Rat Liver Mitochondrial Preparations Research Articles

Antioxidant role of plumbagin in modification of radiation-induced oxidative damage

Objective: Plumbagin is a well-known ingredient of Plumbago zeylanica, which is used in Ayurveda for its multiple therapeutic actions. We have attempted to understand protective effects of plumbagin against gamma radiation-induced damage to lipids, proteins, and enzymes of rat liver mitochondria in vitro. Methods: Rat liver mitochondrial preparation of female Wistar rats has been exposed to gamma radiation (450 Gy) to induce oxidative damage of its components. The protection offered by increasing concentration of plumbagin during irradiation for lipids, proteins and enzymes of rat liver mitochondrial preparation has been studied. The protection exerted by plumbagin (50-150 µM) has been studied using different standard assays for lipids (thiobarbituric acid reactive substance and ferrous oxidation in xylenol orange), proteins (dinitrophenylhydrazine and dithiobisnitrobenzoic acid), glutathione (fluorescence), superoxide dismutase (epinephrine oxidation) and succinate dehydrogenase (dichlorophenolindophenol reduction). Results: Plumbagin has been found to inhibit lipid peroxidation and protein carbonyl formation; protect thiols (glutathione and protein) and enzyme activity (superoxide dismutase and succinate dehydrogenase) in a concentration dependent manner. Conclusion: The protection provided by plumbagin to lipids, proteins and enzymes of rat liver mitochondrial preparation against gamma radiation-induced damage suggest that it can provide radioprotection in the living systems.

Acyl-CoA-induced generation of reactive oxygen species in mitochondrial preparations is due to the presence of peroxisomes

Preparations of rat liver mitochondria, but not of brain and heart mitochondria, produce large quantities of reactive oxygen species (ROS) in the presence of palmitoyl-CoA and other long-chain acyl-CoAs. Palmitoyl-CoA inhibited respiration of rat liver mitochondria with glutamate plus malate or with succinate as substrate. However, ROS production induced by acyl-CoA was independent of respiration inhibition, as it was also observed in antimycin A- and rotenone-inhibited mitochondria and in submitochondrial particles in the absence of respiratory substrates (other than acyl-CoA). Increased ROS production by acyl-CoA in rat liver mitochondrial preparations was observed when measured in the external medium using Amplex red as a probe, but not inside mitochondria using the internal fluorescent probe MitoSOX or aconitase activity as the “intrinsic” indicator of ROS generation in the matrix compartment. Stimulation by acyl-CoA of ROS generation was higher in “light” mitochondrial preparations that were enriched in peroxisomes, as assayed by urate oxidase. It is concluded that stimulation of ROS production in preparations of rat liver mitochondria could be ascribed to contaminating peroxisomes. Preparations of rat brain and heart mitochondria were not or were much less contaminated with peroxisomes, as indicated by low urate oxidase activity.

Curcumin protects against failure to generate a transmembrane potential and protein aggregation of rat liver mitochondria induced by tert-butylhydroperoxides

Curcumin, an active ingredient of curcuma plant has been thought to be beneficial in the treatment of liver diseases. In the previous studies,  we  have  shown  the  protective  effects  of  curcumin  against  lipid  peroxidation  and  swelling  of  the  ratlivermitochondrial preparation induced by tert-butylhydroperoxide (t-BuOOH). In the present study, the administration of t-BuOOH of 90 µM caused the mitochondria failed to generate a transmembrane potential (Ym). Of 3 doses of curcumin administered (0.5µM, 2.5 µM dan 5.0 µM) the maximum protective effect against failure to generate a transmembrane potential caused by t-BuOOH was obtained by 2.5µM of curcumin(79.13+6.28%).Further,  curcumin  of  1000 µM  could  prevent  protein  aggregation  formation  caused  by  t-BuOOH  in  the electrophoretogram. This study shows the protective effects of curcumin against damaged of energy production system and protein of the mitochondria caused by t-BuOOH. (Med J Indones 2007; 16:139-45)

Porphyrin Accumulation in Mitochondria Is Mediated by 2-Oxoglutarate Carrier

Porphyrin Accumulation in Mitochondria Is Mediated by 2-Oxoglutarate Carrier

In vivo and in vitro binding of 2,4-dichlorophenoxyacetic acid to a rat liver mitochondrial protein

2,4-Dichlorophenoxyacetic acid (2,4-D) is a hormonal herbicide widely used in the world because of its efficacy in the control of broadleaf and woody plants. In this study we have demonstrated in vivo covalent binding of the phenoxyherbicide 2,4-D to a single protein of 52 kD (from rat liver mitochondrial preparation) detected through immunoblotting studies with the specific antiserum for 2,4-D. The direct involvement of 2,4-D in the formation of the adduct has also been demonstrated in vitro, using liver mitochondrial preparations exposed to 14C-UL-2,4-D. Radiolabeled protein separated by SDS-PAGE and afterwards electroeluted showed a single labeled protein of 52 kD. When mitochondria exposed to radiolabeled xenobiotic were devoid of their outer membrane, the specific activity observed suggest that protein involved in covalent interaction belongs to the inner mitochondrial membrane. We propose that covalent binding of the phenoxyherbicide 2,4-D to a very specific single protein of 52 kD observed in vitro and in vivo may be related to known alterations of the mitochondrial function.

Evidence for the existence of [3H]-trimetazidine binding sites involved in the regulation of the mitochondrial permeability transition pore.

1. Trimetazidine is an anti-ischaemic drug effective in different experimental models but its mechanism of action is not fully understood. Data indicate that mitochondria could be the main target of this drug. The aim of this work was to investigate the binding of [3H]-trimetazidine on a purified preparation of rat liver mitochondria. 2. [3H]-trimetazidine binds to two populations of mitochondrial binding sites with Kd values of 0.96 and 84 microM. The total concentration of binding sites is 113 pmol mg(-1) protein. Trimetazidine binding sites are differently distributed. The high-affinity ones are located on the outer membranes and represent only a small part (4%) of total binding sites, whereas the low-affinity ones are located on the inner membranes and are more abundant (96%) with a Bmax=108 pmol mg(-1) protein. 3. Drug displacement studies with pharmacological markers for different mitochondrial targets showed that [3H]-trimetazidine binding sites are different from previously described mitochondrial sites. 4. The possible involvement of [3H]-trimetazidine binding sites in the regulation of the mitochondrial permeability transition pore (MTP), a voltage-dependent channel sensitive to cyclosporin A, was investigated with mitochondrial swelling experiments. Trimetazidine inhibited the mitochondrial swelling induced by Ca2+ plus tert-butylhydroperoxide (t-BH). This effect was concentration-dependent with an IC50 value of 200 microM. 5. Assuming that trimetazidine effectiveness may be related to its structure as an amphiphilic cation, we compared it with other compounds exhibiting the same chemical characteristic both for their ability to inhibit MTP opening and to displace [3H]-trimetazidine bound to mitochondria. Selected compounds were drugs known to interact with various biological membranes. 6. A strong correlation between swelling inhibition potency and low-affinity [3H]-trimetazidine binding sites was observed: r=0.907 (n=24; P<0.001). 7. These data suggest that mitochondrial sites labelled with [3H]-trimetazidine may be involved in the MTP inhibiton.

Nonsteroidal antiinflammatory drugs and uncoupling of mitochondrial oxidative phosphorylation.

There is a lack of correlation between cyclooxygenase (COX) inhibition and nonsteroidal anti-inflammatory drug (NSAID)-induced gastrointestinal (GI) damage; it has been suggested that mucosal damage may be initiated by a "topical" action of NSAIDs involving mitochondrial injury. We evaluated the effect of a range of NSAIDs and related compounds on mitochondrial function and assessed the differences between them in relation to their physicochemical properties. Stimulation of respiration, as an indicator of mitochondrial uncoupling, was measured in isolated coupled rat liver mitochondrial preparations, using an oxygen electrode. Conventional NSAIDs and acidic prodrugs all had stimulatory effects on mitochondrial respiration at micromolar concentrations (0.02-2.7 microM); higher concentrations were inhibitory. The uncoupling potency was inversely correlated with drug pKa (r = -0.87, P < 0.001; n = 12). Drugs known to have good GI tolerability, including modified flurbiprofen (dimero-flurbiprofen and nitrobutyl-flurbiprofen), nabumetone (a non-acidic prodrug), and non-acidic highly selective COX-2 inhibitors, did not cause uncoupling. The ability to uncouple mitochondrial oxidative phosphorylation is a common characteristic of antiinflammatory agents with an ionizable group. Modification or absence of an ionizable moiety reduces the effect on mitochondria and could lead to improved NSAID GI safety.

Synthesis and antiviral activity of 3′-azido-3′-deoxythymidine triphosphate distearoylglycerol: a novel phospholipid conjugate of the anti-HIV agent AZT

Phospholipid conjugates of 3′-azido-3′-deoxythymidine (AZT) show activity against the human immunodeficiency virus (HIV) in vitro. In a previous report (K.Y. Hostetler, L.M. Stuhmiller, B.H.M. Lenting, H. van den Bosch and D.D. Richman (1991), J. Biol. Chem. 265, 6112–6117) the syntheses and anti-HIV activities of AZT mono- and diphosphate diglyceride have been described. We now report on the synthesis, characterization and biological activity of 3′-azido-3′-deoxythymidine triphosphate distearoylglycerol (AZTTP-DSG). The compound was prepared by the condensation of AZT diphosphate with distearoylphosphatidic acid morpholidate in anhydrous pyridine at room temperature and purified by means of high-performance liquid chromatography using a silica column. Characterization was performed with 31P-NMR and IR analyses and determination of the fatty acid, phosphorus and nucleoside content of the product. AZTTP-DSG inhibited HIV-1 replication in both CEM and HT4-6C cells at a level intermediate in potency between its mono- and diphosphate analogs. The IC 50 values of AZTTP-DSG were 0.33 and 0.79 μM in these two cell lines, respectively. In addition, AZTTP-DSG was less toxic to CEM cells in vitro than the other AZT liponucleotides and reduced viable cell numbers in this cell type by 50% at 1000 μM. Initial studies on the metabolism of AZTTP-DSG revealed that both AZT and AZT monophosphate were liberated from the lipid pro-drug by a rat liver mitochondrial enzyme preparation. These phospholipid derivatives of AZT nucleotides represent pro-drugs for the intracellular delivery of phosphorylated antiviral nucleoside analogs.

One-step synthesis of radioactive acyl-CoA and acylcarnitines using rat liver mitochondrial outer membrane as enzyme source.

Rat liver mitochondrial outer membrane enriched preparations have proven to be a convenient enzyme source for synthesizing coenzyme A (CoA) and carnitine esters of radioactive fatty acids. These membranes are simple to isolate and they retain acyl-CoA ligase and carnitine palmitoyltransferase activities well upon storage. Enzyme purification is not required. A novel aspect of the present procedure is that the same enzymatic incubation step allows both the acyl-CoA and the acylcarnitine esters to be obtained simultaneously when carnitine is present, but produces acyl-CoA ester only when carnitine is not included. Under the conditions described, the conversion of [1-14C]octanoic acid to the respective esters was about 95%; the corresponding figure for [1-14C]palmitic acids was over 70%. The procedure seems suitable for synthesizing the labeled CoA and carnitine esters from a variety of radioactive fatty acids.

Inhibitors of sterol synthesis: Characterization of side chain oxygenated derivatives formed upon incubation of 3 β-hydroxy-5 α-cholest-8(14)-en-15-one with rat liver mitochondria.

3 β-Hydroxy-5 α-cholest-8(14)-en-15-one, a potent inhibitor of sterol biosynthesis, was incubated with rat liver mitochondrial preparations in the presence of NADPH. The following four major products were isolated and characterized by nuclear magnetic resonance and mass spectrometry: (25R)- and (25S)-3 β,26-dihydroxy-5 α-cholest-8(14)-en-15-one (4:1 ratio), 3 β-hydroxy-15-oxo-5 α-cholest-8(14)-en-26-oic acid, and 3 β,25-dihydroxy-5 α-cholest-8(14)-en-15-one. In addition, 3 α,26-dihydroxy-5 α-cholest-8(14)-en-15-one and 3 β,24-dihydroxy-5 α-cholest-8(14)-en-15-one were identified as minor products by capillary gas chromatography-mass spectrometry.

Enzymes of carnitine acylation. Is overt carnitine palmitoyltransferase of liver peroxisomal carnitine octanoyltransferase?

Liver mitochondria prepared by differential centrifugation are contaminated by significant quantities of peroxisomes and microsomal fractions. 'Easily solubilized carnitine palmitoyltransferase' prepared from liver mitochondria is thought to originate from the outer surface of the mitochondrial inner membrane. We have characterized the carnitine palmitoyltransferase activities of freeze-thaw extracts of rat liver mitochondrial preparations. Chromatography on Sephadex G-100 yields two broad peaks of carnitine decanoyltransferase activity: one eluted at the end of the void volume, which can be removed (precipitated) by ultracentrifugation; the second peak represents the soluble activity and is eluted at an Mr near 70,000. The activity in the soluble peak is precipitated by an antibody raised against carnitine octanoyltransferase purified from mouse liver peroxisomes. In contrast, antibody raised against carnitine palmitoyltransferase purified from liver mitochondrial membranes had no effect (P. Brady & L. Brady, personal communication). The carnitine acyltransferase activities of the Mr-70,000 peak in the presence or absence of Tween 20 showed maximum activity with decanoyl-CoA and about one-third of this activity with palmitoyl-CoA, similar to peroxisomal carnitine octanoyltransferase. These data show that 7500 g preparations of liver mitochondria isolated by differential centrifugation are enriched by peroxisomal carnitine octanoyltransferase (approx. 20% of the protein of the fraction is peroxisomal) and indicate that this enzyme may be the one reported as 'overt' or 'easily solubilized' mitochondrial carnitine palmitoyltransferase.

A sensitive, nonradiometric assay for dihydroorotic acid dehydrogenase using anion-exchange high-performance liquid chromatography

A new method to assay the mitochondrial pyrimidine de novo enzyme, dihydroorotate (DHO) dehydrogenase, which catalyzes the dehydrogenation of DHO, with orotic acid as the product was developed. The assay was optimized using a rat liver mitochondrial preparation. Orotic acid was quantified with high-performance liquid chromatography using an anion-exchange column (Partisil-SAX) with uv detection at 280 nm. Isocratic elution with low phosphate buffer at pH 4.0 was used. The detection limit was 20 pmol per injection, which is comparable to previously described radiometric assays. The HPLC assay was compared with a spectrophotometric assay measuring orotic acid formation in a deproteinized reaction mixture. Absorbance was measured at the optimal wavelength for orotic acid, 278.5 nm. This assay is less sensitive and less specific than the HPLC assay, which can also detect UMP which might be formed from orotic acid in whole homogenates. With both assays kinetic parameters of the enzyme were determined. In the high concentration range (80–1000 μ m) both K m and V max values were comparable. With the HPLC assay the concentration range was extended down to 12 μ m and initial rates could be determined. The apparent K m was about 12 μ m. The HPLC assay is also suitable for use in the study of inhibition of DHO dehydrogenase.

Mitochondrial damage and the subcellular distribution of 2-oxoglutarate:glyoxylate carboligase in normal human and rat liver and in the liver of a patient with primary hyperoxaluria type I.

The subcellular distribution of 2-oxoglutarate:glyoxylate carboligase was investigated in a normal human liver, a liver from a patient with pyridoxine-resistant primary hyperoxaluria type I and rat livers subjected to various degrees and types of trauma. On continuous sucrose gradients most of the carboligase fractionated with a peak equilibrium density of 1.19-1.20 g/cm3 and paralleled the distribution of the major peaks of monoamine oxidase, glutamate dehydrogenase and cytochrome oxidase and can be considered to be mitochondrial. Various proportions of the carboligase and mitochondrial marker enzymes were found to be 'extramitochondrial' (at or near the top of the sucrose gradients), depending on the liver source and the severity of trauma to which they were subjected. Carboligase, monoamine oxidase (outer membrane marker) and glutamate dehydrogenase (matrix marker) were released from mitochondria by the homogenization and centrifugation procedures, to the extent of 19.9%, 32.4% and 11.5% respectively in hyperoxaluric liver, 12.5%, 17.9% and 8.2% in normal human liver and 3.0%, 4.9% and 3.8% in control rat liver. The proportion of extramitochondrial cytochrome oxidase (inner membrane marker) was virtually undetectable in both human and rat livers. However, sonication of rat liver homogenates or the addition of the detergent Triton X-100 caused a massive release of all four enzymes. The extramitochondrial carboligase was probably in the form of a free protein of very high molecular weight or aggregate, rather than associated with a mitochondrion-derived organelle. Subfractionation of a rat liver mitochondrial preparation indicated that most of the carboligase activity paralleled activities of 2-oxoglutarate decarboxylase, citrate synthase and glutamate dehydrogenase and was probably located in the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective toxicity of the antimalarial primaquine-evidence for both uncoupling and inhibitory effects of a metabolite on the energetics of mitochondria and its ATP synthase complex.

A methylene-linked dimeric metabolite of primaquine can exist as an oxidized form (PD(+)) or a reduced form (PDH). Using a rat liver mitochondrial preparation, low concentrations (E 50% = 12 µM) of PD(+) were found to act as a classical uncoupler of oxidative phosphorylation leading to a stimulation of mitochondrial respiration. At higher concentrations, PD(+) was found to inhibit a purified F1-ATPase preparation, while its effects on the complete ATP synthase complex (F0F1-ATPase) was bimodal. A working hypothesis relating F0F1-ATPase and glucose-6-phosphate dehydrogenase activity to the selective toxicity of primaquine on the malaria parasite and host is presented.

Kinetics of conjugation and oxidation of nitrobenzyl alcohols by rat hepatic enzymes

Previous work has suggested that quantitative differences in the in vitro and in vivo metabolism of mononitrotoluene isomers are a result of differences in the hepatic conjugation and oxidation of the first metabolic intermediates, the mononitrobenzyl alcohols. We have determined the steadystate kinetic parameters, V max , K m and V K , for the metabolism of the nitrobenzyl alcohols by rat hepatic alcohol dehydrogenase, glucuronyltransferase, and sulfotransferase. 3-Nitrobenzyl alcohol was the best substrate for cytosolic alcohol dehydrogenase (V max = 1.48 nmoles/min/mg protein, V K = 3.15 × 10 −3 nmoles/min/mg proteinμ M, K m = 503 μ M) . V max and K m values for 4-nitrobenzyl alcohol were similar, but V K was about 60% of that for 3-nitrobenzyl alcohol. 2-Nitrobenzyl alcohol was not metabolized by the alcohol dehydrogenase preparation used here, but it was metabolized to 2-nitro-benzoic acid by a rat liver mitochondrial preparation. 2-Nitrobenzyl alcohol was the best substrate for microsomal glucuronyltransferase ( V max = 3.59 nmoles/min/mg protein, V K = 11.28 × 10 −3 nmoles/min/mg protein/μ M, K m = 373 μ m) . The V max for 3-nitrobenzyl alcohol was similar, but the V K was about half and the K m was about twice that for 2-nitrobenzyl alcohol. The V max for 4-nitrobenzyl alcohol was about 40% and the V K was about half that for 2-nitrobenzyl alcohol. The best substrate for cytosolic sulfotransferase was 4-nitrobenzyl alcohol (V max = 1.69 nmoles/min/mg protein, V K = 37.21 × 10 −3 nmoles/min/mg protein/μ M, K m = 48 μ M) . The V max values for the other two benzyl alcohols were similar, but the V K and K m values were about 11 and 400%, respectively, of those for 4-nitrobenzyl alcohol. These data are in qualitative agreement with results obtained when the nitrobenzyl alcohols were incubated with isolated hepatocytes, but they do not allow quantitative modeling of the data from hepatocytes.

Metabolism of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by liver homogenate fractions.

The metabolic fate of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been examined in rat and rabbit liver mitochondrial and rabbit liver microsomal preparations. The mitochondrial preparations rapidly oxidized MPTP, in a pargyline-sensitive reaction, to a polar material that was shown to contain the 1-methyl-4-phenylpyridinium species as the principal product. NADPH-supplemented microsomal preparations converted MPTP to two principal products: 4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine N-oxide. Carbon monoxide and SKF 525A selectively inhibited the oxidation of MPTP to the nor compound, indicating that this N-demethylation reaction is cytochrome P-450 catalyzed. Attempts to trap possible unstable iminium metabolites of MPTP in microsomal incubation mixtures with sodium cyanide led to the isolation of a monocyano adduct that proved to be the N-cyanomethyl derivative. Thus, hepatic mitochondrial and microsomal enzyme systems catalyze the oxidation of MPTP by different pathways, the former leading to the generation of species that may possess neurotoxic properties.

Carbon tetrachloride activation by highly purified liver mitochondrial preparations.

Highly purified rat liver mitochondrial preparations were found to be able to activate CCl4 to reactive metabolites that bind covalently to lipids. Part of the process is of an enzymatic nature, but most of it is non-enzymatic. The enzymatic mitochondrial CCl4 activation operates more efficiently under anaerobic conditions; it requires NADPH, is CO sensitive, is inducible by phenobarbital pretreatment and is only weakly inhibited by high concentrations of cyanide or azide. The non-enzymatic mitochondrial CCl4 activation is not inhibited by CO and proceeds equally well under air or nitrogen.

Formation of glycine and aminoacetone from L-threonine by rat liver mitochondria.

Threonine is a precursor of glycine in the rat, but the metabolic pathway involved is unclear. To elucidate this pathway, the biosynthesis of glycine, and of aminoacetone, from L-threonine were studied in rat liver mitochondrial preparations of differing integrities. In the absence of added cofactors, intact mitochondria formed glycine and aminoacetone in approximately equal amounts from 20 mM L-threonine, but exogenous NAD+ decreased and CoA increased the ratio of glycine to aminoacetone formed. In intact and freeze-thawed mitochondria, the ratio of glycine to aminoacetone formed was markedly sensitive to the concentration of L-threonine, glycine being the major product at low L-threonine concentrations. Disruption of mitochondrial integrity by sonication (1 min) decreased the ratio of glycine to aminoacetone formed, and in 20000 X g supernatant fractions from sonicated (3 min) mitochondria, aminoacetone was the major product. The main non-nitogenous two-carbon compound detected when intact mitochondria catabolized L-threonine to glycine was acetate, which was probably derived from deacylation of acetyl-CoA. These results suggest that glycine formation from L-threonine in rat liver mitochondria occurred primarily by the coupled activities of threonine dehydrogenase and 2-amino-3-oxobutyrate CoA-ligase, the extent of coupling between the enzymes being dependent upon a close physical relationship and upon the flux through the dehydrogenase reaction. In vivo glycine synthesis would predominate, and aminoacetone would be a minor product.

Spectrofluorimetric assay for acyl CoA-cholesterol acyltransferase

A sensitive assay for acyl CoA-cholesterol acyltransferase (EC 2.3.1.26) in rat liver microsomal and mitochondrial preparations is described. The lowered cholesterol concentration in the enzyme reaction with oleoyl CoA is determined spectrofluorimetrically by using the cholesterol oxidase/peroxidase/ p-hydroxyphenylpropionic acid system. The assay requires as little as 20 μg of protein in the enzyme preparation.

Selective effects on catalysis by the multiple forms of monoamine oxidase produced by interactions of acidic phospholipids with mitochondrial membranes.

The effect of acidic phospholipids on the A and B multiple forms of membrane-bound mitochondrial monoamine oxidase has been investigated by incubating liposomes with isolated rat liver mitochondrial outer membrane preparations at lipid:protein ratios of 0.01 to 1. A strong inhibition of monoamine oxidase B was observed with phosphatidylserine and a moderate activation of monoamine oxidase A with phosphatidylinositol, while cardiolipin had no significant effect on either form. The specificity of phosphatidylserine inhibition for monoamine oxidase B was also confirmed in mitochondrial outer membrane isolated from tissues containing exclusively the A or B form of the enzyme (human placenta and bovine liver). Levels of incorporation were comparable for all the phospholipids and tissues studied and could not account for the different effects observed. Inhibition of monoamine oxidase B was found to be similar in an intact mitochondria preparation to that observed in the isolated outer membrane. A recent report of activation of both monoamine oxidase forms in delipidated whole mitochondria by the acidic phospholipids was re-examined and found to involve release of monoamine oxidase from the mitochondria. The details of the effects of phosphatidylserine and phosphatidylinositol on membrane-bound monoamine oxidase are consistent with the concept of the multiple forms as two distinct peptides, and suggest a second possible mode of in vivo regulation of substrate specificity.