Mitochondrial Isozyme Research Articles

Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with organic phosphates and phosphonates

The interaction of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, that is, hCA I, II, IV, V, and IX with a small library of phosphonic acids/organic phosphates, including methylphosphonic acid, MPA; phenylphosphonic acid, PPA; N-(phosphonoacetyl)- l-aspartic acid, PALA, methylene diphosphonic acid MDPA, the O-phosphates of serine (Ser-OP) and threonine (Thr-OP) as well as the antiviral phosphonate foscarnet has been studied. hCA I was activated by all these compounds, with the best activators being MPA and PPA ( K As of 0.10–1.20 μM). MPA and PPA were on the other hand nanomolar inhibitors of hCA II ( K Is of 98–99 nM). PALA showed an affinity of 7.8 μM, whereas the other compounds were weak, millimolar inhibitors of this isozyme. The best hCA IV inhibitors were PALA (79 nM) and PPA (5.4 μM), whereas the other compounds showed K Is in the range of 0.31–5.34 mM. The mitochondrial isozyme was weakly inhibited by all these compounds ( K Is in the range of 0.09–41.7 mM), similarly to the transmembrane, tumor-associated isozyme ( K Is in the range of 0.86–2.25 mM). Thus, phosphonates may lead to CA inhibitors with selectivity against two physiologically relevant isozymes, the cytosolic hCA II or the membrane-bound hCA IV.

Activities of Hepatic Cytosolic and Mitochondrial Forms of Serine Hydroxymethyltransferase and Hepatic Glycine Concentration Are Affected by Vitamin B-6 Intake in Rats

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate (PLP)–dependent enzyme that exists as cytosolic and mitochondrial isozymes that catalyze the reversible interconversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methyleneTHF. SHMT is a major source of one-carbon units for cellular metabolism, but its sensitivity to various degrees of altered vitamin B-6 nutritional status has not been determined. In this study, cytosolic and mitochondrial SHMT activities were measured in liver from rats fed dietary pyridoxine (PN) ranging from adequate to deficient levels (2, 1, 0.5, 0.1, and 0 mg PN/kg diet; n = 10 per group). Both mitochondrial and cytosolic SHMT activities increased (P < 0.001) with increasing dietary PN over this range, and activities were a linear function of liver PLP concentration. Mitochondrial SHMT comprised ∼70% of total activity. Assays conducted with and without in vitro addition of PLP indicated that total SHMT (apo- and holoenzyme forms) varied with dietary PN for each isoform, but that the proportion of each present as the apoenzyme was not affected by PN intake. This aspect of SHMT nutritional regulation differs from that of many other PLP-dependent enzymes. Hepatic glycine concentration was inversely related to vitamin B-6 intake (P < 0.05), which suggests a functional effect of altered SHMT activity. Overall these results demonstrate the potential for disruption of SHMT-mediated one-carbon metabolism by inadequate vitamin B-6 intake.

Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with carboxylates

A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with carboxylates including aliphatic (formate, acetate), dicarboxylic (oxalate, malonate), hydroxy/keto acids ( l-lactate, l-malate, pyruvate), tricarboxylic (citrate), or aromatic (benzoate, tetrafluorobenzoate) representatives, some of which are important intermediates in the Krebs cycle, is presented. The cytosolic isozyme hCA I was strongly activated by acetate, oxalate, pyruvate, l-lactate, and citrate ( K A around 0.1 μM), whereas formate, malonate, malate, and benzoate were weaker activators ( K A in the range 0.1–1 mM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with inhibition constants in the range of 0.03–24 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by carboxylates, showing a K I of 99 nM for citrate and oxalate, of 2.8 μM for malonate and of 14.5 μM for pyruvate among others. The mitochondrial isozyme hCA V was weakly inhibited by all these carboxylates ( K Is in the range of 1.67–25.9 mM), with the best inhibitor being citrate ( K I of 1.67 mM), whereas this is the most resistant CA isozyme to pyruvate inhibition ( K I of 5.5 mM), which may be another proof that CA V is the isozyme involved in the transfer of acetyl groups from the mitochondrion to the cytosol for the provision of substrate(s) for de novo lipogenesis. Furthermore, the relative resistance of CA V to inhibition by pyruvate may be an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of this anion within this organelle. The transmembrane, tumor-associated isozyme hCA IX was similar to isozyme II in its slight inhibition by all these anions ( K I in the range of 1.12–7.42 mM), except acetate, lactate, and benzoate, which showed a K I > 150 mM. The lactate insensitivity of CA IX also represents an interesting finding, since it is presumed that this isozyme evolved in such a way as to show a high catalytic activity in hypoxic tumors rich in lactate, and suggests a possible metabolon in which CA IX participates together with the monocarboxylate/H + co-transporter in dealing with the high amounts of lactate/H + present in tumors.

Carbonic anhydrase inhibitors. Inhibition of isozymes I, II, IV, V, and IX with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate

The inhibition of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes; the cytosolic hCA I and II, the membrane-bound hCA IV, the mitochondrial hCA V, and the tumor-associated, transmembrane hCA IX, with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate; such as chlorate, perchlorate, bromate, iodate, periodate, silicate, bismuthate, vanadate, molybdate, and wolframate is reported. Apparently, the geometry of the inhibitor (tetrahedral or trigonal) does not influence its binding to the Zn(II) ion of the enzyme active site, but the nature of the central element is the most important factor influencing potency. Isozymes hCA I and II are best inhibited by chlorate, perchlorate, and silicate, together with the anions structurally related to sulfate, sulfamate, and sulfamidate, but sulfate itself is a weak inhibitor (inhibition constant of 74 mM against hCA I and 183 mM against hCA II). Molybdate is a very weak hCA I inhibitor ( K I of 914 mM) but it interacts with hCA II ( K I of 27.5 mM). Isozyme IV is well inhibited by sulfate ( K I of 9 mM), sulfamate, and sulfamidate (in the low micromolar range), but not by perchlorate ( K I of 767 mM). The mitochondrial isozyme V has the lowest affinity for sulfate ( K I of 680 mM) and carbonate ( K I of 95 mM) among all the investigated isozymes, suggesting on one hand its possible participation in metabolon(s) with sulfate anion exchanger(s), and on the other hand an evolutionary adaptation to working at higher pH values (around 8.5 in mitochondria) where rather high amounts of carbonate in equilibrium with bicarbonate may be present. Metasilicate, isosteric to carbonate, is also about a 10 times weaker inhibitor of this isozyme as compared to other CAs investigated here ( K I of 28.2 mM). Surprisingly, the tumor-associated isozyme IX is resistant to sulfate inhibition ( K I of 154 mM) but has affinity in the low micromolar range for carbonate, sulfamate, and sulfamidate ( K I in the range of 8.6–9.6 μM). This constitutes another proof that this isozyme best works at acidic pH values present in tumors, being inhibited substantially at higher pH values when more carbonate may be present. Bromate and chlorate are quite weak CA IX inhibitors ( K I s of 147–274 mM).

Carbonic anhydrase inhibitors: inhibition of the membrane-bound human isozyme IV with anions

The membrane-associated human isozyme of carbonic anhydrase, hCA IV, has been investigated for its interaction with anion inhibitors, for the CO 2 hydration reaction catalyzed by this enzyme. Surprisingly, halides were observed to act as potent hCA IV inhibitors, with inhibition constants in the range of 70–90 μM, although most of these ions, and especially fluoride, the best hCA IV inhibitor among the halides, are weak inhibitors of other isozymes, such as hCA I, II and V. The metal poisons cyanate, cyanide and hydrogen sulfide were weaker hCA IV inhibitors ( K i’s in the range of 0.6–3.9 mM), whereas thiocyanate, azide, nitrate and nitrite showed even weaker inhibitory properties ( K i’s in the range of 30.8–65.1 mM). Sulfate was a good hCA IV inhibitor ( K i of 9 mM), although it is a much weaker inhibitor of isozymes I, II, V and IX. Excellent hCA IV inhibitory properties showed sulfamic acid, sulfamide, phenylboronic acid and phenylarsonic acid, with K i’s in the range of 0.87–0.93 μM, whereas their affinities for the other investigated isozymes were in the millimolar range. The interaction of some anions with the mitochondrial isozyme hCA V has also been investigated for the first time here. It has been observed that among all these isozymes, hCA V has the lowest affinity for bicarbonate and carbonate ( K i’s in the range of 82–95 mM), which may represent an evolutionary adaptation of this isozyme to the rather alkaline environment (pH 8.5) within the mitochondria, where hCA V plays important functions in some biosynthetic reactions involving carboxylating enzymes (pyruvate carboxylase and acetyl coenzyme A carboxylase). There are important differences of affinity for anions between the two membrane-associated isozymes, hCA IV and hCA IX.

Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with phosphates, carbamoyl phosphate, and the phosphonate antiviral drug foscarnet

A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with inorganic phosphates, carbamoyl phosphate, the antiviral phosphonate foscarnet as well as formate is reported. The cytosolic isozyme hCA I was weakly inhibited by neutral phosphate, strongly inhibited by carbamoyl phosphate ( K I of 9.4 μM), and activated by hydrogen- and dihydrogenphosphate, foscarnet and formate (best activator foscarnet, K A = 12 μM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with carbamoyl phosphate showing a K I of 0.31 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by phosphates/phosphonates, showing a K I of 84 nM for PO 4 3−, of 9.8 μM for HPO 4 2−, and of 9.9 μM for carbamoyl phosphate. Foscarnet was the best inhibitor of this isozyme ( K I of 0.82 mM) highly abundant in the kidneys, which may explain some of the renal side effects of the drug. The mitochondrial isozyme hCA V was weakly inhibited by all phosphates/phosphonates, except carbamoyl phosphate, which showed a K I of 8.5 μM. Thus, CA V cannot be the isozyme involved in the carbamoyl phosphate synthetase I biosynthetic reaction, as hypothesized earlier. Furthermore, the relative resistance of CA V to inhibition by inorganic phosphates suggests an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of such anions in these energy-converting organelles, where high amounts of ATP are produced by ATP synthetase, from ADP and inorganic phosphates. The transmembrane, tumor-associated isozyme hCA IX was on the other hand slightly inhibited by all these anions.

Carbonic anhydrase inhibitors: inhibition of human cytosolic isozyme II and mitochondrial isozyme V with a series of benzene sulfonamide derivatives

Among the 14 human isozymes of carbonic anhydrase (CA, EC 4.2.1.1) presently known, the cytosolic hCA II is the most active and plays a host of physiological functions, whereas the mitochondrial hCA V is unique due to its role in several biosynthetic reactions. An inhibition study of these isozymes with a series of sulfonamides is reported here, with the scope to detect lead molecules for the design of isozyme-specific CA inhibitors (CAIs) targeting the mitochondrial isoform. Indeed, recently it has been shown that CA V is a novel target for the drug design of anti-obesity agents among others. Compounds included in this study were mainly ortho-, meta-, and para-substituted-benzenesulfonamides, together with several halogeno-substituted sulfanilamides and disubstituted-benzene-1,3-disulfonamide derivatives. Isozyme V showed an inhibition profile with these sulfonamides different of that of hCA II. Thus, IC 50 values in the range of 80 nM to 74 μM against hCA II, and 0.78–63.7 μM against hCA V with these derivatives have been obtained. Only one compound, 2-carboxymethyl-benzenesulfonamide, was more active against hCA V over hCA II (selectivity ratio of 1.39), whereas all other derivatives investigated here were much better hCA II inhibitors (selectivity ratios CA II/CA V in the range of 0.0008–0.73) than hCA V inhibitors.

Carbonic anhydrase inhibitors. Inhibition of the newly isolated murine isozyme XIII with anions

The inhibition of the newly discovered cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozyme XIII of murine origin (mCA XIII) has been investigated with a series of anions, such as the physiological ones (bicarbonate, chloride), or the metal complexing anions (cyanate, cyanide, azide, hydrogen sulfide, etc), nitrate, nitrite, sulfate, sulfamate, sulfamide as well as with phenylboronic and phenylarsonic acids. The best mCA XIII inhibitors were cyanate, thiocyanate, cyanide and sulfamide, with K I-s in the range of 0.25 μM–0.74 mM, whereas fluoride, iodide, azide, carbonate and hydrogen sulfide were less effective ( K I-s in the range of 3.0–5.5 mM). The least effective inhibitors were sulfate, chloride and bicarbonate ( K I-s in the range of 138–267 mM). The affinity of mCA XIII for anions is very different from that of the other cytosolic isozymes (hCA I and II) or the mitochondrial isozyme hCA V. This resistance to inhibition by the physiological anions bicarbonate and chloride suggests an evolutionary adaptation of CA XIII to the presence of high concentrations of such anions (e.g., in the reproductive tract of both female and male), and the possible participation of this isozyme (similarly to CA II, CA IV and CA V) in metabolons with proteins involved in the anion exchange and transport, such as the anion exchangers (AE1-3) or the sodium bicarbonate co-transporter (NBC1 and NBC3) proteins, which remain to be identified.

Expression in multigene families. Analysis of chloroplast and mitochondrial proteases.

The proteolytic machinery of chloroplasts and mitochondria in Arabidopsis consists primarily of three families of ATP-dependent proteases, Clp, Lon, and FtsH, and one family of ATP-independent proteases, DegP. However, the functional significance of the multiplicity of their genes is not clear. To test whether expression of specific isomers could be differently affected by growth conditions, we analyzed transcript abundance following short-term exposure to different environmental stimuli, using 70-mer oligonucleotide arrays. This analysis revealed variability in the response to high light and different temperatures within members of each family. Thirty out of the 41 tested genes were up-regulated in response to high light, including both chloroplast and mitochondrial isozymes, whereas only six and five genes responded to either high or low temperature, respectively. The extent of response was variable, ranging from 2- to 20-fold increase in the steady-state levels. Absolute transcript levels of the tested genes, compiled from one-channel arrays, were also variable. In general, transcripts encoding mitochondrial isozymes were accumulated to a lower level than chloroplastic ones. Within the FtsH family, transcript abundance of most genes correlated with the severity of mutant phenotypes in the relevant genes. This correlation was also evident at the protein level. Analysis of FtsH isozymes revealed that FtsH2 was the most abundant species, followed by FtsH5 and 8, with FtsH1 being accumulated to only 10% of FtsH2 level. These results suggest that, unlike previous expectations, the relative importance of different chloroplast protease isozymes, evidenced by mutant phenotypes at least in the FtsH family, is determined by their abundance, and not necessarily by different specific functions or specialized expression under certain conditions.

Carbonic anhydrase inhibitors. Inhibition of mitochondrial isozyme V with aromatic and heterocyclic sulfonamides.

The first inhibition study of the mitochondrial isozyme carbonic anhydrase (CA) V (of murine origin) with a series of aromatic and heterocyclic sulfonamides is reported. Inhibition data of the cytosolic isozymes CA I and CA II and the membrane-bound isozyme CA IV with these inhibitors are also provided for comparison. Several low nanomolar CA V inhibitors were detected (KI values in the range of 4-15 nM), most of them belonging to the acylated sulfanilamide, ureido-benzenesulfonamide, 1,3,4-thiadiazole-2-sulfonamide, and aminobenzolamide type of compounds. The clinically used inhibitors acetazolamide, methazolamide, ethoxzolamide, dorzolamide, brinzolamide, and topiramate on the other hand were less effective CA V inhibitors, showing inhibition constants in the range of 47-63 nM. Some of the investigated sulfonamides, such as the ureido-benzenesulfonamides and the acylated sulfanilamides showed higher affinity for CA V than for the other isozymes, CA II included, which is a remarkable result, since most compounds investigated up to now inhibited the cytosolic isozyme CA II better. These results prompt us to hypothesize that the selective inhibition of CA V, or the dual inhibition of CA II and CA V, may lead to the development of novel pharmacological applications for such sulfonamides, for example in the treatment or prevention of obesity, by inhibiting CA-mediated lipogenetic processes.

Trichinella spiralis: activity of the cerebral pyruvate recycling pathway of the host (mouse) in hypoglycemia induced by the infection

The activity of the cerebral pyruvate recycling pathway and energy metabolism in mice infected with Trichinella spiralis were investigated using 13C-NMR and in vivo 31P-NMR spectroscopy, respectively. The 13C-NMR analysis, using [1,2- 13C 2] acetate as a substrate, of whole-brain extracts demonstrated that activity of the pathway increased when T. spiralis infection induced hypoglycemia in the host. The in vivo 31P-NMR observation showed that the cerebral ATP in normal level sustained throughout this experiment. These findings indicate that the pyruvate recycling pathway plays a role in the energy supply to the host in hypoglycemia induced by T. spiralis infection. Index Descriptors and Abbreviations: Trichinella spiralis; Nematode; Pyruvate recycling; Hypoglycemia; Brain; PECPK, phosphoenolpyruvate carboxykinase; PK, pyruvate kinase; ME, malic enzyme; mME, mitochondrial isozyme of ME; NMR, nuclear magnetic resonance; ML, muscle larvae; TSP, 3-(trimethylsilyl)- d 4-propionate; ATP, adenosine triphosphate; ADP, adenosine diphosphate; PCr, phosphocreatine; Pi, inorganic phosphate; PME, phosphomonoesters

Human Mitochondrial C1-Tetrahydrofolate Synthase: GENE STRUCTURE, TISSUE DISTRIBUTION OF THE mRNA, AND IMMUNOLOCALIZATION IN CHINESE HAMSTER OVARY CELLS

C1-tetrahydrofolate (THF) synthase is a trifunctional enzyme found in eukaryotes that contains the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. The cytoplasmic isozyme of C1-THF synthase is well characterized in a number of mammals, including humans; but a mitochondrial isozyme has been previously identified only in the yeast Saccharomyces. Here, we report the identification and characterization of the human gene encoding a functional mitochondrial C1-THF synthase. The gene spans 236 kilobase pairs on chromosome 6 and consists of 28 exons plus one alternative exon. The gene encodes a protein of 978 amino acids, including an N-terminal mitochondrial targeting sequence. The mitochondrial isozyme is 61% identical to the human cytoplasmic isozyme. Expression of the gene was detected in most human tissues, but transcripts were highest in placenta, thymus, and brain. Two mRNAs were detected, a 3.6-kb transcript and a 1.1-kb transcript, and both transcripts were observed in varying ratios in each tissue. The shorter transcript results from an alternative splicing event, where exon 7 is spliced to exon 8a instead of exon 8. Exon 8a is derived from an exonized Alu sequence, sharing no homology with exon 8 of the long transcript, and encodes just 15 amino acids followed by a stop codon and a polyadenylation signal. This short transcript potentially encodes a bifunctional enzyme lacking 10-formyl-THF synthetase activity. Both transcripts initiate at the same 5'-site, 107 nucleotides up-stream of the ATG start codon. The full-length (2934 bp) cDNA fused to a C-terminal V5 epitope tag was expressed in Chinese hamster ovary cells. Immunoblots of subfractionated cells revealed a 107-kDa protein only in the mitochondrial fractions of these cells, confirming the mitochondrial localization of the protein. Yeast cells expressing the full-length human cDNA exhibited elevated 10-formyl-THF synthetase activity, confirming its identification as the human mitochondrial C1-THF synthase.

Carbonic anhydrase inhibitors: Inhibition of human and murine mitochondrial isozymes V with anions

In addition to sulfonamides, metal complexing anions represent the second class of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The first inhibition study of the mitochondrial isozyme CA V (of murine and human origin) with anions is reported here. Inhibition data of the cytosolic isozymes CA I and CA II as well as the membrane-bound isozyme CA IV with a large number of anionic species such as halides, pseudohalides, bicarbonate, nitrate, hydrosulfide, arsenate, sulfamate, and sulfamidate and so on, are also provided for comparison. Isozyme V has an inhibition profile by anions completely different to those of CA I and IV, but similar to that of hCA II, which may have interesting physiological consequences. Similarly to hCA II, the mitochondrial isozymes show micro-nanomolar affinity for sulfonamides such as sulfanilamide and acetazolamide.

Human mitochondrial and cytosolic branched-chain aminotransferases are cysteine S-conjugate β-lyases, but turnover leads to inactivation

The mitochondrial and cytosolic branched-chain aminotransferases (BCAT m and BCAT c) are homodimers in the fold type IV class of pyridoxal 5′-phosphate-containing enzymes that also contains d-amino acid aminotransferase and 4-amino-4-deoxychorismate lyase (a β-lyase). Recombinant human BCAT m and BCAT c were shown to have β-lyase activity toward three toxic cysteine S-conjugates [ S-(1,1,2,2-tetrafluoroethyl)- l-cysteine, S-(1,2-dichlorovinyl)- l-cysteine, and S-(2-chloro-1,1,2-trifluoroethyl)- l-cysteine] and toward β-chloro- l-alanine. Human BCAT m is a much more effective β-chloro- l-alanine β-lyase than two aminotransferases (cytosolic and mitochondrial isozymes of aspartate aminotransferase) previously shown to possess this activity. BCAT m, but not BCAT c, also exhibits measurable β-lyase activity toward a relatively bulky cysteine S-conjugate [benzothiazolyl- l-cysteine]. Benzothiazolyl- l-cysteine, however, inhibits the l-leucine–α-ketoglutarate transamination reaction catalyzed by both enzymes. Inhibition was more pronounced with BCAT m. In the presence of β-lyase substrates and α-ketoisocaproate (the α-keto acid analogue of leucine), no transamination could be detected. Therefore, with an amino acid containing a good leaving group in the β position, β-elimination is greatly preferred over transamination. Both BCAT isozymes are rapidly inactivated by the β-lyase substrates. The ratio of turnover to inactivation per monomer in the presence of toxic halogenated cysteine S-conjugates is ∼170–280 for BCAT m and ∼40–50 for BCAT c. Mitochondrial enzymes of energy metabolism are especially vulnerable to thioacylation and inactivation by the reactive fragment released from toxic, halogenated cysteine S-conjugates such as S-(1,1,2,2-tetrafluoroethyl)- l-cysteine. The present results suggest that BCAT isozymes may contribute to the mitochondrial toxicity of these compounds by providing thioacylating fragments, but inactivation of the BCAT isozymes might also block essential metabolic pathways.

Zidovudine Phosphorylation and Mitochondrial Toxicity in Vitro

Zidovudine (ZDV) is a thymidine analogue activated to its triphosphate (ZDVTP) by the host's intracellular enzymes. The initial phosphorylation step is conversion to ZDV monophosphate (ZDVMP). The poor affinity of ZDVMP for thymidylate kinase results in intracellular accumulation of ZDVMP. Clinical use of ZDV is associated with cytotoxicity, thought to be mediated through mitochondrial damage. It has been suggested that ZDV cytotoxicity correlates with intracellular ZDVMP. Here we have further studied the role of ZDVMP in cytotoxicity and some of the mechanisms involved. Intracellular metabolism of ZDV in five lymphocyte/monocyte cell lines, U937, BSM, MOLT 4, JJAHN, and RAJI (4 × 106 cells), was investigated following 24 h incubation with [3H]ZDV (1.2 μCi; 0.1 μM) and cytotoxicity was determined by the MTT assay. Cytotoxicity was closely related to intracellular concentrations of the major metabolite (ZDVMP) but not with the active metabolite ZDVTP. ZDVMP was the only metabolite detected following incubation of viable mitochondria isolated from U937 cells with ZDV (1.2 μCi; 0.1 μM; 24 h) with mitochondrial levels of 0.27 ± 0.11 pmol/μg protein (mean ± SD; n = 3). No MTT toxicity was seen in isolated mitochondria. Following phytohemagglutinin (PHA) stimulation of peripheral blood mononuclear cells there was an increase in ZDV cytotoxicity compared to unstimulated cells. The results suggest that the mitochondrial isozyme of thymidine kinase (TK2) plays only a minor part in ZDVMP formation. Following PHA stimulation, activation of the cytosolic thymidine kinase isozyme (TK1) is associated with increased toxicity of ZDV. We conclude that ZDVMP responsible for mitochondrial toxicity is formed in the cytosol.

Three Arabidopsis genes encoding proteins with differential activities for cysteine synthase and beta-cyanoalanine synthase.

Three cDNA clones encoding putative cysteine synthases (O-acetylserine (thiol) lyase, EC 4.2.99.8) were isolated from Arabidopsis thaliana and designated AtcysC1, AtcysD1 and AtcysD2, respectively. Southern blot analyses suggested that the corresponding genes were present as a single copy, or at most two copies, in the A. thaliana genome. Escherichia coli complementation analyses confirmed that the cDNAs encode cysteine synthase and the corresponding proteins produced in E. coli clearly showed cysteine synthase activity. In addition, AtcysC1 protein showed beta-cyanoalanine synthase (EC 4.4.1.9) activity, but the other two did not. Kinetic analysis suggests that AtcysC1 actually functions as beta-cyanoalanine synthase rather than cysteine synthase in vivo. The mRNA accumulation of AtcysC1, AtcysD1 and AtcysD2 differed in various organs, but did not change markedly when A. thaliana seedlings were subjected to various stresses, including nutrient deprivation. In vivo targeting experiments indicated that AtcysD1 and AtcysD2 are cytoplasmic isozymes, and AtcysC1 is a mitochondrial isozyme.

Evolution of a glucose-regulated ADH gene in the genus Saccharomyces.

To determine when a glucose-repressed alcohol dehydrogenase isozyme and its regulatory gene, ADR1, arose during evolution, we surveyed species of the genus Saccharomyces for glucose-repressed ADH isozymes and for ADR1 homologues. Glucose-repressed ADH isozymes were present in all species of Saccharomyces sensu strictu and also in Saccharomyces kluyveri, the most distant member of the Saccharomyces clade. We cloned and characterized ADH promoters from S. bayanus, S. douglasii, and S. kluyveri. The ADH promoters from S. bayanus and S. douglasii had conserved sequences, including upstream regulatory elements, and an extended polydA tract. The expression of a reporter gene driven by the S. bayanus promoter was glucose-repressed and dependent on the major activator of transcription, ADR1, when it was introduced into S. cerevisiae. One S. kluyveri promoter was also glucose-repressed and ADR1-dependent in S. cerevisiae. The other S. kluyveri ADH promoter was expressed constitutively and was ADR1-independent. Although showing little sequence conservation with the S. cerevisiae ADH2 promoter, the glucose-repressed S. kluyveri promoter contains numerous potential binding sites for Adr1. The glucose-repressed ADH from S. kluyveri is a mitochondrial isozyme most closely related to S. cerevisiae ADHIII. ADR1 homologues from S. douglasii and S. paradoxus contain a trinucleotide repeat encoding polyAsn that is lacking in S. cerevisiae and S. bayanus. No ADR1 homologue could be detected in S. kluyveri, suggesting that the potential for Adr1 regulation may have arisen first, before ADR1 evolved.

X-ray crystallographic studies of mammalian carbonic anhydrase isozymes.

The year 1997 marked the 25th year of x-ray crystallographic studies of the mammalian carbonic anhydrase (CA) isozymes. These remarkable enzymes catalyze the reversible hydration of carbon dioxide (H2O + CO2 ⇔ HCO– 3 + H+) via a zinc-hydroxide mechanism (Coleman, 1986; Silverman and Lindskog, 1988; Christianson and Fierke, 1996). To date, five of the seven known isozymes have yielded x-ray crystal structures: three cytosolic isozymes, human CAI (Kannan et al., 1984), human CA II ( Liljas et al., 1972; Eriksson et al., 1988; Halansson, 19929, and bovine CA III (Eriksson and Liljas, 1993); a membrane-associated isozyme, human CA IV (Stams et al., 1996); and a mitochondrial isozyme, murine CA V (Boriack-Sjodin et al., 1995). X-ray crystallographic studies of cytosolic isozymes I, II, and III have been previously reviewed by Eriksson and Liljas (1991). In this review, we extend structural comparisons within the CA family to include the membrane-associated isozyme IV and the mitochondrial isozyme V. We note that to date, the structures of two mammalian CA isozymes remain undetermined: CA VI, a secretory protein found in saliva, and CA VII, a cytosolic isozyme found primarily in salivary glands.

Sorting of peroxisomal and mitochondrial carnitine acetyltransferase isozymes in the diploid yeast, Candida tropicalis.

Peroxisomal and mitochondrial carnitine acetyltransferase (CAT; EC 2.3.1.7) isozymes are synthesized from the first and second ATG codons of the open reading frame of one gene, Candida tropicalis CAT. Primer extension analysis and RNase protection assay revealed that the peroxisomal CAT, initiating at the second AUG codon of the transcripts, was synthesized by a translational readthrough of the first AUG codon of the open reading frame. When C. tropicalis CAT was introduced into the other yeast, Saccharomyces cerevisiae, 5' ends of transcripts were similar to those observed in C. tropicalis. Peroxisomal and mitochondrial CAT isozymes were strongly suggested to occur by the alternative initiation of translation, chiefly dependent on the structure or sequence context of the region from the 5' end to the second AUG codon; their transcripts harbored sufficient information to bring about alternative initiation of translation in both yeasts. Sorting of peroxisomal and mitochondrial CAT isozymes to their own compartment was carried out by their own targeting sequences, but, before transportation to their destination, their biosyntheses were regulated by alternative initiation of translation.

The membrane carbonic anhydrases: from CO2 transport to tumor markers.

The growing carbonic anhydrase family includes at least 11 enzymatically active isozymes (CAs) and several carbonic anhydrase-related proteins (CARPs) (Sly and Hu, 1995; Hewett-Emmett and Tashian, 1996). CAs I, II III, and VII are cytosolic isozymes covered extensively elsewhere in this volume. CA V is a mitochondrial isozyme expressed in the mitochondrial matrix (Dodgson et al., 1980It is thought to play a role in ureagenesis, providing HCO – 3 for carbamoyl phosphate synthase, and in gluconeogenesis, providing HCO –3 for pyruvate carboxylase (Dodgson and Cherian, 1989; Dodgson et al., 1993). There is emerging evidence for a second mitochondrial enzyme which has not yet been characterized. CA VI is the isozyme expressed in saliva (Murakami and Sly, 1987; Feldstein and Silverman, 1984; Fernley et al., 1989) that has been suggested to protect gastroesophageal mucosa from acid injury (Parkkila et al., 1997). The three known non-enzymatic CA-RPs CA VII, CA X, and CA XI are covered elsewhere in this volume. The focus of this report is on CA IV, CA IX, and CA XII, the three membrane CAs which were discovered in that order.KeywordsCarbonic AnhydraseHuman Carbonic AnhydraseCrab GillHuman Male Reproductive TractThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.