High-level expression of three members of the murine angiogenin family in Escherichia coli and purification of the recombinant proteins.

Angiogenin (Ang) is a small basic protein which belongs to the pancreatic ribonuclease superfamily. It potently induces the formation of new blood vessels and has emerged as a promising anticancer target. Mice possess genes encoding one ortholog (mAng) and three homologs of Ang, designated angiogenin-related protein (mAngrp), angiogenin-3 (mAng-3), and angiogenin-4 (mAng-4). Structural and functional study of these homologs has been hampered by the low yield of protein from the existing heterologous expression system. In the experiments described, we used a pET expression vector to express these proteins in the cytoplasm of Escherichia coli BL21-CodonPlus(DE3)-RIL cells, whereupon substantial amounts of each accumulated in the form of insoluble aggregates. The proteins were renatured using an arginine-assisted procedure and subsequently purified by cation-exchange chromatography and reversed-phase HPLC; each purified protein was shown to be enzymatically active toward tRNA. The yields of pure mAngrp and mAng-3 were 7.6 and 12 mg/liter culture, respectively, representing substantial increases over previously reported experiments. This is also the first report of the expression and purification of mAng-4, obtained here in a yield of 30 mg/liter culture. The ready availability of milligram quantities of these proteins will enable further functional studies and high-resolution structural analyses to be conducted.

Technical advance: application of high-performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids.

The application of high-performance liquid chromatography (HPLC) using a C30 reverse-phase stationary matrix has enabled the simultaneous separation of carotenes, xanthophylls, ubiquinones, tocopherols and plastoquinones in a single chromatogram. Continuous photodiode array (PDA) detection ensured identification and quantification of compounds upon elution. Applications of the method to the characterization of transgenic and mutant tomato varieties with altered isoprenoid content, biochemical screening of Arabidopsis thaliana, and elucidation of the modes of action of bleaching herbicides are described to illustrate the versatility of the procedure.

Isomerization of dietary lycopene during assimilation and transport in plasma.

Diets of individuals were supplemented with tomatoes, either cooked or as tomato pureé in order to compare uptake of lycopene from intact and homogenized fruit tissue matrices. Following a diet containing cooked tomatoes over three consecutive 7-day periods, little change in the carotenoid levels in plasma lipoproteins occurred. In contrast, a diet supplemented with concentrated tomato pureé, over a 2 week period, caused a significant (p < 0.05) increase in lycopene levels in plasma, showing that the lycopene within intact cells is less bioavailable than that from processed tissue. The isomeric composition of plasma lycopene was significantly different to that of the ingested pureé. A number of cis-isomers (predominantly 5-cis, 13-cis and 9-cis-) were detected in plasma, that are not present in the lycopene from pureé. The significance of the increase in lycopene following dietary supplementation with respect to bioavailability and the causes of isomerization are discussed.

Adenosylcobalamin-dependent glutamate mutase: properties of a fusion protein in which the cobalamin-binding subunit is linked to the catalytic subunit.

Adenosylcobalamin-dependent glutamate mutase (EC 5.4.99.1) from Clostridium tetanomorphum comprises two protein components, MutE and MutS. The formation of the holoenzyme is a kinetically complex process that involves the co-operative association of MutS, MutE and adenosylcobalamin. The MutS portion of the cobalamin-binding site is conserved within a group of adenosylcobalamin-dependent enzymes that catalyse similar isomerizations. However, in contrast with glutamate mutase, in these other enzymes the cobalamin-binding region represented by MutS is present as a C-terminal domain. We have investigated the effect on the structural and kinetic properties of glutamate mutase of linking MutS to the C-terminus of MutE. Kinetic analysis of this protein, MutES, showed, unexpectedly, that enzyme activity was still co-operatively dependent on protein concentration. The Km for L-glutamate was unchanged from the wild type, whereas Vmax was decreased to approx. one-thirtieth and the Km for coenzyme increased approx. 10-fold. Investigation of the quaternary structure of MutES by equilibrium ultra-centrifugation indicated that the protein existed in equilibrium between monomeric and dimeric forms. Thus linking MutE and MutS together seems to substantially weaken the contacts that are responsible for the dimerization of MutE. The two domains of the MutES monomer seem unable to communicate, so that active enzyme is formed by the intermolecular association of two MutES subunits in a co-operative manner.

Carboxymethylation of MutS-cysteine-15 specifically inactivates adenosylcobalamin-dependent glutamate mutase. Examination of the role of this residue in coenzyme-binding and catalysis.

The sensitivity of adenosylcobalamin (AdoCbl)-dependent glutamate mutase toward thiol-directed reagents has been investigated. Iodoacetate specifically alkylates one cysteine residue, Cys-15, in MutS with concomitant irreversible loss of enzyme activity. Cys-15 lies between the conserved residues Asp-14 and His-16, that are believed to coordinate cobalt to form a Co-His-Asp hydrogen-bonded "triad" when AdoCbl is bound by the enzyme. Although inactive, carboxymethylated MutS still bound AdoCbl with only a 5-fold increase in apparent Kd. To determine whether Cys-15 plays an essential role in catalysis, it was mutated to serine and to alanine. These mutants were active, but both exhibited decreased Vmax and increased apparent Km and Kd for AdoCbl. To mimic the effect of carboxymethylation, Cys15 was mutated to aspartate and, as an isosteric control, to asparagine. Neither of these mutants was active: MutS-C15N bound AdoCbl approximately 10-fold weaker than wild type, whereas MutS-C15N bound AdoCbl over 100 times less strongly than wild type. The results demonstrate both coenzyme-binding and catalysis to be very sensitive to mutations at position 15 that could potentially perturb the Co-His-Asp hydrogen-bonding network.

Adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum. Overexpression in Escherichia coli, purification, and characterization of the recombinant enzyme.

The genes encoding both components, MutE and MutS, of adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum have been over-expressed in Escherichia coli. This has allowed MutE to be obtained in homogeneous form, free of inhibiting cobamides and traces of MutS. MutE binds MutS cooperatively, with a Hill coefficient of 1.3. The recombinant enzyme has an unchanged Km for L-glutamate, but a much higher specific activity than those previously reported for preparations from clostridia. The apparent Km for adenosylcobalamin was dependent upon the concentration of MutS and varied between 18 microM with equimolar concentrations of MutS and MutE and 5.8 microM with a 5-fold molar excess of MutS over MutE present in the assay. The dissociation constant for adenosylcobalamin was measured directly using equilibrium gel filtration. In the presence of equimolar amounts of MutE and MutS, the apparent Kd was 5.4 microM, but this decreased to 1.8 microM when MutS was present at a 5-fold molar excess. No binding of adenosylcobalamin to MutE was observed in the absence of MutS. This suggests that the (minimal) function for MutS, whose role in the reaction has been unclear until now, is to form part of the adenosylcobalamin-binding site. It seems likely that MutS is representative of a cobalamin-binding domain conserved across several cobalamin-dependent enzymes.

Cloning and sequencing of glutamate mutase component E from Clostridium tetanomorphum. Organization of the mut genes.

The gene encoding component E, the large subunit, of adenosylcobalamin (coenzyme B12)-dependent glutamate mutase from Clostridium tetanomorphum has been cloned and sequenced. The mutE gene encodes a protein of 485 amino acid residues, with M(r) 53,708. The mutE gene is situated some 1,400 bp downstream of the mutS gene, which encodes the small subunit of glutamate mutase. Between the two is an open reading frame encoding a protein of 462 amino acids, with M(r) 50,171, and of unknown function. All three genes appear to be transcribed as an operon and lie immediately upstream of the gene encoding beta-methylaspartase, the next enzyme in the pathway of glutamate fermentation. Local homology exists between mutE and a region of beta-methylaspartase which contains an active-site serine residue.

Cloning and sequencing of glutamate mutase component S from Clostridium tetanomorphum. Homologies with other cobalamin-dependent enzymes.

The gene encoding component S, the small subunit, of glutamate mutase, an adenosylcobalamin (coenzyme B12)-dependent enzyme from Clostridium tetanomorphum has been cloned and its nucleotide sequence determined. The mutS gene encodes a protein of 137 amino acid residues, with M(r) 14,748. The deduced amino acid sequence showed homology with the C-terminal portion of adenosylcobalamin-dependent methylmalonyl-CoA mutase [1989, Biochem. J. 260, 345-352] and a region of cobalamin-dependent methionine synthase which has been shown to bind cobalamin [1989, J. Biol. Chem 264, 13888-13895].

Long-term effects of inadequate and excessive dietary ascorbate on bile acid metabolism in the guinea pig.

The effects of long-term chronic ascorbic acid deficiency and excessive ascorbic acid consumption on bile acid metabolism and biliary lipid composition were studied in guinea pigs. Male, weanling guinea pigs were fed a cereal-based scorbutigenic diet for 19 or 21 weeks. Ascorbic acid was administered either orally at 0.15 (group A) or 2.0 (group B) mg/100 g body weight, or it was mixed in the diet at levels of 500 (group C), 16-22 (group D), or 20,000 mg/kg (group E). Chronic ascorbic acid deficiency (groups A and D) caused depression of hepatic cytochrome P-450 levels and elevation of plasma cholesterol. Excessive ascorbate consumption did not alter these parameters relative to control levels. In contrast to results obtained in guinea pigs fed low or high amounts of ascorbate for 7-9 weeks, prolonged consumption of inadequate or excessive ascorbate resulted in little or no change in bile acid metabolism and biliary lipid composition except that bile acid pool size was increased 12% as a result of excessive ascorbate ingestion. Results of the present study suggest that there may be important differences in the guinea pig's metabolic response to ascorbic acid deficiency and ascorbic acid excess, depending on the length of the experimental period.

Influence of dietary ascorbic acid on cholesterol 7 alpha-hydroxylase activity in the rat.

An experiment was carried out to determine whether dietary excess of ascorbic acid inhibits cholesterol 7 alpha-hydroxylase activity and elevates cholesterol levels in the rat, as previously observed in the guinea pig. Male, weanling Sprague-Dawley rats were fed a cereal-based diet supplemented with 0, 0.5, 10.0, or 20.0 g ascorbate/kg for 45 days. Ascorbate supplementation did not alter plasma ascorbate levels in the rat, but did increase hepatic ascorbate at the highest dietary intakes (10.0 and 20.0 g ascorbate/kg diet). Ascorbate supplementation had no effect upon plasma and liver cholesterol levels or cholesterol 7 alpha-hydroxylase activity. Under the experimental conditions employed, the rat appears resistant to ascorbate-induced alteration of sterol metabolism.

Dietary ascorbic acid and hepatic mixed function oxidase activity in the guinea pig.

Studies were carried out to characterize the response of hepatic mixed function oxidase (MFO) activity to chronic ascorbic acid deficiency and excessive ascorbic acid intake in the guinea pig. When guinea pigs were fed excessive ascorbic acid, there was a small increase in hepatic cytochrome P-450 which was unaccompanied by any alteration in drug-metabolizing enzyme activity. Similarly, induction of MFO activity by phenobarbital was not modified by excessive ascorbic acid administration. Chronic ascorbic acid deficiency resulted in depressed metabolism of aniline, aminopyrine, ethoxycoumarin and benzphetamine, but not of ethylmorphine, in comparison with animals fed diets containing control and/or excessive amounts of ascorbic acid. In contrast to the metabolism of all drugs studied, the 7 alpha-hydroxylation of cholesterol was depressed by both inadequate and excessive vitamin C intake, demonstrating the unique sensitivity of cholesterol 7 alpha-hydroxylase to dietary ascorbate.

Human T-cell function in experimental ascorbic acid deficiency and spontaneous scurvy.

Studies in animal models suggest that ascorbic deficiency impairs T-cell-mediated immunity. We studied five normal volunteers hospitalized on a metabolic unit and consuming a strictly controlled diet deficient in ascorbic acid I) after a 5-wk control period of ascorbic acid supplementation (75 mg/day) and 2) after a 9-wk period of no supplementation. Three of the subjects were restudied after a 5-wk period of ascorbic acid supplementation after the deficient period. At the end of both control periods ascorbic acid levels in plasma ranged from 0.9 to 1.3 mg/dl and in leukocytes from 19 to 30 microgram/10(8) cells. At the end of the deficient period levels of ascorbic acid in plasma ranged from 0.09 to 0.15 mg/dl and in leukocytes from 6.2 to 10 microgram/10(8) cells, levels at or below those frequently found in frank scurvy. None of the T-cell parameters tested including mitogen responsiveness to phytohemagglutinin and percentage of T-cells bearing receptors for IgM (helper cells) and IgG (suppressor cells) was different in the deficient period compared to the control periods. One patient with spontaneous scurvy (plasma ascorbic acid 0.07 mg/dl, leukocytic ascorbic acid 4.9 microgram/10(8) cells) was studied at the time of admission and after vigorous ascorbic acid repletion. All T-cell parameters after repletion were unchanged from admission. We conclude that in man ascorbic acid deficiency, even at the scorbutic level, does not alter T-cell numbers or impair in vitro T-cell function.

Lack of effect of subclinical ascorbic acid deficiency upon antipyrine metabolism in man.

The influence of experimentally induced subclinical ascorbic acid deficiency upon antipyrine metabolism was assessed in five healthy male volunteers maintained in a hospital metabolic ward and fed a controlled diet deficient in ascorbic acid. Antipyrine pharmacokinetic parameters were determined four times during the study: at the end of an initial control period, after 28 and 63 days of depletion, and at the end of a second control period. No differences in antipyrine metabolism were observed despite the fact that the subjects had plasma ascorbate levels indicative of vitamin C deficiency (i.e., plasma levels less than 0.3 mg/dl) for 5 days (28 day-depletion) or 40 days (63 day-depletion). This experiment demonstrates that pronounced ascorbic acid deficiency of relatively short duration does not alter antipyrine metabolism in man.

Comparison of bile acid synthesis determined by isotope dilution versus fecal acidic sterol output in human subjects.

Fecal acidic sterol output has been found to be much lower than bile acid synthesis determined by isotope dilution (J. Lipid Res. 17: 17, 1976). Because of this confusing discrepancy, we compared these 2 measurements done simultaneously on 13 occasions in 5 normal volunteers. In contrast to previous findings, bile acid synthesis by the Lindstedt isotope dilution method averaged 16.3% lower than synthesis simultaneously determined by fecal acidic sterol output (95% confidence limit for the difference - 22.2 to -10.4%). When one-sample determinations of bile acid pools were substituted for Lindstedt pools, bile acid synthesis by isotope dilution averaged 5.6% higher than synthesis by fecal acidic sterol output (95% confidence limits -4.9 to 16.1%). These data indicate that the 2 methods yield values in reasonably close agreement with one another. If anything, fecal acidic sterol outputs are slightly higher than synthesis by isotope dilution.

Influence of chronic ascorbic acid deficiency and excessive ascorbic acid intake on bile acid metabolism and bile composition in the guinea pig.

The influence of chronic ascorbic acid (AA) deficiency and excessive ascorbate consumption on bile acid metabolism, liver and plasma cholesterol levels, hepatic microsomal cytochromes and biliary lipid composition was investigated. Male weanling guinea pigs were fed a cereal-based scorbutigenic diet supplemented with four levels of AA for 7 weeks: deficient, 15 and 30 mg/kg; control, 500 mg/kg; and excess, 20,000 mg/kg. Bile acid kinetic parameters were determined following the intraperitoneal administration of [24-14C] chenodeoxycholic acid. Dietary extremes of AA caused similar alterations in the parameters studied. Relative to the control group, the deficient and excess groups exhibited reduced cytochrome P-450 concentration, lower cholesterol 7 alpha-hydroxylase activity, lower bile acid turnover rate, prolonged bile acid half-life and increased plasma and liver cholesterol concentrations. Deficient and excess groups also exhibited lower biliary cholesterol saturation (i.e., increased bile acid-neutral sterol ratios) than controls. Urinary bile acid excretion was 2- to 3-fold higher in excess guinea pigs than in the other three groups. The data demonstrate the exceptional susceptibility of cholesterol 7 alpha-hydroxylase activity to alteration by dietary extremes of AA, resulting in marked inhibition of bile acid synthesis and elevation of cholesterol levels by both inadequate and excessive AA intake.