and maximal thickness of 90 A. Monomers are crystallographically equivalent. Each is folded into two unequal alpha/beta domains connected by an alpha-helix to give a comma-like shape with approximate maximal dimensions of 90 A x 55 A x 55 A. The secondary structural composition is 35% alpha-helix and 23% beta-strand. The N-terminal domain (160 amino acid residues) mediates trimer-trimer interactions and does not appear to participate directly in catalysis, while the C-terminal domain (327 amino acid residues) is responsible for catalysis and binds the two zinc ions, which are less than 3 A apart. These two metal ions are located near the edge of an eight-stranded, saddle-shaped beta-sheet. The zinc ion that has the lower temperature factor is co-ordinated by one carboxylate oxygen atom from each of Asp255, Asp332 and Glu334, and the carbonyl oxygen of Asp332. The other zinc ion, presumed to be readily exchangeable, is co-ordinated by one carboxylate oxygen atom of each of Asp273 and Glu334 and the side-chain amino group of Lys250. The active site also contains two positively charged residues, Lys262 and Arg336. The six active sites are themselves located in the interior of the hexamer, where they line a disk-shaped cavity of radius 15 A and thickness 10 A. Access to this cavity is provided by solvent channels that run along the 2-fold symmetry axes. Bestatin binds to one of the active site zinc ions, and its phenylalanine and leucine side-chains occupy hydrophobic pockets adjacent to the active site. Finally, the relationship between bovine lens leucine aminopeptidase and the homologous enzyme pepA from Escherichia coli is discussed.     

Exploration of disorder in protein structures by X-ray restrained molecular dynamics

Conformational disorder in crystal structures of ribonuclease-A and crambin is studied by including two independent structures in least-squares optimizations against X-ray data. The optimizations are carried out by X-ray restrained molecular dynamics (simulated annealing refinement) and by conventional least-squares optimization. Starting from two identical structures, the optimizations against X-ray data lead to significant deviations between the two, with rms backbone displacements of 0.45 A for refinement of ribonuclease at 1.53 A resolution, and 0.31 A for crambin at 0.945 A. More than 15 independent X-ray restrained molecular dynamics runs have been carried out for ribonuclease, and the displacements between the resulting structures are highly reproducible for most atoms. These include residues with two or more conformations with significant dihedral angle differences and alternative hydrogen bonding, as well as groups of residues that undergo displacements that are suggestive of rigid-body librations. The crystallographic R-values obtained are approximately 13%, as compared to 15.3% for a comparable refinement with a single structure. Least-squares optimization without an intervening restrained molecular dynamics stage is sufficient to reproduce most of the observed displacements. Similar results are obtained for crambin, where the higher resolution of the X-ray data allows for refinement of unconstrained individual anisotropic temperature factors. These are shown to be correlated with the displacements in the two-structure refinements.