The effect of mixed-function oxidation of enzymes on their susceptibility to degradation by a nonlysosomal cysteine proteinase

Mixed-function oxidation of Escherichia coli glutamine synthetase by ascorbate, oxygen, and iron has previously been shown to cause inactivation of the enzyme and enhanced susceptibility to proteolytic attack by a variety of proteases. One of these proteases, from rat liver, is a high molecular weight cysteine proteinase which does not degrade native glutamine synthetase at neutral pH. Although inactive, the oxidized glutamine synthetase preparations used in this study were only partially degraded by this proteinase. Some of the subunits were degraded to acid soluble products with no detectable intermediates; the remaining subunits had not become susceptible to proteolytic attack during the limited exposure to the ascorbate mixed-function oxidation system. Several mammalian enzymes which are known to be inactivated by mixed-function oxidation were tested as substrates for the proteinase. Native rabbit muscle enolase and pyruvate kinase were resistant to degradation, but their oxidatively inactivated forms were degraded. Oxidized phosphoglycerate kinase and creatine kinase were also preferentially degraded. Moreover, trypsin degraded oxidized preparations of all of these enzymes faster than control preparations. Oxidative inactivation of superoxide dismutase by hydrogen peroxide caused a slight increase in susceptibility to proteolytic attack, but the enzyme was still relatively resistant to degradation both by the cysteine proteinase and by trypsin. Although oxidation conditions may not have been optimal for demonstrating enhanced proteolytic susceptibility, the results do indicate that mixed-function oxidation can render some mammalian enzymes, as well as bacterial glutamine synthetase, susceptible to degradation. Mixed-function oxidation of these proteins may be a mechanism of marking them for intracellular turnover.     

Preferential degradation of the oxidatively modified form of glutamine synthetase by intracellular mammalian proteases

Four intracellular proteases partially purified from liver preferentially degraded the oxidatively modified (catalytically inactive) form of glutamine synthetase. One of the proteases was cathepsin D which is of lysosomal origin; the other three proteases were present in the cytosol. Two of these were calcium-dependent proteases with different calcium requirements. The low-calcium-requiring type (calpain I) accounted for most of the calcium-dependent activity of both mouse and rat liver. The calcium-independent cytosolic protease, referred to as the alkaline protease, has a molecular weight of 300,000 determined by gel filtration. Native glutamine synthetase was not significantly degraded by the cytosolic proteases at physiological pH, but oxidative modification of the enzyme caused a dramatic increase in its susceptibility to attack by these proteases. In contrast, trypsin and papain did degrade the native enzyme and the degradation of modified glutamine synthetase was only 2- to 4-fold more rapid. Adenylylation of glutamine synthetase had little effect on its susceptibility to proteolysis. Although major structural modifications such as dissociation, relaxation, and denaturation also increased the rate of degradation, the oxidative modification is a specific type of covalent modification which could occur in vivo. Oxidative modification can be catalyzed by a variety of mixed function oxidase systems present within cells and causes inactivation of a number of enzymes. Moreover, the presence of cytosolic proteases which recognize the oxidized form of glutamine synthetase suggests that oxidative modification may be involved in intracellular protein turnover.     

Modulation of the hydrophobicity of glutamine synthetase by mixed-function oxidation

Oxidative modification of Escherichia coli glutamine synthetase renders the enzyme susceptible to proteolytic degradation by a specific protease purified from the bacterium; native enzyme is not a substrate for the protease. A model oxidizing system consisting of ascorbate, iron, and oxygen was used to generate a series of glutamine synthetases of increasing oxidative modification. We assessed the effect of oxidative modification on the surface hydrophobicity of the glutamine synthetases, utilizing hydrophobic chromatography on a phenyl matrix. Initial exposure to the oxidizing system caused inactivation of the enzyme and generated a protein that was more hydrophilic than the native form; it was not a substrate for the protease. Continued exposure to the oxidizing system yielded a protein with additional oxidative modification. This form was distinctly more hydrophobic than the native form and it was very susceptible to proteolytic attack by the purified protease. Thus, oxidative modification modulates the surface hydrophobicity of glutamine synthetase, and this modulation can control susceptibility to proteolysis.     

Oxidative modification of glutamine synthetase. II. Characterization of the ascorbate model system

The first step in the proteolytic degradation of bacterial glutamine synthetase is a mixed function oxidation of one of the 16 histidine residues in the glutamine synthetase subunit (Levine, R.L. (1983) J. Biol. Chem. 258, 11823-11827). A model system, consisting of oxygen, a metal ion, and ascorbic acid, mimics the bacterial system in mediating the oxidative modification of glutamine synthetase. This model system was studied to gain an understanding of the mechanism of oxidation and of factors which control the susceptibility of the enzyme to oxidation. Availability of substrates and the extent of covalent modification of the enzyme (adenylylation) interact to modulate susceptibility of the enzyme to oxidation. This interaction provides the biochemical basis for physiologic regulation of intracellular proteolysis of glutamine synthetase. The oxidative modification requires hydrogen peroxide. While the reaction may involve Fenton chemistry, the participation of free radicals, superoxide anion, and singlet oxygen could not be demonstrated.     

Purification of a protease from Escherichia coli with specificity for oxidized glutamine synthetase

A soluble Escherichia coli protease has been identified and purified to homogeneity. The protease cleaves glutamine synthetase which has been modified by mixed function oxidation; native glutamine synthetase is not a substrate. Using [14C]glutamine synthetase as a substrate (prepared by growing E. coli on 14C-labeled amino acids), protease activity was assayed by determining the release of trichloroacetic acid-soluble material. The pure protease cleaves glutamine synthetase near the carboxyl terminus yielding 4,500 and 47,000 Mr products. The characteristics of this enzyme distinguish it from proteases previously purified from E. coli. These characteristics include a molecular weight of 75,000, alkaline pH optimum, lack of inhibition by serine protease inhibitors, and the ability to degrade insulin and casein. Oxidation of glutamine synthetase and other enzymes can be catalyzed by a variety of mixed function oxidase systems from bacterial and mammalian sources. Mixed function oxidation may be a "signal" or "marker" which consigns a protein for proteolytic degradation. Susceptibility to oxidation is subject to metabolic regulation, thereby providing control of proteolytic turnover. Isolation of a protease specific for modified glutamine synthetase provides the enzymatic basis for the specificity of this scheme.     

Protein oxidation and proteolysis during aging and oxidative stress

Previous studies in this laboratory have shown that glutamine synthetase (GS) and other key metabolic enzymes are inactivated by metal-catalyzed oxidation reactions in vitro. Oxidative inactivation renders these proteins highly susceptible to proteolysis, especially to a class of newly identified alkaline proteases which exhibit little or no activity against the native enzymes. These studies have suggested that oxidative inactivation may be an important marking step for intracellular protein degradation. Because many of the enzymes which have been shown to accumulate as inactive or less active forms during aging are readily inactivated by metal-catalyzed oxidation reactions in vitro, we have investigated the possible relationship between protein oxidation and proteolysis during aging and oxidative stress in vivo. Oxidized proteins accumulate in hepatocytes of rats exposed to 100% oxygen during the first 48 h of oxygen treatment. In the interval between 48 and 54 h the levels of oxidized proteins decline sharply. The specific activities of at least two liver enzymes, glutamine synthetase and glucose-6-phosphate dehydrogenase (G-6-PDH), decrease during the 54-h experiment. GS and G-6-PDH specific immunological cross-reactivity remains high during the first 48 h of oxygen treatment and then declines in the interval between 48 and 54 h. During this same interval the levels of alkaline proteases which degrade oxidized proteins increase, indicating that these activities are induced or activated in response to oxidative stress and subsequently degrade the proteins which have become oxidized during the initial phase of oxygen treatment. Oxidized proteins accumulate progressively during aging in hepatocytes from rats 3 to 26 months old, with the largest incremental increase between 20 and 26 months. The increase in protein oxidation is correlated with a loss of specific activity of GS and G-6-PDH without a concomitant loss of immunological cross-reactivity. The levels of alkaline proteases which degrade oxidized proteins in hepatocytes from 26-month-old rats is only 20% that of 3-month-old rats, suggesting that oxidized proteins accumulate in hepatocytes from old rats, in part, because the proteases which degrade them are deficient or defective. moreover, when old rats are subjected to treatment with 100% oxygen, the levels of oxidized proteins continue to increase and the alkaline protease activity remains low, indicating that these protease activities are not increased in response to oxidative stress in old rats.     

Inactivation of Bacillus subtilis glutamine synthetase by metal-catalyzed oxidation.

Instability of Bacillus subtilis glutamine synthetase in crude extracts was attributed to site-specific oxidation by a mixed-function oxidation, and not to limited proteolysis by intracellular serine proteases (ISP). The crude extract from B. subtilis KN2, which is deficient in three intracellular proteases, inactivated glutamine synthetase similarly to the wild-type strain extract. To understand the structural basis of the functional change, oxidative modification of B. subtilis glutamine synthetase was studied utilizing a model system consisting of ascorbate, oxygen, and iron salts. The inactivation reaction appeared to be first order with respect to the concentration of unmodified enzyme. The loss of catalytic activity was proportional to the weakening of subunit interactions. B. subtilis glutamine synthetase was protected from oxidative modification by either 5 mM Mn2+ or 5 mM Mn2+ plus 5 mM ATP, but not by Mg2+. The CD-spectra and electron microscopic data showed that oxidative modification induced relatively subtle changes in the dodecameric enzyme molecules, but did not denature the protein. These limited changes are consistent with a site-specific free radical mechanism occurring at the metal binding site of the enzyme. Analytical data of the inactivated enzyme showed that loss of catalytic activity occurred faster than the appearance of carbonyl groups in amino acid side chains of the protein. In B. subtilis glutamine synthetase, the catalytic activity was highly sensitive to minute deviations of conformation in the dodecameric molecules and these subtle changes in the molecules could be regarded as markers for susceptibility to proteolysis.     

Protease So from Escherichia coli preferentially degrades oxidatively damaged glutamine synthetase

After oxidative damage (e.g. induced with iron, ascorbate, and oxygen), the inactivated glutamine synthetase is selectively hydrolyzed in extracts of Escherichia coli. We therefore tested if glutamine synthetase treated with this system is hydrolyzed preferentially by any of the known E. coli proteases. Protease So, a cytoplasmic serine protease, was found to degrade the oxidized form of glutamine synthetase to acid-soluble peptides 5-10 times faster than the native glutamine synthetase. Degradation of the oxidized glutamine synthetase was inhibited by EDTA and stimulated 5-10-fold by Mg2+, Ca2+, or Mn2+, even though casein hydrolysis by protease So is not affected by divalent cations. Apparently, these cations affect the conformation of this substrate, making it more susceptible to proteolytic attack. Protease Re, another cytoplasmic protease, also degrades preferentially the oxidized form of glutamine synthetase and seems to correspond to the glutamine synthetase-degrading activity recently described by Roseman and Levine [1987) J. Biol. Chem. 262, 2101-2110). However, it is much less active in this reaction than protease So. No other soluble E. coli protease, including Do, Ci, Mi, Fa, Pi, or the ATP-dependent proteases Ti and La (the lon product), appears to degrade this oxidized protein. These results suggest that protease So participates in the hydrolysis of oxidatively damaged proteins and that E. coli has multiple systems for degrading different types of aberrant proteins.     

Metal-catalyzed oxidation of Escherichia coli glutamine synthetase: correlation of structural and functional changes

Metal-catalyzed oxidation of proteins has been implicated in a variety of biological processes, particularly in the marking of proteins for subsequent proteolytic degradation. The metal-catalyzed oxidation of bacterial glutamine synthetase causes conformational, covalent, and functional alterations in the protein. To understand the structural basis of the functional changes, the time course of oxidative modification of glutamine synthetase was studied utilizing a nonenzymic model oxidation system consisting of ascorbate, oxygen, and iron. The structural modifications induced included: decreased thermal stability; weakening of subunit interactions; decrease in isoelectric point; introduction of carbonyl groups into amino acid side chains; and loss of two histidine residues. These changes did not denature the protein, but instead induced relatively subtle changes. Indeed, even the most extensively modified protein had a sedimentation velocity which was identical to that of the native enzyme. Comparison of the time courses of the structural and functional changes established that: (i) Loss of the metal binding site and of catalytic activity occurred with loss of one histidine per subunit; (ii) increased susceptibility to proteolysis occurred with loss of two histidine residues per subunit. Thus, oxidation at one site suffices to inactivate the enzyme, but two sites must be modified to induce susceptibility to proteolysis. The limited and specific changes induced by metal-catalyzed oxidation are consistent with a site-specific free radical mechanism.     

Glutamine synthetase from the green alga Monoraphidium braunii is regulated by oxidative modification

Monoraphidium braunii glutamine synthetase is inactivated by several mixed-function oxidation systems. Inactivation requires oxygen and a metal cation as it does not take place under anaerobic conditions or in the presence of EDTA. Glutamine synthetase can be protected against that inactivation by peroxidase and catalase but not by superoxide dismutase indicating that hydrogen peroxide is involved in the process, although hydrogen peroxide is not itself effective. The oxidative modification of glutamine synthetase renders the protein more sensitive to temperature and susceptible to proteolytic attack. This has been demonstrated by measuring by quantitative immunoelectrophoresis the levels of glutamine synthetase antigen, in enzymatic preparations treated with different oxidation systems. Besides, immunoblotting of crude extracts in the presence of mixed-function oxidation systems shows the disappearance of material cross-reacting with anti-glutamine synthetase antibodies. Other results show that glutamine synthetase from Chlamydomonas reinhardtii could be subjected to the same kind of oxidative inactivation. The possible regulatory role of oxidative modification of glutamine synthetase in green algae is discussed.     

Oxidation of Neurospora crassa glutamine synthetase.

The glutamine synthetase of Neurospora crassa, either purified or in cell extracts, was inactivated by ascorbate plus FeCl3 and by H2O2 plus FeSO4. The inactivation reaction was oxygen dependent, inhibited by MnCl2 and EDTA, and stimulated in cell extracts by sodium azide. This inactivation could also be brought about by adding NADPH to the cell extract. The alpha and beta polypeptides of the active glutamine synthetase were modified by these inactivating reactions, giving rise to two novel acidic polypeptides. These modifications were observed with the purified enzyme, with cell extracts, and under in vivo conditions in which glutamine synthetase is degraded. The modified glutamine synthetase was more susceptible to endogenous phenylmethylsulfonyl fluoride-insensitive proteolytic activity, which was inhibited by MnCl2 and stimulated by EDTA. The possible physiological relevance of enzyme oxidation is discussed.     

Oxidation increases the proteolytic susceptibility of a localized region in rhodanese

For the first time, the enzyme rhodanese has been proteolytically cleaved to give species that most likely correspond to individual domains. This indicates cleavage can occur in the interdomain tether. Further, the conditions for cleavage show that availability of the susceptible bond(s) depends on conformational changes triggered by oxidative inactivation. Rhodanese, without persulfide sulfur (E), was oxidized consequent to incubation with phenylglyoxal, NADH, or hydrogen peroxide. The oxidized enzyme (Eox) was probed using the proteolytic enzymes endoproteinase glutamate C (V8), trypsin, chymotrypsin, or subtilisin. The proteolytic susceptibility of Eox, formed using hydrogen peroxide, was compared with that of E and the form of the enzyme containing transferred sulfur, ES. ES was totally refractory to proteolysis, while E was only clipped to a small extent by trypsin or V8 and not at all by chymotrypsin or subtilisin. Eox was susceptible to proteolysis by all the proteases used, and, although there were some differences among the proteolytic patterns, there was always a band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to Mr = 16,500. This was the only band observed in addition to the parent species (Mr = 33,000) when Eox was digested with chymotrypsin, and conservation of total protein was observed after digestion up to 90 min. No additional species were observable on silver staining, although there was some indication that the band at 16,500 might be a doublet. The results are consistent with the occurrence of a conformational change after oxidation that results in increased exposure and/or flexibility of the interdomain tether which contains residues that meet the specificity requirements of the proteases used.     

Degradation of the DNA-binding domain of wild-type and i-d lac repressors in Escherichia coli.

It has been shown that 28 transdominant mutant lac repressors which have lost operator DNA-binding ability in vivo and in vitro, but still bind inducer and are able to form tetramers (i-d repressors), could be divided into two groups by their capacity or incapacity to bind non-specifically to the phosphate groups of the DNA backbone. All but one of 15 analysed i-d repressors with amino acid substitutions to the C-terminal of residue 52 showed uneffected non-specific DNA binding. All 13 tested i-d repressors with amino acid substitutions to the N-terminal of residue 53 did not bind to double-stranded DNA, and 11 of these repressors derived from missense mutations in the lacI gene were endogenously degraded. The degradation in vivo only affects the amino-terminal 50-60 residues producing a mutant-specific pattern of stable repressor fragments. These fragments are tetrameric and capable of binding inducer in vivo and in vitro. The proteolytic attack presumably takes place during synthesis of the i-d repressors, since the resulting fragments are stable, both in vivo (as shown by a pulse-chase experiment) and in vitro. The proteolysis in vivo depends on the growth conditions of the bacteria and is higher in cells grown in minimal media than in rich media. Wild-type repressor is only susceptible to limited proteolysis in cells grown in minimal media but not in cells grown in rich media. The results suggest that the majority of the sequence alterations before residue 53 in missense mutant i-d lac repressor proteins affect the three-dimensional structure of the amino-terminal DNA-binding domain of the repressor protein, making it susceptible to proteolytic attack by one or several intracellular proteases.     

Specificity of the glutamine-binding site involved in the reguation of glutamine-synthetase activity in hepatoma tissue-culture cells.

Glutamine accelerates the degradation of glutamine synthetase in hepatoma tissue culture cells. Compounds structurally related to glutamine were tested for their ability to mimic or antagonize this effect of glutamine. 6-Diazo-5-oxo-L-norleucine, like glutamine depressed the activity of glutamine synthetase in hepatoma tissue culture cells. L-Methionine sulfone, albizzine, L-methionine sulfoxide, L-gamma-glutamyl hydrazide and gamma-N-methyl-L-glutamine (listed in order of decreasing potency) were antagonists which prevented the effect of glutamine on glutamine synthetase activity. These antagonists had little effect on glutamine transport or protein synthesis of hepatoma tissue culture cells and their effects were reversible. The effects of compounds on gluatmine synthetase activity in cell-free extracts of the cells were examined. Diazo-oxonorleucine and albizzine inhibited neither the transferase nor the synthetase activity of glutamine synthetase. This observation is interpreted to mean that the glutamine-binding site involved in the regulation of glutamine synthetase activity of hepatoma tissue culture cells is not the active site of the enzyme.     

Regulation of glutamine synthetase by dexamethasone in hepatoma tissue culture cells.

In certain lines of hepatoma tissue culture (HTC) cells, glutamine synthetase (EC 6.3.1.2) specific activity is increased 2.5- to 3-fold by the addition of glucocorticoids to the growth media. Actinomycin D blocks both the induction and deinduction of glutamine synthetase by glucocorticoids, suggesting a requirement of RNA synthesis for both processes. Using an antiserum raised against purified rat liver glutamine synthetase, we have precipitated radiolabeled glutamine synthetase from HTC cells. Electrophoresis of the immunoprecipitates on sodium didecyl sulfate-acrylamide gels isolates the subunit of glutamine synthetase and permits the radioactivity in the glutamine synthetase band to be quantitated. Using this technique, we have investigated the effect of dexamethasone, a synthetic glucocorticoid, on the rates of synthesis and degradation of glutamine synthetase. Dexamethasone (10(-7) M) increases the rate of synthesis of glutamine synthetase 2- to 3-fold but has no effect on the rate of glutamine synthetase degradation. The rates of total cell protein synthesis and degradation are not significantly affected by dexamethasone. The presence of actinomycin D at the time of removal of dexamethasone from induced cells prevents the fall in the induced rate of synthesis of glutamine synthetase normally seen when the inhibitor is removed from the culture medium. The regulation of glutamine synthetase by dexamethasone has been compared to the regulation of another dexamethasone-inducible enzyme in HTC cells, tyrosine aminotransferase, and been found to be similar in all parameters studied.     

Degradation of proteins microinjected into HeLa cells. The role of substrate flexibility

Increasing the flexibility of a protein enhances its susceptibility to defined proteases in vitro. To ascertain whether flexibility also affects protein stability in vivo, radioiodinated proteins with similar structures, but dissimilar flexibilities, were introduced into HeLa cells using red cell-mediated microinjection. Intracellular proteolysis was then measured as the rate of release of 125I-tyrosine into the medium. Ribonuclease A was considerably more resistant to degradation by purified proteases or in reticulocyte lysate than its flexible derivatives ribonuclease S and S-protein. In contrast, all three proteins were equally stable within HeLa cells. Like the results obtained for RNases, the rates of degradation of trypsin inhibitors, trypsin analogs, and their complexes correlated with flexibility in reticulocyte lysate. However, the intracellular half-lives of anhydrotrypsin and various proteinaceous trypsin inhibitors were not affected upon formation of enzyme-inhibitor complexes. Furthermore, trypsinogen was degraded more slowly than the structurally similar anhydrotrypsin in HeLa cells, although trypsinogen has additional segmental flexibility in its activation domain. Electrophoretic analyses revealed that trypsin-inhibitor complexes remained intact following injection into HeLa cells, and that neither free inhibitors nor anhydrotrypsin formed Triton-stable complexes with soluble cytoplasmic proteins. The observation that the components of the trypsin-inhibitor complexes were degraded simultaneously indicates that neither constituent unfolded prior to the onset of proteolysis. These studies provide evidence that RNases, trypsin, and trypsin inhibitors are degraded by an intracellular proteolytic pathway(s) which recognizes surface features of the folded proteins.     

Methionine sulfoxide and proteolytic cleavage contribute to the inactivation of cathepsin G by hypochlorous acid: an oxidative mechanism for regulation of serine proteinases by myeloperoxidase

Using myeloperoxidase and hydrogen peroxide, activated neutrophils produce high local concentrations of hypochlorous acid (HOCl). They also secrete cathepsin G, a serine protease implicated in cytokine release, receptor activation, and degradation of tissue proteins. Isolated cathepsin G was inactivated by HOCl but not by hydrogen peroxide in vitro. We found that activated neutrophils lost cathepsin G activity by a pathway requiring myeloperoxidase, suggesting that oxidants generated by myeloperoxidase might regulate cathepsin G activity in vivo. Tandem mass spectrometric analysis of oxidized cathepsin G revealed that loss of a peptide containing Asp108, which lies in the active site, associated quantitatively with loss of enzymatic activity. Catalytic domain peptides containing Asp108 were lost from the oxidized protein in concert with the conversion of Met110 to the sulfoxide. Release of this peptide was blocked by pretreating cathepsin G with phenylmethylsulfonyl fluoride, strongly implying that oxidation introduced proteolytic cleavage sites into cathepsin G. Model system studies demonstrated that methionine oxidation can direct the regiospecific proteolysis of peptides by cathepsin G. Thus, oxidation of Met110 may contribute to cathepsin G inactivation by at least two distinct mechanisms. One involves direct oxidation of the thioether residue adjacent to the aspartic acid in the catalytic domain. The other involves the generation of new sites that are susceptible to proteolysis by cathepsin G. These observations raise the possibility that oxidants derived from neutrophils restrain pericellular proteolysis by inactivating cathepsin G. They also suggest that methionine oxidation could render cathepsin G susceptible to autolytic cleavage. Myeloperoxidase may thus play a previously unsuspected role in regulating tissue injury by serine proteases during inflammation.     

Modification of the proteolytic fragmentation pattern upon oxidation of cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase

The proteolytic susceptibility of the native CO(2)-fixing photosynthetic enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco) has been shown to increase in vitro after oxidative treatments that affect cysteine thiols. A limited incubation of oxidized (pretreated with the disulfide cystamine) Rubisco from Chlamydomonas reinhardtii with subtilisin or proteinase K generated fragments of molecular mass about 53 kDa (band I in SDS-PAGE) and 47 kDa (band II) derived from the large subunit (55 kDa) of the enzyme. In contrast, proteolysis of the reduced Rubisco (pretreated with the free thiol cysteamine) produced only the 53 kDa band. The same fragmentation pattern was reproduced with Rubiscos from other algae and higher plants, as well as with other chemical modifications of protein cysteines. N-terminal sequencing of the fragments showed that band I arised from clipping the unstructured N-terminal stretch of the large subunit up to Lys18. Band II was generated by a cleavage close to Val69. The increased susceptibility of the oxidized form resulted from proteases gaining access to a loop (from Ser61 to Thr68) located between stretches of secondary structure that form the N-terminal domain. Native electrophoresis and kinetic analysis of fragment accumulation during subtilisin digestion demonstrated that subunit dissociation was induced by the proteolytic processing at the Ser61-Thr68 loop, which is characteristic of the oxidized Rubisco. Holoenzyme dissasembly was readily followed by the full degradation of the released subunits. In contrast, the limited processing to band I observed with the reduced enzyme did not compromise the quaternary structure of the Rubisco hexadecamer, thus preventing further proteolysis.     

Effect of glutamine on the degradation of glutamine synthetase in hepatoma tissue-culture cells.

In certain lines of hepatoma tissue-culture cells, the extracellular glutamine concentration regulates the specific activity of glutamine synthetase. By quantifying the radioactivity in immunoprecipitated glutamine synthetase on polyacrylamide gels, we found that the rate of degradation, but not of synthesis, of glutamine synthetase is a sensitive function of extracellular glutamine. The activiy that degrades this enzyme appears to be labile.     

Effects of glutamine,methionine sulfone and dexamethasone on rates of synthesis of glutamine synthetase in cultured hepatoma cells

Glutamine synthetase (EC 6.3.1.2) activity of hepatoma tissue culture cells is elevated by cortocisteroids and depressed by glutamine (Kulka, R.G., Tomkins, G.M. and Crook, R.B. (1972) J. Cell Biol., 54, 175–179). The transfer of cells from high (1–5 mM) to low (0.2–0.4 mM) concentrations of glutamine causes a marked increase in glutamine synthetase activity. The addition of a glutamine antagonist, methionine sulfone (1 mM) to cells suspended in high (1 mM) concentrations of glutamine also causes an increase of glutamine synthetase activity which is greater than that elicited by the transfer of cells to low concentrations of glutamine. Rates of synthesis of glutamine synthetase have been measured by radioimunoprecipitation in hepatoma tissue culture cells incubated under various conditions. Incubation of cells with the synthetic corticosteroid hormone, dexamethasone, markedly stimulates the relative rate of glutamine synthetase biosynthesis. Glutamine, or its analogue, methionine sulfone, have no effect on the relative rate of synthesis of the enzyme. However, total protein and RNA synthesis increase markedly with increasing external glutamine concentration in the range 0–1 mM. Methionine sulfone (1 mM) inhibits the degradation of glutamine synthetase in the presence of 1 mM glutamine. The data are consistent with the conclusion that the corticosteroid, dexamethasone, elevates glutamine synthetase activity by stimulating its rate of synthesis, whereas methionine sulfone elevates glutamine synthetase activity by inhibiting the glutamine-stimulated degradation of preformed enzyme.