Mild oxidation of human serum low-density lipoprotein (LDL) converts the apoprotein from a nearly homogeneous component of high apparent molecular weight to a mixture of apparently lower mo ecular weight polypeptide components, as characterized by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis. This protein alteration, which correlates temporally with increases in the formation of lipid oxidation products and in the fluorescence of the apoprotein, is markedly reduced when oxygen is excluded or when EDTA or the free-radical-scavenging antioxidants, butylated hydroxytoluene or propyl gallate, are added. The conversion thus appears to be due to a reaction between the protein moiety and auto-oxidizing lipid. The presence of the antibacterial agent sodium azide markedly accelerates the oxidation, suggesting that it should only be used with caution in lipid-containing solutions. Structural and functional characterization of serum lipoproteins has received attention because molecular or metabolic abnormalities associated with this class of molecules might be significant in the development of atherosclerosis (1). The lowdensity lipoprotein fraction (LDL) has been of particular interest because of its apparent role in atherogenesis. Such a role is indicated by a correlation between high serum levels of LDL and clinical atherosclerosis (2), as well as the accelerated atherosclerosis in patients who have an inherited abnormality in LDL catabolism (familial hypercholesterolemia) (3). LDL is catabolized after it is bound through its protein moiety (apo-B or apo-LDL) to a cell-surface receptor (4).Although characterization of the apoprotein of LDL has been difficult (5), studies on the lipid moiety appear to be less controversial. LDL contains a large proportion of unsaturated lipids (somewhat variable on diet) (6) and is unique among the serum lipoproteins in its susceptibility for undergoing auto-oxidation in vitro (7-9). This finding has led some authors to speculate that such auto-oxidation plays a role in atherogenesis (7,10,11). Although structural (7-13), spectrophotometric (9,13,14), and lipid compositional (8, 11) changes in LDL have been observed upon oxidation, its polypeptide chain has not been characterized. Model studies with lipid-protein mixtures have shown that oxidized lipid can lead to major modification of protein components; such modifications include crosslinking (15), polypeptide scission (16), and loss of amino acids (17, 18). It thus seems possible that some of the difficulties encountered in characterizing apo-B are associated with partial oxidation of LDL during purification and/or storage. The observation that plasma lipids from human subjects contain significant diene conjugation (an early manifestation of lipid auto-oxidation) suggests that oxidation of LDL can be initiated in vivo (19).We show here that mild oxidation of LDL in vitro results in altering the sodium dodecyl sulfate (NaDodSO4)-apoprotein electrophoretic pattern from a single major band of low mobility into nume...

Carbamyl phosphate synthetase (from Escherichia coli) consists of a 7.3S protomeric unit that contains one heavy polypeptide chain (molecular weight about 130,000) and one light chain (molecular weight about 42,000). The heavy and light chains were separated by gel filtration in the presence of 1 M potassium thiocyanate. In contrast to the native enzyme and the reconstituted enzyme (prepared by mixing the separated heavy and light chains), the heavy chain does not catalyze glutamine-dependent carbamyl phosphate synthesis, although it does catalyze the synthesis of carbamyl phosphate from ammonia. The heavy chain also catalyzes two of the partial reactions catalyzed by the intact enzyme; i.e., the bicarbonate-dependent cleavage of ATP and the synthesis of ATP from ADP and carbamyl phosphate. Both positive (ammonia, ornithine, IMP) and negative (UMP) allosteric regulatory sites are located on the heavy chain. The only catalytic activity exhibited by the light chain is the hydrolysis of glutamine. A model is presented according to which glutamine binds to the light chain, which is followed by release of nitrogen from the amide group for use by the heavy chain. The findings suggest that glutamine-dependent carbamyl phosphate synthetase (and perhaps other glutamine amidotransferases) arose in the course of evolution by a combination of a primitive ammonia-dependent synthetic enzyme and a glutaminase; this combination may have been associated with a change from ammonia to glutamine as the principal source of nitrogen.Carbamyl phosphate, a metabolite of broad biological significance, is required for the biosynthesis of the pyrimidines and of the amino acid arginine. There are two types of carbamyl phosphate synthetases (1-3). One of these uses ammonia and requires N-acetylglutamate as a cofactor. The other can use both glutamine and ammonia and does not require N-acetylglutamate; glutamine is thought to be the "natural" substrate because it exhibits a higher affinity for the enzyme. Enzymes of the latter type resemble the other glutamine amidotransferases, which catalyze reactions in which the amide group of glutamine is used for various synthetic purposes; these enzymes can also use ammonia in place of glutamine (4).The glutamine-dependent carbamyl phosphate synthetase of Escherichia coli is subject to positive and negative feedback control by products of the purine and pyrimidine biosynthetic pathways, respectively (5), as well as by positive regulation by ornithine (6) and ammonia (unpublished It has recently been demonstrated that the enzyme can be dissociated into two kinds of polypeptide chains under extreme denaturing conditions*. We have independently made similar observationst, and now report that a relatively mild solvent perturbation promotes a reversible dissociation into nonidentical subunits. We were thus able to physically separate the two subunits and to compare their regulatory and catalytic properties with those of the recombined system and of the original enzyme.

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SummaryBackground: Since the introduction of exogenous factor VIII therapy, several studies have explored the clinical benefits of prophylactic use of factor VIII. Little research, though, has focused on the economic aspects of this regimen. We conducted a cost analysis using data from the Orthopedic Outcomes Study, a prospective, cross-national study of the clinical outcomes associated with different patterns of factor VIII utilization to examine the health care costs incurred and expenditures averted in patients receiving on-demand versus prophylactic use of factor VIII in hemophilia. Methods and Analysis: 831 patients with severe hemophilia aged 1 to 31 years, from 19 centers around the world were included in the cost analysis. Patients were categorized into three groups according to the number of weeks during the study years in which they received prophylactic regimens of factor VIII. For each subject, we estimated the costs of hospitalization, surgery, days lost from school or work, and factor VIII utilization. Costs were then stratified by age and by joint score to assess confounding, and a multivariate model developed to determine the relationship between use of factor VIII prophylaxis and total costs, while controlling for potential confounders.Results: Patients who received factor VIII episodically incurred substantially greater disability-related costs (days lost from school or work, days hospitalized due to hemophilia, surgery) than patients who received factor VIII prophylactically for some or all of the study period. For all treatment regimens, most disability-related costs were accounted for by hospitalization for hemophilia-related conditions. The cost of factor VIII itself was substantial in all treatment categories but was highest among patients who received year-round prophylaxis, exceeding the savings resulting from reduced disability and other health care expenditures.Conclusions: Reductions in non-factor health care costs and disability associated with prophylactic use of factor VIII in hemophilia were substantial and helped somewhat to offset the much higher costs of this regimen. For certain subgroups, frequent episodic treatment may be more expensive than full-time prophylaxis. However, because of the very high cost of year-round prophylactic use of factor VIII, total health care expenditures were highest among patients receiving this therapeutic regimen. However, because prophylaxis clearly offers important clinical benefits, this approach may be warranted on medical rather than economic grounds.

In the course of studies on glutaminedependent carbamyl phosphate synthetase from Aerobacter aerogenes, we purified another protein which was found to be glutamate synthase (EC 2.6.1.53). The enzyme, obtained in apparently homogeneous form (monomer molecular weight about 227,000; s2ow = 17.6 S), was found to be a typical glutamine amidotransferase in that it exhibits glutaminase activity and can utilize ammonia in place of glutamine as a nitrogen donor. Earlier work in this laboratory showed that glutamine-dependent carbamyl phosphate synthetase from Escherichia coli consists of a heavy subunit which can catalyze the synthesis of carbamyl phosphate from ATP, bicarbonate, and ammonia (but not from glutamine), and a light subunit which functions to bind glutamine (1, 2). In subsequent hybridization experiments with glutamine dependent carbamyl phosphate synthetase from Aerobacter aerogenes, it was found that the light subunit of the A. aerogenes enzyme could combine with the heavy subunit of the E. coli enzyme to form an active glutamine-dependent hybrid enzyme; similarly, an active hybrid enzyme could be prepared from the light subunit of the E. coli enzyme and the heavy subunit of the A. aerogenes enzyme.* In the course of isolating glutamine-dependent carbamyl phosphate synthetase from A. aerogenes, we noted during a gel filtration step in the procedure that another protein had also been purified (see first peak, Fig. 1). The catalytic properties of this protein were initially obscure, but we decided to pursue study of this apparently homogeneous protein after we found that it exhibited glutaminase activity and that it could be split to a heavy and a light subunit when subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate.These observations led us to think that the new protein might be a glutanmine amidotransferase, and after testing it for the activities of the several known enzymes in this category we discovered that the new protein is glutamate synthase (EC 2.6.1.53) whose major catalytic activity is to form glutamate from a-ketoglutarate and glutamine according to the following reaction (3-5): a-ketoglutarate + TPNH + H+ + L-glutamine 2 L-glutamate + TPN+ Several glutamine amidotransferases [e.g., carbamyl phosphate synthetase (1, 2), anthranilate synthetase (6), p-aminobenzoate synthetase (7) ] are composed of a heavy and a light subunit, and there is strong evidence in each case that the light subunit has the function of binding glutarmine; the amide nitrogen is then transferred to the heavy subunit for use in the corresponding synthesis reactions. We therefore expected that the light subunit of A. aerogenes glutamate synthase would contain the glutamine binding site of this enzyme. However, as described here, we found that the glutamine binding site of glutamate synthase is located on the heavy subunit. Miller and Stadtman (8,9) had previously shown that glutamate synthase from E. coli is an iron-sulfide flavoprotein. We have found that the A. aerogenes glutamate synthase is a...