Several molecular mechanisms for cleavage of the oxalate carbon-carbon bond by manganese-dependent oxalate decarboxylase have recently been proposed involving high oxidation states of manganese. We have examined the oxalate decarboxylase from Bacillus subtilis by electron paramagnetic resonance in perpendicular and parallel polarization configurations to test for the presence of such species in the resting state and during enzymatic turnover. Simulation and the position of the half-field Mn(II) line suggest a nearly octahedral metal geometry in the resting state. No spectroscopic signature for Mn(III) or Mn(IV) is seen in parallel mode EPR for samples frozen during turnover, consistent either with a large zero-field splitting in the oxidized metal center or undetectable levels of these putative high-valent intermediates in the steady state. A narrow, featureless g ‫؍‬ 2.0 species was also observed in perpendicular mode in the presence of substrate, enzyme, and dioxygen. Additional splittings in the signal envelope became apparent when spectra were taken at higher temperatures. Isotopic editing resulted in an altered line shape only when tyrosine residues of the enzyme were specifically deuterated. Spectral processing confirmed multiple splittings with isotopically neutral enzyme that collapsed to a single prominent splitting in the deuterated enzyme. These results are consistent with formation of an enzyme-based tyrosyl radical upon oxalate exposure. Modestly enhanced relaxation relative to abiological tyrosyl radicals was observed, but site-directed mutagenesis indicated that conserved tyrosine residues in the active site do not host the unpaired spin. Potential roles for manganese and a peripheral tyrosyl radical during steady-state turnover are discussed.The oxalate decarboxylase (OxDC, 1 or YvrK based on the corresponding open reading frame) from Bacillus subtilis has recently garnered attention because of its mechanistically intriguing reaction (1-4) (Scheme 1). Purified OxDC crystallizes as a hexamer of 43-kDa subunits, each of which is a member of the "bicupin" structural family (5), and contains two mononuclear manganese centers with amino acid ligands that are functionally similar to those of manganese superoxide dismutase (MnSOD) (6); namely three histidine residues and one carboxylic acid residue. Oxalate 13 C and 18 O isotope effects on V max /K m of YvrK suggest the presence of a slow, partially rate-determining step prior to the irreversible C-C bond cleavage (4), which was proposed to be a proton-coupled electron transfer.The requirement for and substoichiometric consumption of dioxygen in the YvrK catalytic process, involvement of open shell manganese (2), and chemical precedent for oxalate decarboxylation in the Kolbe electrolysis (7,8) and the BorodinHunsdiecker reaction (9 -11) have led to various mechanistic proposals involving radical intermediates, and enzymic manganese complexes with formal oxidation states of either Mn(III) (2, 4) or Mn(IV) (3). A previously published electron paramagneti...

A variant of apolipoprotein B has been observed in the lymph lipoproteins [chylomicrons, very low density lipoproteins (VLDL), and low density lipoproteins (LDL)J of rats, in the plasma VLDL of fed rats, and in the plasma VLDL and LDL of rats fed a high-fat, high-cholesterofdiet. It is the sole apolipoprotein B in the chylomicrons and VLDL of lymph. It differs from the apolipoprotein B of normal plasma LDL in its immunological properties and in its apparent molecular weight from electrophoresis on 3.5% NaDodSO,/polyacrylamide gel. The liver and the intestine are primarily responsible for producing the apolipoprotein components of the plasma lipoproteins (1-6). However, these two organs do not produce an identical spectrum of apolipoproteins. The liver apparently contributes most of the apolipoproteins C and E (7-9). On the other hand, both the liver and the intestine provide significant quantities of apolipoproteins A and B (7-12). It has been assumed that the structures of the apolipoproteins that originate in the intestine and first appear in lymph were very similar, if not identical, to the structures of corresponding apolipoprotein species found in the major plasma lipoproteins and presumed to be of hepatic origin. In this report we present evidence that the apolipoprotein B made by the rat intestine and incorporated into chylomicrons differs in a number of properties from the apolipoprotein B isolated as the major constituent of normal circulating plasma low density lipoprotein (LDL) in the rat. MATERIALS AND METHODSMale Sprague-Dawley rats, weighing 200-250 g, were maintained on Rockland-Purina Rat and Mouse Chow and water ad lib. For some experiments, rats were fasted at least 16 hr prior to the collection of blood or prior to gastric intubation and the collection of mesenteric duct lymph. Some animals were fed a semisynthetic diet containing 1% orotic acid (13) or a high-fat, high-cholesterol, sodium-cholate diet (13).Blood and lymph serum were adjusted to final concentrations of 0.05% EDTA (pH 7.0) and 0.02% sodium azide. Chylomicrons were removed by flotation in a Beckman L2-65 ultracentrifuge, SW 27 rotor (82,000 X g; 30 min). The remaining lipoprotein fractions were collected by ultracentrifugation in 1 mM EDTA/0.02% sodium azide at densities of 1.006 g/ml (very low density lipoprotein, VLDL), 1.019-1.063 g/ml (LDL), and 1.063-1.21 g/ml (high density lipoprotein, HDL), essentially as described (14). In some cases LDL was obtained in the density range 1.019-1.050 g/ml. Lipoprotein fractions were dialyzed at 40C against 0.01 M sodium phosphate buffer, pH 7.6/1 mM EDTA/0.02% sodium azide.Lymph was collected over a period of 24-48 hr from both normally fed and orotic acid-fed rats whose thoracic ducts wereThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. cannulated (15). Prior to lymph-duct cannulation, rats had been given 2 ml of corn ...

The lipoproteins isolated from rat plasma by flotation in the density range 1.019-1.063 g/ml were further characterized. Using rate zonal ultracentrifugation, we isolated two lipoproteins in almost equal proportions from this density range. Similar isolations may be accomplished with density gradients in a swinging-bucket rotor. On isopycnic-density-gradient ultracentrifugation one component banded at rho = 1.031 g/ml and the other at rho = 1.054 g/ml. More that 98% of the apoprotein of the lighter component was B protein, and hence this particle is LD (low-density) lipoprotein. Of the apoproteins of the rho = 1.054 g/ml particles, designated lipoprotein HDL1, over 60% was arginine-rich peptide, and the remainder was A-I, A-IV and C peptides. The molecular weight of these lipoproteins determined by agarose column chromatography was 2.36 x 10(6) for LD lipoprotein and 1.30 x 10(6) for lipoprotein HDL1. On electron microscopy the radius of LD lipoprotein was 14.0 nm and that of lipoprotein HDL1 was 10.0 nm, in contrast with molecular radii of 10.4 nm and 8.4 nm respectively determined from the gel-permeation-chromatography data. The lipid and phospholipid composition of both particles was determined. Lipoprotein HDL1 was notable for both the concentration of its esterified cholesterol, which was similar to that of LD lipoprotein, and the low triacylglycerol content, resembling that of HD lipoprotein. The possible origin of lipoprotein HDL1 is discussed.