Immobilized-metal affinity chromatography of serum proteins on gel-immobilized group III A metal ions

The potential pharmacologic use of enzymes has long been considered. Practical applications, however, have been limited by the toxicity and allergic response to administered foreign proteins. A simple in vitro modification that allows the intraperitoneal administration of large doses of L-gulonolactone oxidase to guinea pigs is described. The enzyme is precipitated by guinea pig antisera and reacted with glutaraldehyde (0.125%). The product is comparatively nontoxic in guinea pigs. Administration of this enzyme enables guinea pigs to synthesize ascorbic acid. Success of this approach may depend on reinforcement by the bifunctional reagent of the enzyme-antibody complex.     

Ontogenetic development of L-gulonolactone oxidase activity in several vertebrates

Activity of L-gulonolactone oxidase (EC 1.1.3.8) in livers of fetal Rattus norvegicus and Mus musculus was detectable on the 18th day of gestation, increased rapidly to maxima at 15 and 5 days postpartum for the two species, respectively, and thereafter declined to adult levels. L-Gulonolactone oxidase was not detectable in liver or kidney of fetal guinea pigs at any stage of development. Near-term fetal snowshoe hares had higher activities of liver L-gulonolactone oxidase than observed in a large sample of adults. L-Gulonolactone oxidase was detectable in chicken (Gallus gallus) embryos by the sixth day of incubation, increased rapidly in the kidney with no discontinuity at hatching, reached a maximum at about the 35th day from the beginning of incubation, and then declined to adult levels. Barn swallow (Hirundo rustica) embryos appeared to synthesize little if any L-ascorbic acid; nestlings had considerably higher levels of L-gulonolactone oxidase than adults. Tadpoles of three species of frogs had appreciable levels of L-gulonolactone oxidase activity.     

Is L-gulonolactone-oxidase the only enzyme missing in animals subject to scurvy?

Evidence has recently appeared implicating an unusual microsomal D-glucuronolactone reductase, which requires carbonyl reagents for activity, in the biosynthesis of ascorbic acid. It was also shown that this microsomal enzyme activity was missing in guinea pigs and primates suggesting that L-gulonolactone oxidase deficiency was not the only defect in animals subject to scurvy. However, we have shown that highly purified L-glulonolactone oxidase catalyzes the conversion of the oxime and semicarbazone of D-glucuronolactone to the corresponding ascorbic acid derivative. There is, therefore, no need to propose a second pathway to ascorbic acid, nor is there evidence for more than the one enzyme defect in scurvy-prone animals.     

Heterologous immunoprecipitates also have potential for therapeutic use

Potential therapeutic usefulness of administered enzymes is limited by toxicity and allergenicity. To overcome these problems we are using scurvy to test various enzyme modifications that may be suitable for therapy. L-Gulonolactone oxidase, which catalyzes the final step in ascorbic acid biosynthesis, is immunoprecipitated with specific antisera from rabbits and then cross-linked with glutaraldehyde. The modified enzyme retains activity sufficient to elicit ascorbic acid synthesis in scorbutic guinea pigs. Intraperitoneal injection of this altered enzyme to animals supplemented with L-gulonolactone increases plasma concentrations of the vitamin. Importantly, multiple doses of the complex are tolerated. Therefore, it is possible to prolong survival time of animals fed an ascorbic acid-deficient diet by this enzyme replacement therapy. This procedure can also be applied to other enzymes that have potential therapeutic use. Serum cholinesterase and asparaginase both retain activity after this modification and are tolerated in single or in weekly repeated injections. Following three or four weekly injections, an anaphylactic reaction to serum but not to enzyme can be elicited if they are injected intravascularly. We conclude that the stability of the immobilized foreign enzyme is a critical factor in lessening the toxicity to multiple injections of these foreign proteins.     

Evaluation of gene expression profiling in a mouse model of L-gulonolactone oxidase gene deficiency

Humans and guinea pigs are species which are unable to synthesize ascorbic acid (vitamin C) because, unlike rodents, they lack the enzyme L-gulonolactone oxidase (Gulo). Although the phenotype of lacking vitamin C in humans, named scurvy, has long been well known, information on the impact of lacking Gulo on the gene expression profiles of different tissues is still missing. This knowledge could improve our understanding of molecular pathways in which Gulo may be involved. Recently, we discovered a deletion that includes all 12 exons in the gene for Gulo in the sfx mouse, characterized by spontaneous bone fractures. We report here the initial analysis of the impact of the Gulo gene deletion on the murine gene expression profiles in the liver, femur and kidney.     

L-gulonolactone oxidase activity and vitamin C status in riboflavin-deficient rats

The effect of riboflavin deficiency on the activity of L-gulonolactone oxidase [L-gulono-γ-lactone : oxygen 2-oxidoreductase, EC 1.1.3.8] and on vitamin C status was studied. A marked decrease in the specific activity of L-gulonolactone oxidase was observed in the liver microsomes isolated from riboflavin-deficient rats: the specific activity was approx. one-third of that in the microsomes isolated from control rats. The L-ascorbic acid content in the liver of the riboflavin-deficient rats was approx. one-half of that in the liver of the control rats. It seems that the rate of production of L-ascorbic acid in the riboflavin-deficient rats is limited by the decreased level of L-gulonolactone oxidase activity. Immunotitration using rabbit antiserum directed to L-gulonolactone oxidase revealed that a substantial amount of an inactive form of this enzyme is present in the liver microsomes of the riboflavin-deficient rats. L-Gulonolactone oxidase activity in the microsomes of these rats increased by approx. 35% upon addition of FAD, but it was slightly decreased by the addition of FMN or riboflavin. These results indicate that the liver microsomes of the riboflavin-deficient rats contain a protein which exhibits L-gulonolactone oxidase activity upon addition of FAD.     

Scurvy-prone animals, including man, monkey, and guinea pig, do not express the gene for gulonolactone oxidase

Man, monkeys, and guinea pigs cannot synthesize ascorbic acid due to a lack of gulonolactone oxidase activity. Recently, using two immunological methods, immunoprecipitation and microcomplement fixation, we reported that guinea pigs do not contain antigenic material related to gulonolactone oxidase. Now, using such immunologie techniques as double immunodiffusion, microcomplement fixation, antibody affinity chromatography, and a more sensitive radioimmunoassay, we have found that all three of these species do not contain immunologically cross-reacting material to gulonolactone oxidase. On the other hand, comparable extracts from tissues of all other species that were investigated and that do possess gulonolactone oxidase did cross-react with antiserum to enzyme from two widely differing species, rat and goat. We conclude that the gene for gulonolactone oxidase is not expressed in these scurvy-prone animals.     

Occurrence in humans and guinea pigs of the gene related to their missing enzyme L-gulono-gamma-lactone oxidase

Humans, other primates, and guinea pigs are missing an enzyme L-gulono-gamma-lactone oxidase which catalyzes the last step of L-ascorbic acid biosynthesis. We have recently isolated a cDNA encoding this enzyme of the rat (T. Koshizaka, M. Nishikimi, T. Ozawa, and K. Yagi (1988) J. Biol. Chem. 263, 1619-1621). Northern blot hybridization using this cDNA as a probe demonstrated that guinea pigs lack mRNA for L-gulono-gamma-lactone oxidase. Nevertheless, existence of a DNA sequence related to this enzyme in the genome of this animal was shown by Southern blot hybridization. The human genome was also found to contain a sequence that is hybridizable with the cDNA probe; however, the degree of hybridization was less than those of hybridization with the L-gulono-gamma-lactone oxidase genes of animals possessing the enzyme, suggesting that the human L-gulono-gamma-lactone oxidase gene has diverged more rapidly than the genes of L-ascorbic acid-synthesizing species. This hypothesis was confirmed by comparison of a partial nucleotide sequence of the human gene with that of the rat one. The L-gulono-gamma-lactone oxidase-related sequences in the guinea pig and human genomes may represent the remnants of the gene of the enzyme that were once active but became nonfunctional during the course of evolution.     

Administration of isolated chicken L-gulonolactone oxidase to guinea pigs evokes ascorbic acid synthetic capacity

l-Gulonolactone oxidase was purified from chicken kidney microsomes in order to test whether this enzyme had potential advantages in our enzyme therapy studies. Chicken was selected because it has an enzyme that is structurally distinct from the enzyme in mammals and has high enzyme activity. An essentially homogeneous preparation of chicken l-gulonolactone oxidase is obtained by a seven-step procedure. Certain characteristics of this enzyme are presented. The enzyme was found to be quite unstable. However, immunoprecipitates of the enzyme are greatly stabilized. Therefore, this form was administered to young ascorbic acid-deficient guinea pigs that had been supplemented with l-gulonolactone. These animals showed a marked increase in plasma ascorbic acid concentrations.     

Guinea pigs possess a highly mutated gene for L-gulono-gamma-lactone oxidase, the key enzyme for L-ascorbic acid biosynthesis missing in this species.

Guinea pigs cannot synthesize L-ascorbic acid because of their deficiency in L-gulono-gamma-lactone oxidase, a key enzyme for the biosynthesis of this vitamin in higher animals. In this study we isolated the L-gulono-gamma-lactone oxidase gene of the rat and the homologue of this gene of the guinea pig by screening rat and guinea pig genomic DNA libraries in lambda phage vectors, respectively, using a rat L-gulono-gamma-lactone oxidase cDNA as a probe. Sequencing analysis showed that the amino acid sequence of the rat enzyme is encoded by 12 exons and that all the intron/exon boundaries follow the GT/AG rule. On the other hand, regions corresponding to exons I and V were not identified in the guinea pig L-gulono-gamma-lactone oxidase gene homologue. Other defects found in this gene homologue are a deletion of the nucleotide sequence corresponding to a 3' 84-base pair part of rat exon VI, a 2-base pair deletion in the remaining exon VI-related region, and nonconformance to the GT/AG rule at one of the putative intron/exon boundaries. Furthermore, a large number of mutations were found in the amino acid-coding regions of the guinea pig sequence; more than half of them lead to nonconservative amino acid changes, and there are three stop codons as well. Thus it is clear that the guinea pig homologue of the L-gulono-gamma-lactone oxidase gene exists as a pseudogene that randomly accumulated a large number of mutations without functional constraint since the gene ceased to be active during evolution. On the basis of the neutral theory of evolution, the date of the loss of L-gulono-gamma-lactone oxidase in the ancestors of the guinea pig was roughly calculated to be less than 20 million years ago.     

Ascorbic acid and L-gulonolactone oxidase in lagomorphs.

1. The activity of L-gulonolactone oxidase (EC 1.1.3.8) in the liver of eastern cottontail rabbits (Sylvilagus floridanus) is about 10-fold greater in winter than in summer. 2. L-gulonolactone oxidase activity is low and tissue ascorbate high during all seasons in snowshoe hares (Lepus americanus). 3. Liver contents of ascorbate fall to low levels in L. americanus fed on rabbit chow in the laboratory. 4. The activity of L-gulonolactone oxidase in liver of Sylvilagus and Oryctolagus is depressed by feeding high levels of L-ascorbic acid. 5. The New Zealand White breed of domestic rabbit (Oryctolagus cuniculus) has considerably higher levels of L-gulonolactone oxidase and liver ascorbate than does the Dutch breed. 6. In a wild population of Oryctolagus sampled in Australia L-gulonolactone oxidase levels were intermediate between those of the two domestic breeds and more variable than either.     

Differences in activities of antioxidant superoxide dismutase, glutathione peroxidase and prooxidant xanthine oxidoreductase/xanthine oxidase in the normal corneal epithelium of various mammals

Under normal conditions, antioxidants at the corneal surface are balanced with the production of reactive oxygen species without any toxic effects. Danger from oxidative stress appears when natural antioxidants are overwhelmed leading to antioxidant/prooxidant imbalance. The aim of the present study was to examine the activities of enzymes contributing to the antioxidant/prooxidant balance in normal corneal epithelium of various mammals. The enzyme activities of antioxidant superoxide dismutase and glutathione peroxidase, as well as prooxidant xanthine oxidoreductase/xanthine oxidase were examined using biochemical methods. Results show that superoxide dismutase activity is high in rabbits and guinea pigs, whereas in pigs the activity is low and in cows it is nearly absent. In contrast, glutathione peroxidase activity is high in cows, pigs and rabbits, whereas in guinea pigs the activity is low. As far as prooxidant enzymes are concerned, elevated xanthine oxidoreductase/xanthine oxidase activities were found in rabbits, lower activities in guinea pigs, very low activity in cows and no activity in pigs. In conclusion, the above results demonstrate inter-species variations in activities of enzymes participating in antioxidant/prooxidant balance in the corneal epithelium. It is suggested that the levels of antioxidant and prooxidant enzymes studied in the corneal epithelium might be associated with the diurnal or nocturnal activity of animals. UV rays decompose hydrogen peroxide to damaging hydroxyl radicals and perhaps for this reason large animals with diurnal activity (cow, pig) require more effective peroxide removal (high glutathione peroxidase activity) together with the suppression of peroxide production (low superoxide dismutase activity, low xanthine oxidoreductase activity).     

Hydrazonopropionic acids, a class of hypoglycemic substances. 1. Hypoglycemic effect of 2-(phenylethylhydrazono)- and 2-(2-cyclohexyl-ethylhydrazono)-propionic acid

The two hydrazone-compounds 2-(phenylethylhydrazono)-propionic acid (PEHP) and 2-(2-cyclohexyl-ethylhydrazono)-propionic acid (CHEHP) significantly lowered the blood glucose level in several laboratory animals fasted 48 hours (guinea pigs, mice, hamsters and rats). In the guinea pig, PEHP produced a three times stronger hypoglycemic effect than phenelzine, its corresponding hydrazine. Conversely both hydrazono compounds decreased the monoamine oxidase activity much less, than phenelzine. CHEHP (145 mumol/kg) inhibited this enzyme by less than 14%. After oral administration both hydrazones (200 mumol/kg) also produced a distinct hypoglycemic effect. The blood glucose lowering properties of the two hydrazones were most manifest in fasted guinea pigs, diabetic mice and rats with streptozotozin diabetes.     

Progressive pseudogenization: vitamin C synthesis and its loss in bats

For the past 50 years, it was believed that all bats, like humans and guinea pigs, did not synthesize vitamin C (Vc) because they lacked activity of L-gulonolactone oxidase (GULO) in their livers. Humans and guinea pigs lack the activity due to pseudogenization of GULO in their genomes, but there is no genetic evidence to show whether such loss in bats is caused by pseudogenization. Unexpectedly, our successful molecular cloning in one frugivorous bat (Rousettus leschenaultii) and one insectivorous bat (Hipposideros armiger) ascertains that no pseudogenization occurs in these species. Furthermore, we find normal GULO protein expression using bat-specific anti-GULO polyclonal antibodies in bats, evaluated by Western blotting. Most surprisingly, GULO activity assays reveal that these two bat species have retained the ability to synthesize Vc, but at low levels compared with the mouse. It is known that bats in the genus Pteropus have lost GULO activity. We then found that functional constraints acting on the GULO of Pteropus vampyrus (which lost its function) are relaxed. These results imply that the ability to synthesize Vc in bats has not been lost completely in species as previously thought. We also suggest that the evolution of bat GULO genes can be a good model to study genetic processes associated with loss-of-function.     

Cynomolgus monkey liver aldehyde oxidase: extremely high oxidase activity and an attempt at purification

Aldehyde oxidase (EC 1.2.3.1) in monkey (Macaca fascicularis) liver was characterized. Liver cytosol exhibited extremely high benzaldehyde and phthalazine oxidase activities based on aldehyde oxidase, compared with those of rabbits, rats, mice and guinea pigs. Monkey liver aldehyde oxidase showed broad substrate specificity distinct from that of the enzyme from other mammals. Purified aldehyde oxidase from monkey liver cytosol showed two major bands and two minor bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These bands were also observed in Western blotting analysis using anti-rat aldehyde oxidase. The molecular mass of the enzyme was estimated to be 130-151 kDa by SDS-PAGE, and to be about 285 kDa by HPLC gel filtration. The results suggest that isoforms of aldehyde oxidase exist in monkey livers.     

Catalytic activity of administered gulonolactone oxidase polyethylene glycol conjugates

Scurvy in guinea pigs provides a convenient model of inborn metabolic disease for the investigation of enzyme therapy protocols. Gulonolactone oxidase, the enzyme in ascorbic acid biosynthesis that is missing from the scurvy-prone species, was modified by attachment of polyethylene glycol. The catalytic properties of this enzyme were affected little by the modification. Intravenous injection of this modified form of the enzyme elicited ascorbic acid synthesis in a dose-dependent manner. The modified enzyme was stabilized to incubation at 37 degrees C but was not protected from inactivation by trypsin. The circulating half-life of enzyme activity was not prolonged by this modification. Further, attachment of polyethylene glycol did neither abolish the enzyme's ability to react with preformed antibodies nor eliminate its immunogenicity.     

Participation of liver aldehyde oxidase in reductive metabolism of hydroxamic acids to amides

The liver enzyme responsible for the reduction of aromatic and heterocyclic hydroxamic acids to the corresponding amides was investigated with salicylhydroxamic acid, benzohydroxamic acid, anthranilhydroxamic acid, and nicotinohydroxamic acid. Rabbit liver cytosol exhibited significant reductase activities toward the hydroxamic acids under anaerobic conditions when supplemented with an electron donor of aldehyde oxidase. Similarly, rabbit liver aldehyde oxidase reduced these compounds to amides in the presence of its own electron donor, indicating that the reductase activities observed in the liver cytosol are due mainly to the cytosolic molybdoflavin enzyme. Furthermore, a significant reduction of salicylhydroxamic acid and nicotinohydroxamic acid was also observed, when an electron donor of aldehyde oxidase was added, with liver cytosols from hamsters, guinea pigs, rats, and mice. The cytosolic reductase activities toward salicylhydroxamic acid were markedly inhibited by menadione, an inhibitor of aldehyde oxidase.     

The activities and intracellular distributions of enzymes of carbohydrate, lipid and ketone-body metabolism in lactating mammary glands from ruminants and non-ruminants.

1. The activities of several enzymes of carbohydrate, lipid, acetate and ketone-body metabolism were measured in lactating mammary glands from rats, mice, rabbits, guinea pigs, sows, sheep, cows and goats. The intracellular distributions of many of the enzymes were measured by fractional extraction. 2. Acetyl-CoA synthetase was predominantly cytoplasmic in rats and guinea pigs, but was more mitochondrial in the other species. The different location of this enzyme in rats and mice is discussed in relation to the disposal of reducing equivalents. 3. 3-Oxo acid CoA-transferase and acetoacetyl-CoA thiolase assayed at 600 microM-CoA were predominantly mitochondrial in all species investigated. Acetoacetyl-CoA thiolase assayed at 8 microM-CoA was predominantly cytoplasmic, except in rabbits and guinea pigs. Ruminants appeared to possess little, if any, of the cytoplasmic enzyme. 4. The activities and distributions of NADP-isocitrate dehydrogenase were consistent with a role in supplying cytoplasmic NADPH in ruminant tissue, and indicated that this system may also occur in guinea pigs.     

Intravascularly administered crosslinked immunoprecipitates of gulonolactone oxidase are toxic and rapidly cleared from the circulation

Glutaraldehyde crosslinked, immunoprecipitated gulonolactone oxidase, injected intraperitoneally, has significant catalytic activity and is capable of providing long-term therapeutic benefit for the enzyme deficiency disease scurvy. The enzyme is tolerated even in repetitive doses. In the present study, however, we have found that when administered intra-arterially this modified enzyme is quite toxic even in single doses. Prior to administration the enzyme complex was filtered through a 5-microns filter. When administered intravascularly the enzyme is not nearly as active catalytically. In spite of this, activity can be detected in vivo as an elevation of plasma ascorbic acid and prolonged survival of guinea pigs fed without the vitamin. Following administration both activity and the enzyme complex are rapidly removed from the circulation. Liver and spleen are largely responsible for this uptake. Because of its toxicity intra-arterial injection of this form of the enzyme does not appear suitable for enzyme therapy.     

Immunological studies on the respiratory burst oxidase of pig blood neutrophils

Recently, a flavin enzyme (pI 5.0), that is probably responsible for superoxide (O2-)-generated oxidase activity, was separated by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) from neutrophil membranes in our laboratory [(1987) J. Biol. Chem. 262, 12316-12322]. In the present work, we performed immunological studies on this enzyme derived from pig blood neutrophils. The enzyme extract obtained on IEF-PAGE was injected into guinea pigs to raise antibodies. IgG antibody against the pI 5.0 protein inhibited maximally 54% of the O2- -generating activity of the membrane-solubilized oxidase, whereas the normal serum IgG was not inhibitory at all. Our results further confirmed that the enzyme (PI 5.0) is one of the component(s) of the O2- -generating system. The enzyme gave rise to a band corresponding to a major protein of 72 +/- 4 kDa on both non-denaturing and SDS-PAGE. Immunoblotting after SDS-PAGE demonstrated labelling of peptides of 70-72, 28-32 and 16-18 kDa.