Argininosuccinate Lyase Activity Research Articles

Mutational analysis of amino acid residues involved in argininosuccinate lyase activity in duck delta II crystallin.

Delta-crystallins are the major structural eye lens proteins of most birds and reptiles and are direct homologues of the urea cycle enzyme argininosuccinate lyase. There are two isoforms of delta-crystallin, delta Iota and delta IotaIota, but only delta IotaIota crystallin exhibits argininosuccinate lyase (ASL) activity. At the onset of this study, the structure of argininosuccinate lyase/delta IotaIota crystallin with bound inhibitor or substrate analogue was not available. Biochemical and X-ray crystallographic studies had suggested that H162 may function as the catalytic base in the argininosuccinate lyase/delta IotaIota crystallin reaction mechanism, either directly or indirectly through the activation of a water molecule. The identity of the catalytic acid was unknown. In this study, the argininosuccinate substrate was modeled into the active site of duck delta IotaIota crystallin, using the coordinates of an inhibitor-bound Escherichia coli fumarase C structure to orient the fumarate moiety of the substrate. The model served as a means of identifying active site residues which are positioned to potentially participate in substrate binding and/or catalysis. On the basis of the results of the modeling, site-directed mutagenesis was performed on several amino acids, and the kinetic and thermodynamic properties of each mutant were determined. Kinetic studies reveal that five residues, R115, N116, T161, S283, and E296, are essential for catalytic activity. Determination of the free energy of unfolding/refolding of wild-type and mutant delta II crystallins revealed that all constructs exhibit similar thermodynamic stabilities. During the course of this work, the structure of an inactive delta IotaIota crystallin mutant with bound substrate was solved [Vallee et al. (1999) Biochemistry 38, 2425-2434], which has allowed the kinetic data to be interpreted on a structural basis.

Domain exchange experiments in duck delta-crystallins: functional and evolutionary implications.

Delta-crystallin, the major soluble protein component of the avian and reptilian eye lens, is homologous to the urea cycle enzyme argininosuccinate lyase (ASL). In duck lenses there are two delta crystallins, denoted delta1 and delta2. Duck delta2 is both a major structural protein of the lens and also the duck orthologue of ASL, an example of gene recruitment. Although 94% identical to delta2/ASL in the amino acid sequence, delta1 is enzymatically inactive. A series of hybrid proteins have been constructed to assess the role of each structural domain in the enzymatic mechanism. Five chimeras--221, 122, 121, 211, and 112, where the three numbers correspond to the three structural domains and the value of 1 or 2 represents the protein of origin, delta1 or delta2, respectively--were constructed and thermodynamically and kinetically analyzed. The kinetic analysis indicates that only domain 1 is crucial for restoring ASL activity to delta1 crystallin, and that amino acid substitutions in domain 2 may play a role in substrate binding. These results confirm the hypothesis that only one domain, domain 1, is responsible for the loss of catalytic activity in delta1. The thermodynamic characterization of human ASL (hASL) and duck delta1 and delta2 indicate that delta crystallins are slightly less stable than hASL, with the delta1 being the least stable. The deltaGs of unfolding are 57.25, 63.13, and 70.71 kcal mol(-1) for delta1, delta2, and hASL, respectively. This result was unexpected, and we speculate that delta crystallins have adapted to their structural role by adopting a slightly less stable conformation that might allow for enhanced protein-protein and protein-solvent interactions.

Chemical mechanism of the endogenous argininosuccinate lyase activity of duck lens delta2-crystallin.

The endogenous argininosuccinate lyase activity of duck delta2-crystallin was specifically inactivated by the histidine-specific reagent, diethyl pyrocarbonate. The protein was protected by l-citrulline or l-arginine from the diethyl pyrocarbonate inactivation. To characterize further the chemical mechanism of the delta2-crystallin-catalysed reaction, deuterium-labelled argininosuccinate was enzymically synthesized from fumarate and l-arginine with delta2-crystallin in 2H2O. The argininosuccinate synthesized contained about 19% of the anhydride form; however, the deuterium was clearly demonstrated to be incorporated enantioselectively. Only the pro-HR atom at C-9 of the succinate moiety was labelled in the [2H]argininosuccinate-9-d synthesized, which indicates an anti-elimination mechanism for the endogenous argininosuccinate lyase activity of delta2-crystallin. The enzymic activity of duck lens delta2-crystallin in the pH range 5.5-8.5 was investigated using both protium- and deuterium-labelled argininosuccinate as the substrate. From the logkcat versus pH plot, two molecular pKa values of 6.18+/-0.02 and 8.75+/-0.03 were detected in the delta2-crystallin-argininosuccinate binary complex. The former must be dehydronated and the latter hydronated to achieve an optimum reaction rate. The logkcat/Km versus pH plot suggested two molecular pKa values of 5.96+/-0.09 and 8.29+/-0.10 for the free delta2-crystallin to be involved in the substrate binding. Small kinetic isotope effects of 1.17+/-0.02 and 1.05+/-0.09 were found for kcat and kcat/Km respectively. Combining results from labelling and kinetic analysis indicates that the endogenous argininosuccinate lyase activity of duck delta2-crystallin is compatible with a stepwise E1cB mechanism, the rate-limiting step probably at the C-N bond-cleavage step.

Intragenic complementation at the argininosuccinate lyase locus: reconstruction of the active site.

Intragenic complementation has been observed at the argininosuccinate lyase (ASL) locus and the ASL alleles in the ASL-deficient cell strains of two complementation phenotypes have been identified. The frequent complementers, strains that participate in the majority of the complementation events, were found to be either homozygous or heterozygous for the Q286R allele, while the high-activity complementers, those strains in which complementation is associated with a high restoration of activity, were found to be either homozygous or heterozygous for the D87G allele. Direct proof of the intragenic complementation observed at the ASL locus has been obtained with the co-expression of the D87G and Q286R alleles in COS cells. A significant increase in the ASL activity was observed when the two alleles were co-expressed relative to the expression of each mutant allele alone. The increase in activity was comparable to that observed previously in the fibroblast complementation studies. The structure determinations of ASL and the homologous eye lens protein, duck delta II crystallin, have revealed that the active site of ASL is made up of residues from three different monomers. The structural mapping of the Q286 and D87 residues shows that both are located near the active site but that, in any one active site, each is contributed by a different monomer. The molecular symmetry of the ASL protein is such that when mutant monomers combine randomly, one active site will contain both mutations and at least one active site will contain no mutations at all. It is these 'native' active sites in the hybrid Q286R/D87G proteins that give rise to the partial recovery of enzymatic activity observed during intragenic complementation.

Compartmentation and kinetics of urea cycle enzymes in porcine enterocytes

We have recently reported the synthesis of urea from ammonia, glutamine and arginine in enterocytes of postweaning pigs. The present study was conducted to determine the compartmentation and kinetics of urea cycle enzymes in these cells. Carbamoyl phosphate synthase I (CPS I) and ornithine carbamoyltransferase (OCT) were located exclusively in mitochondria, whereas argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) were found in the cytosol. Arginase isozymes were present in both the cytosol and mitochondria of enterocytes, and differed in their sensitivity to heat inactivation. Except for OCT, V max values of urea cycle enzymes were much lower in enterocytes than in the liver of pigs, and vice versa for their K m values. Because of a low rate of ureagenesis in enterocytes compared with the liver, intestinal urea cycle enzymes may function primarily to synthesize citrulline. The co-localization of CPS I and OCT and a high activity of OCT in enterocyte mitochondria favors the intestinal synthesis of citrulline from ammonia, HCO 3 − and ornithine. Low activities of cytosolic ASS and ASL minimize the conversion of citrulline into arginine and therefore, the recycling of citrulline into ornithine via arginase in postweaning-pig enterocytes. These kinetic properties of intestinal urea cycle enzymes maximize the net synthesis of citrulline from glutamine and explain the release of large amounts of citrulline by the pig small intestine. The two compartmentally separated arginase isozymes in enterocytes may play an important role in regulating the intestinal metabolism of proline, nitric oxide and polyamines.

Developmental Control of Argininosuccinate Lyase Gene by Methylation

The gene of argininosuccinate lyase (ASL) is expressed in a developmental specific manner in the liver and is regulated by hormones, namely glucocorticoids, glucagon and insulin. To assess the role of DNA methylation in the developmental pattern of ASL gene expression, we analyzed the restriction profile obtained by cleavage of genomic DNA with MspI and HpaII in fetal and adult rat liver, two developmental stages with different levels of expression of the ASL gene. Southern analysis showed that the 5′ region of this gene appeared more methylated in the fetal liver which expressed ASL at a low level than in the adult liver where the ASL gene is highly expressed. Moreover, treatment of fetuses of various gestational stages with the hypomethylating agent 5-azacytidine for 18 h caused an increase of the hepatic ASL activity and mRNA level. The stimulating effect of this drug could be also observed in vitro in cultured fetal hepatocytes. These results suggest a developmental control of the ASL gene by the DNA methylation status.

A novel approach to practical enzymology teaching: a conductimetric investigation of arginase, inorganic pyrophosphatase, aliphatic esterase, ornithine carbamyl transferase and argininosuccinate lyase activities from mammalian liver

Kinetic conductivity measurement, run as a microcomputer-controlled workstation with eight independent reaction cells, is shown here to be a simple, cost-effective method for assaying enzymes. The technique is demonstrated with enzymes that can be used as markers for the major subcellular compartments of hepatocytes as a one-day student laboratory class with great potential for variation and development. The method is virtually unknown in enzymology, but could be envisaged as standard apparatus for all biochemical laboratories and teaching classes.

PH-Induced Reversible Dissociation of Tetrameric Duck Lens Delta-Crystallin

Animal lenses constitute many soluble proteins, which play a prominent role in eyes' light transparency. δ2-Crystallin, one of the major taxon-specific crystallins in duck lens, is a tetrameric protein consisting of four identical subunits, which contain endogenous argininosuccinate lyase activity. Under a neutral pH environment in this work, the protein was cross-linked with glutaraldehyde as tetrameric and dimeric forms with tetramer as the major form. Under acidic conditions, the protein was time-dependently dissociated into monomers with amino acid residues of pKavalues 6.29±0.45 and 7.17±0.49 being involved in the monomer-monomer interactions and 6.20±0.10 and 8.88±0.07 in the dimer-dimer interactions. Duck lens δ2-crystallin thus possesses a double dimer structure (α2)2with stronger monomer-monomer interactions than the dimer-dimer interactions. The acidic protein solution's reneutralization caused rapid reassociation of monomers into dimers and tetramers. The tetramer-dimer-monomer dissociation-reassociation thus is a pH-dependent freely interconvertible process.

Ornithine cycle in Nostoc PCC 73102: presence of an in vitro functional argininosuccinate lyase

The presence of argininosuccinate lyase (ASL), an enzyme catalyzing the final step of arginine biosynthesis, was demonstrated in the heterocystous cyanobacterium Nostoc PCC 73102 by measuring in vitro enzyme activity and by visualization of ASL in native protein gels. Activity staining of a native PAGE gel revealed one ASL-dependent band with a molecular mass of about 240 kDa. A colorimetric assay for ASL based on the measurement of urea produced from arginine in the presence of an excess of arginase was further used to analyze the cyanobacterial ASL. The in vitro ASL activity was highest in cells in exponential growth phase and decreased significantly during the stationary phase of growth, ranging from 4.4 to 0.8 nmol of product formed (mg protein) −1 min −1. Including arginine, citrulline or ornithine in the growth medium resulted in no significant change in the ASL activities, indicating that Nostoc PCC 73102 ASL is not regulated by metabolites of the ornithine cycle.

Glucocorticoids Play an Important Role in Mediating the Enhanced Metabolism of Arginine and Glutamine in Enterocytes of Postweaning Pigs

Weaning is associated with increased intestinal metabolism of glutamine and arginine as well as elevated plasma concentrations of cortisol (the major circulating glucocorticoid) in pigs. The objective of this study was to determine if cortisol plays an important role in mediating the enhanced amino acid metabolism in enterocytes of weaned pigs by administering RU486 (a glucocorticoid receptor antagonist). Eighteen 21-d-old pigs were randomly assigned to three groups of six. Two of these groups received intramuscular injections of 0 or 10 mg RU486 per kg body weight 5 min before and 24 and 72 h after weaning to a corn-soybean meal–based diet. The third group was allowed to suckle freely from sows. When the pigs were 29 d old, jugular venous blood was obtained and pigs were killed for preparation of jejunal enterocytes. The activities of arginase, argininosuccinate synthase (ASS), argininosuccinate lyase (ASL) and pyrroline-5-carboxylate (P5C) synthase were measured. For metabolic studies, cells were incubated for 0 or 30 min at 37°C in 2 mL of Krebs-bicarbonate buffer (pH 7.4) containing 0 or 2 mmol/L L-[U-14C]arginine or 2 mmol/L L-[U-14C]glutamine. In comparison with suckling pigs, weaning resulted in increases in the following: 1) the activities of arginase, ASS, ASL and P5C synthase, 2) the metabolism of arginine to CO2, proline and ornithine, and 3) the conversion of glutamine to ornithine, citrulline and CO2. The effects of the administration of RU486 were as follows: 1) attenuation of the increase in arginase activity and the production of ornithine from arginine, 2) abolition of the induction of ASL and P5C synthase, and 3) prevention of the increase in glutamine metabolism and the production of proline and CO2 from arginine in enterocytes of weaned pigs. These data suggest that glucocorticoids play an essential role in mediating the enhanced intestinal degradation of arginine and glutamine during weaning.

Enhanced metabolism of arginine and glutamine in enterocytes of cortisol-treated pigs.

This study was designed to determine whether cortisol plays a role in arginine and glutamine metabolism in enterocytes and, more specifically, whether cortisol regulates metabolic changes in these cells during weaning. Twenty-eight 21-day-old suckling pigs were randomly assigned to one of four groups (7 animals in each) and received intramuscular injections of vehicle solution (sesame oil) (control group), hydrocortisone 21-acetate (HYD) (25 mg/kg body wt), RU-486 (10 mg/kg body wt) (a potent blocker of glucocorticoid receptors), or HYD plus RU-486. At 29 days of age, pigs were killed for preparation ofjejunal enterocytes. During the entire experimental period, pigs were nursed by sows. Activities of argininosuccinate synthase, argininosuccinate lyase (ASL), arginase, and pyrroline-5-carboxylate (P5C) synthase were measured. For metabolic studies, enterocytes were incubated for 30 min at 37 degrees C in 2 ml of Krebs-bicarbonate buffer (pH 7.4) containing 0, 0.5, or 2 mM [U-(14)C]arginine or [U-(14)C]glutamine. Compared with control, cortisol administration increased 1) the activities of ASL and arginase and the production of CO(2), ornithine, and proline from arginine, and 2) P5C synthase activity and the formation of glutamate, alanine, aspartate, ornithine, citrulline, proline, and CO(2) from glutamine in enterocytes. The stimulating effects of cortisol on the enzyme activities and the metabolism of arginine and glutamine were abolished by coadministration of RU-486. Our data suggest that cortisol plays an important role in regulating arginine and glutamine metabolism in enterocytes via a glucocorticoid receptor-mediated mechanism.

Intragenic Complementation at the Human Argininosuccinate Lyase Locus: IDENTIFICATION OF THE MAJOR COMPLEMENTING ALLELES

To determine the molecular and biochemical basis of intragenic complementation observed at the human argininosuccinate lyase (ASL) locus, we identified the ASL alleles in ASL-deficient cell strains with two unique complementation phenotypes: (i) frequent complementers, strains that participated in the majority of complementation events, and (ii) high activity complementers, strains in which complementation was associated with a relatively high level of restoration of ASL activity. Four mutations (Q286R, D87G, A398D, and a deletion of exon 13) were identified in the four strains examined. One of the two frequent complementers was homozygous, and the other heterozygous, for the Q286R allele. Similarly, one of the two high activity complementers was homozygous, and the other heterozygous, for the D87G allele. When the Q286R and D87G mutations were introduced by site-directed mutagenesis into wild-type ASL cDNA, each conferred loss of ASL activity in COS cell transfection assays. To test directly the hypothesis that intragenic complementation occurs at the ASL locus, one of the major complementation events observed previously, between strains carrying the Q286R and D87G alleles, was reconstructed in COS cell transfection assays. A partial restoration of ASL activity, comparable with the increase seen in the fibroblast complementation analysis, was observed on joint cotransfection of these two alleles. The results provide molecular confirmation of the major features of the ASL mutant complementation map, identify the Q286R and D87D alleles as the frequent and high activity complementing alleles, respectively, and provide direct proof of intragenic complementation at the ASL locus.

Induction of astrocyte argininosuccinate synthetase and argininosuccinate lyase by dibutyryl cyclic AMP and dexamethasone.

Arginine is an intermediate in the elimination of excess nitrogen and is the substrate for nitric oxide synthesis. Arginine synthesis has been reported in brain tissue. We have studied the activity of the arginine biosynthetic enzymes argininosuccinate synthetase and argininosuccinate lyase in dexamethasone and/or dibutyryl cyclic AMP treated rat astrocyte cultures. Argininosuccinate lyase activity was stimulated by treatment with either effector and an additive effect was obtained when both agents were added simultaneously. Argininosuccinate synthetase was also increased in dexamethasone treated astrocytes. The effect of dibutyryl cyclic AMP on argininosuccinate synthetase was variable, suggesting a role for additional factors in its regulation as compared to argininosuccinate lyase. Regulation of arginine synthesis in astrocytes may be important to insure that arginine is not limiting for nitric oxide synthesis in neural tissue.

Characterization and enzyme activity of argininosuccinate lyase/δ-crystallin of the embryonic duck lens

Argininosuccinate lyase (ASL)/δ-crystallin, a major soluble protein of the transparent eye lens of birds and reptiles, is a mixture of tetramers comprising all possible combinations of two similar polypeptides (δ1 and δ2). Only the δ2 polypeptide has ASL activity. In the present investgation we have purified each of the 5 major isoforms (δA to δE, p I 5.2 to 5.8) of δ-crystallin tetramers from the embryonic duck lens by isoelectric focussing and established by peptide sequencing that the δ1 and δ2 polypeptides are encoded in the previously identified, linked δ1 and δ2 genes, respectively. The relative amounts of the different tetramers in the 14-day-old embryonic lens were consistent with equal expression of the 2 δ-crystallin genes and no preference for assembly of the 2 δ polypeptides. The relative amount of ASL activity of the tetramers was a linear function of the relative amount of their δ2 polypeptides, with δA (only δ1) lacking enzymatic activity altogether. δB (3 δ1:1 δ2), δC (2 δ1:2 δ2), δD (1 δ1:3 δ2) and δE (4 δ2) all gave normal Michaelis-Menten kinetics for fumarate production from argininosuccinate at 40°C and had a similar K m (average K m for mixture was 0.15 mM). δE had a K m of 0.187 mM and a V max of 9 μmol/min per mg protein. Unlike bovine and like human ASL, both reported previously, embryonic duck ASL/δ-crystallin showed no evidence of cooperativity or activation by GTP. Each isoform had a similar far ultraviolet circular dichroism spectrum and thermal stability between 20°C and 60°C, with denturation occurring at 65°C. Our data suggest that gene duplication, structural modifications leading to greater thermal stability of the δ1 and δ2 polypeptides, and selective loss of ASL activity in the δ1 polypeptide all occurred during the recruitment of ASL for a refractive role in the duck lens, resulting in the generation of ASL isoenzymes.

Arginine metabolism in cat kidney.

1. Arginine is essential for growth in the kitten and, because of the resulting hyperammonaemia, in the adult cat an arginine-free diet is life threatening. 2. The kidney is the main site of arginine synthesis. 3. This study was performed to determine whether the cat kidney synthesizes arginine and to establish which factors, such as low citrullinaemia, defects of argininosuccinate synthase and lyase activities or high renal arginase activity, might limit renal arginine production. 4. Identified nephron segments were isolated by microdissection from collagenase-treated cat kidney. 5. Arginine metabolism was studied by incubating the nephron segments with either physiological concentrations of L-[ureido-14C]citrulline (anabolism) or L-[guanido-14C]-arginine (catabolism). Arginine and urea were measured by a micro-enzymatic method. Amino acids were measured by HPLC. 6. In cat blood, the citrulline, but not the arginine, concentration was very low by comparison with other species. 7. Arginine synthesis occurred almost entirely in the proximal tubule, the highest rate occurring in the proximal convoluted tubule and the lowest in the medullary straight proximal tubule. 8. Arginase activity was restricted to the proximal tubule. Urea production increased from the convoluted towards the medullary straight tubule. 9. The limited capacity of the cat kidney to produce arginine in vivo may result from the low blood concentration of citrulline and from the high arginase activity in the various proximal cells with the ability to synthesize arginine.

Enzyme activities of canavanine metabolism in Canavalia lineata L. Callus

Summary When ornithine, citrulline, and argininosuccinic acid were added to the suspension cultures of Canavalia lineata callus, the arginine content was increased by 47%, 44%, and 75% in white callus, and 24%, 28 %, and 26 % in green callus, respectively. Feeding of canaline or ureidohomoserine increased canavanine content in green callus but not in white callus. However, when canavaninosuccinic acid was added to the medium, canavanine was measured in the white and green calli. The activities of ornithine carbamyitransferase, agininosuccinate synthetase, and argininosuccinate lyase were higher in the green callus than in the white callus. The activities of canavanine-dependent arginase and canaline reductase in the white and green calli were similar. Activity ratios (green/white) of ornithine carbamyitransferase, argininosuccinate synthetase, and argininosuccinate lyase were 1.45, 2.83, and 2.76 in canavanine accumulation and 1.35, 1.32, and 2.40 in arginine accumulation, respectively. The favorable Cascade effect of canavanine accumulation was observed in the green callus as compared with the white callus. Among the three enzymes, ureidohomoserine-dependent argininosuccinate synthetase activity of green callus was significantly higher than that of white callus. White callus had one peak of argininosuccinate synthetase activity (ASS-2) and green callus had two peaks of argininosuccinate synthetase activity (ASS-1 and ASS-2) from the DEAE-Sephacel column chromatography. Citrulline-dependent and ureidohomoserine-dependent catalytic activities of argininosuccinate synthetase were detected at the same peaks. It is concluded that the synthesis of canavanine is regulated through the coordination of ornithine carbamyitransferase, argininosuccinate synthetase, and argininosuccinate lyase.

Linkage and expression of the argininosuccinate lyase/δ-crystallin genes of the duck: insertion of a CR1 element in the intergenic spacer

δ-Crystallin is the major component of the lenses of most birds and reptiles. In the chicken there are two closely linked, tandemly oriented genes. Almost all of the δ-crystallin of the embryonic chicken lens is produced by the 5′ δ1 gene. This high lens activity has been attributed to an enhancer in intron 3. The 3′ δ2 gene encodes the enzyme argininosuccinate lyase (ASL) which is expressed at a low level in the chicken lens. Both chicken 8-crystallin genes are also expressed slightly in heart and brain, with ASL/δ2 predominating over δ1. In the duck ( Anas platyrhynchos) , ASL/δ2-crystallin serves as both enzyme and crystallin, resulting in very high levels of ASL activity in the lens. Here we show by genomic cloning that the ASL/δ-crystallin locus is highly conserved between duck and chicken, with the two duck δ-crystallin genes closely linked in tandem. The 4.6 kbp intergenic spacer in the duck locus is 79% identical to the 4 kbp chicken spacer, except for the existence of a 615 by CR1 element, highly reiterated in the duck genome, 1.8 kbp upstream of the duck ASL/δ2 gene. The CR1 sequence is a truncated LINE element containing the 3′ half of an open reading frame for a retroviral pol-like reverse transcriptase. Sequence analysis revealed (i) that intron 3 of the duck ASL/δ2 gene is very similar (80%) to intron 3 of the chicken δ1 and ASL/δ2 genes, especially in the region of the chicken δ1 enhancer core (93% identical) and (ii) that the 3′ boundary of exon 2 of the duck ASL/δ2 gene has undergone a recent splice-site slippage event, resulting in a two amino acid insertion in the encoded polypeptide. Finally, reverse transcription/polymerase chain reaction experiments established that both δ-crystallin genes are equally expressed to a high level in the embryonic duck lens; by contrast, both δ-crystallin genes produce a low amount of mRNA in the heart and brain of the embryonic duck, with the enzymatically active ASL/δ2 being preferentially expressed.

Glucocorticoid-Dependent Induction of the mRNA Coding for Argininosuccinate Lyase in Cultured Fetal Rat Hepatocytes

Dexamethasone increased both argininosuccinate lyase (ASL) activity and specific mRNA level in cultured fetal hepatocytes. Addition of various inhibitors of RNA synthesis showed that the increase in ASL mRNA may be related to an enhancement of ASL gene transcription, but not to a specific messenger stabilization. An apparent half-life of about 12 h for ASL mRNA was found in both untreated and dexamethasone-treated hepatocytes. About 30 h were necessary to observe the maximal effect of dexamethasone, and, in addition, both puromycin and cycloheximide (two inhibitors of protein synthesis) blocked the inducing effect of the steroid. These results suggested the involvement of intermediary protein(s) in the mechanism of induction of ASL mRNA by glucocorticoids.

Characterization of the Multiple Forms of Duck Lens δ-Crystallin with Endogenous Argininosuccinate Lyase Activity

Duck lens δ-crystallins were the translational products of two tandem arranged genes, δ1 and δ2. Two fractions of the δ-crystallin (δa and δb) were resolved when the duck eye ball lens extract was passed through a Pharmacia-LKB Q-Sepharose column. These fractions were further separated by chromatofocusing into multiple forms with p I value ranging from 5.05 to 5.45. The charge heterogeneity of δ-crystallins was also demonstrated on the polyacrylamide gel when electrophoresis was performed under nonreducing conditions. All of those multiple forms possessed endogenous argininosuccinate lyase activity with activation energy ∼12.5 ± 1.6 kcal/mol. δb-Crystallins showed higher enzyme activity than δa-crystallins. Slightly different kinetic parameters were observed for the multiple forms of δb-crystallins. On the other hand, δa-crystallins could be further divided into two subgroups according to their kinetic parameters. There is 12-fold difference in k cat value between these two subgroups. δa-Crystallins also have lower K m values than the δb-crystallins. When examined by polacrylamide gel electrophoresis under reducing conditions in the presence of sodium dodecyl sulfate, the multiple forms were shown to be composed of subunits with similar M r of 55,000. All of these forms showed same antigenicity toward the rabbit anti-duck δ-crystallin antiserum; however, different carbonyl contents were observed for these forms, indicating that the origin of these multiple forms was due to post-translational oxidative modification of the protein molecules. Two δ-crystallin forms were demonstrated to be the N-truncated δ2-crystallin, lacking the first eight amino acid residues from N-terminus. Modification and/or truncation of the proteins resulted in changing of the intrinsic tryptophan and tyrosine fluorescence. Those forms with higher p I values were shown to be much more thermostable than those with lower p I values. Our system may represent the first in vivo information that oxidation does not always lead to inactivation of an enzyme.

Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library.

We report the rescue of an arginine-requiring mutant (arg7-8) of Chlamydomonas reinhardtii by complementation using total DNA from a genomic cosmid library. Using the glass-bead transformation method of Kindle [8] four putative transformants able to grow in the absence of exogenous arginine were obtained from 3 x 10(9) treated cells. Southern blot analysis reveals that at least three of the clones have acquired an additional copy of the gene (ARG7) encoding argininosuccinate lyase (ASL). The arginine-independent phenotype is stable in the absence of selective pressure and high levels of ASL activity are detected in all four clones. We conclude that these represent true transformants and that any stable nuclear mutant of Chlamydomonas could be rescued using this approach.