Biomedical subjects

N P Curthoys

Publications and source records attributed to N P Curthoys.

At least 19 recordsLinked to original sources

PubMed2001

Identification of zeta-crystallin/NADPH:quinone reductase as a renal glutaminase mRNA pH response element-binding protein.

Increased renal ammoniagenesis and bicarbonate synthesis from glutamine during chronic metabolic acidosis facilitate the excretion of acids and partially restore normal acid-base balance. This adaptation is sustained, in part, by a cell-specific stabilization of the glutaminase mRNA that leads to an increased synthesis of the mitochondrial glutaminase. A direct repeat of an 8-base AU sequence within the 3'-nontranslated region of the glutaminase mRNA binds a unique protein with high affinity and specificity. Expression of various chimeric mRNAs in LLC-PK(1)-FBPase(+) cells demonstrated that a single 8-base AU sequence is both necessary and sufficient to function as a pH response element (pH RE). A biotinylated oligoribonucleotide containing the direct repeat was used as an affinity ligand to purify the pH RE-binding protein from a cytosolic extract of rat renal cortex. The purified binding activity retained the same specific binding properties as observed with crude extracts and correlated with the elution of a 36-kDa protein. Microsequencing by mass spectroscopy and Western blot analysis were used to identify this protein as zeta-crystallin/NADPH:quinone reductase. The purified protein contained eight tryptic peptides that were identical to sequences found in mouse zeta-crystallin and three peptides that differed by only a single amino acid. The observed differences may represent substitutions found in the rat homolog. A second protein purified by this protocol was identified as T-cell-restricted intracellular antigen-related protein (TIAR). However, the purified TIAR neither bound nor affected the binding of zeta-crystallin/NADPH:quinone reductase to the pH RE. Furthermore, specific antibodies to zeta-crystallin, but not TIAR, blocked the formation of the complex between the pH RE and either the crude cytosolic extract or the purified protein. Thus, zeta-crystallin/NADPH:quinone reductase is a pH response element-binding protein.

Animals↗

PubMed2001

Isolation and characterization of the promoter region of the rat kidney-type glutaminase gene.

A lambdaEMBL3 rat genomic library was screened to clone a phage that contained the promoter region of the kidney-type mitochondrial glutaminase gene. The resulting lambdaGA1 phage contained 13.7 kb of genomic DNA that was mapped by Southern blotting and restriction analysis. The 2.22 kb and 0.83 kb SacI fragments of lambdaGA1 were sequenced and the transcription initiation site was identified by RNase mapping. The reported sequence contains 2287 bp of the promoter, the entire exon 1 (542 bp), and 223 bp of the initial intron of the glutaminase gene. The initial exon contains 141 bp of 5'-nontranslated sequence and 401 bp of coding sequence that encodes the 72-amino acid mitochondrial targeting presequence and 61 amino acids from the N-terminus of the mature 66 kDa glutaminase subunit. Various segments of the GA promoter were cloned into a chloramphenicol acetyltransferase (CAT) expression vector. The resulting GA-CAT constructs were transfected into LLC-PK(1)-F(+) kidney cells to assess the promoter function of the isolated genomic DNA. The GA(-402)CAT construct produced a 10-fold greater CAT activity than the promoter-less pCAT vector. Analysis of various deletion constructs indicated that elements located between -402 and -63 bp must act in synergy with more proximal elements to create a functional promoter. The initial 402 bp segment lacks a TATA sequence but is GC-rich and contains two CCAAT boxes and two Sp1 sites.

Amino Acid Sequence↗

PubMed2001

pH regulation of renal gene expression.

The increase in intracellular pH (pHi) associated with various tumour cells triggers changes in gene expression. Similar adaptations also occur as part of the physiological response to changes in acid base balance. For example, during metabolic acidosis, increased renal ammoniagenesis and bicarbonate synthesis are sustained by the increased expression of various transport proteins and key enzymes of glutamine metabolism. In rat kidney, increased expression of the mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH) results from stabilization of their respective mRNAs. The 3'-untranslated region (UTR) of the GA mRNA contains a direct repeat of an 8-base AU sequence that functions as a pH-response element. This sequence exhibits a high affinity and specificity for z-crystallin. The same protein binds to two separate, but homologous, 8-base AU sequences within the 3'-UTR of the GDH mRNA. The apparent binding activity of z-crystallin is increased significantly during onset of metabolic acidosis. Thus, increased binding of z-crystallin may initiate the pH-responsive stabilization of the two mRNAs. In contrast, induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene occurs at the transcriptional level. In LLC-PK1-FBPase+ kidney cells, a decrease in pHi leads to activation of the p38 stress-activated protein kinase and subsequent phosphorylation of ATF-2. This transcription factor binds to the CRE-1 element within the promoter of the PEPCK gene to enhance transcription. Similar mechanisms may contribute to altered gene expression in tumour cells.

Animals↗

PubMed2001

Role of mitochondrial glutaminase in rat renal glutamine metabolism.

During normal acid-base balance, the kidney extracts very little of the plasma glutamine. However, during metabolic acidosis, as much as one third of the plasma glutamine is extracted and metabolized in a single pass through this organ. The substantial increase in renal utilization occurs solely within the proximal convoluted tubule and is sustained by compensating adaptations in the intraorgan metabolism of glutamine. The primary pathway for renal glutamine metabolism involves its transport into mitochondria and its deamidation and deamination by glutaminase (GA) and glutamate dehydrogenase (GDH), respectively. The resulting ammonium ions are excreted predominantly in the urine where they function as expendable cations to facilitate the excretion of acids. The resulting alpha-ketoglutarate is further metabolized to phosphoenolpyruvate and subsequently to glucose or CO2. The intermediate steps yield two bicarbonate ions that are selectively transported into the venous blood to partially compensate the metabolic acidosis. In rat kidney, this adaptation is sustained in part by the cell-specific induction of the glutaminase that results primarily from stabilization of the GA mRNA. The 3'-nontranslated region of the GA mRNA contains a direct repeat of an 8-base AU-sequence that functions as a pH-response element. This sequence exhibits a high affinity and specificity for zeta (z)-crystallin. The same protein binds to two separate, but homologous, 8-base AU-sequences within the 3'-nontranslated region of the GDH mRNA. The apparent binding activity of z-crystallin is increased significantly during onset of metabolic acidosis. Thus, increased binding of z-crystallin may initiate the pH-responsive stabilization of the two mRNAs.

Acid-Base Equilibrium↗

PubMed2001

Mechanism of increased renal gene expression during metabolic acidosis.

Increased renal catabolism of plasma glutamine during metabolic acidosis generates two ammonium ions that are predominantly excreted in the urine. They function as expendable cations that facilitate the excretion of acids. Further catabolism of alpha-ketoglutarate yields two bicarbonate ions that are transported into the venous blood to partially compensate for the acidosis. In rat kidney, this adaptation is sustained, in part, by the induction of multiple enzymes and various transport systems. The pH-responsive increases in glutaminase (GA) and phosphoenolpyruvate carboxykinase (PEPCK) mRNAs are reproduced in LLC-PK(1)-fructose 1,6-bisphosphatase (FBPase) cells. The increase in GA activity results from stabilization of the GA mRNA. The 3'-untranslated region of the GA mRNA contains a direct repeat of an eight-base AU sequence that functions as a pH-response element. This sequence binds zeta-crystallin/NADPH:quinone reductase with high affinity and specificity. Increased binding of this protein during acidosis may initiate the pH-responsive stabilization of the GA mRNA. In contrast, induction of PEPCK occurs at the transcriptional level. In LLC-PK(1)-FBPase(+) kidney cells, a decrease in intracellular pH leads to activation of the p38 stress-activated protein kinase and subsequent phosphorylation of transcription factor ATF-2. This transcription factor binds to cAMP-response element 1 within the PEPCK promoter and may enhance its transcription during metabolic acidosis.

Acidosis↗

PubMed2001

C/EBPbeta contributes to cAMP-activated transcription of phosphoenolpyruvate carboxykinase in LLC-PK(1)-F+ cells.

Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme in renal gluconeogenesis. Activation of various PEPCK(-2300)Luc reporter constructs in LLC-PK(1)-F+ cells, a gluconeogenic line of porcine renal proximal tubule-like cells, by protein kinase A (PKA) is mediated, in part, through the cAMP-response element (CRE)-1 of the PEPCK promoter. Incubation of a CRE-1 containing oligonucleotide with nuclear extracts from LLC-PK(1)-F+ cells produced multiple bands, all of which were blocked by antibodies that are specific for C/EBPbeta but not for C/EBPalpha or C/EBPdelta. Treatment of cells with cAMP did not affect the expression of C/EBPbeta, but the observed binding activity was increased nearly threefold. Mutation of CRE-1 to a Gal-4 binding site reduced the PKA-dependent activation of PEPCK(-2300)Luc to 40% of that observed with the wild-type construct. Coexpression of a chimeric protein containing a Gal-4 binding domain and the transactivation domain of C/EBPbeta, but not of C/EBPalpha or CRE binding protein (CREB), restored full activation by PKA. A deletion construct that lacks the activation domain of C/EBPbeta functions as a dominant negative inhibitor. Thus the binding of C/EBPbeta to the CRE-1 may contribute to the cAMP-dependent activation of the PEPCK promoter in kidney cells.

Animals↗

PubMed2000

Isolation, characterization and expression of a human brain mitochondrial glutaminase cDNA.

Various cDNAs that encode overlapping portions of the full-length human brain glutaminase (GA) cDNA were cloned and sequenced. The overall nucleotide sequence of hGA has a very high degree of identity with that of the rat kidney-type GA cDNA (77.4%) and the known portion of the cDNA that encodes the 5.0-kb porcine GA mRNA (81.1%). The identity is even more remarkable at the amino acid level, particularly in the C-terminal half where the three proteins share a 99.7% sequence identity. The hGA cDNA encodes a 73,427-Da protein that contains an N-terminal mitochondrial targeting signal and retains the primary proteolytic cleavage site characterized for the cytosolic precursor of the rat renal mitochondrial glutaminase. The entire coding region was assembled through the use of unique restriction sites and cloned into a baculovirus. Sf9 cells infected with the recombinant virus express high levels of properly processed and active glutaminase. Thus, expression of the isolated hGA cDNA should provide a means to purify large amounts of the mitochondrial glutaminase, a protein that catalyzes a key reaction in the metabolism of glutamine and the synthesis of important excitatory and inhibitory neurotransmitters.

Amino Acid Sequence↗

PubMed2000

Differential expression and acid-base regulation of glutaminase mRNAs in gluconeogenic LLC-PK(1)-FBPase(+) cells.

LLC-PK(1)-FBPase(+) cells, which are a gluconeogenic substrain of porcine renal LLC-PK(1) cells, exhibit enhanced oxidative metabolism and increased levels of phosphate-dependent glutaminase (PDG) activity. On adaptation to acidic medium (pH 6.9, 9 mM HCO(-)(3)), LLC-PK(1)-FBPase(+) cells also exhibit a greater increase in ammonia production and respond with an increase in assayable PDG activity. The changes in PDG mRNA levels were examined by using confluent cells grown on plastic dishes or on permeable membrane inserts. The latter condition increased the state of differentiation of the LLC-PK(1)-FBPase(+) cells. The levels of the primary porcine PDG mRNAs were analyzed by using probes that are specific for the 5.0-kb PDG mRNA (p2400) or that react equally with both the 4.5- and 5.0-kb PDG mRNAs (p930 and r1500). In confluent dish- and filter-grown LLC-PK(1)-FBPase(+) cells, the predominant 4.5-kb PDG mRNA is increased threefold after 18 h in acidic media. However, in filter-grown epithelia, which sustain an imposed pH and HCO(-)(3) gradient, this adaptive increase is observed only when acidic medium is applied to both the apical and the basolateral sides of the epithelia. Half-life experiments established that induction of the 4. 5-kb PDG mRNA was due to its stabilization. An identical pattern of adaptive increases was observed for the cytosolic PEPCK mRNA. In contrast, no adaptive changes were observed in the levels of the 5. 0-kb PDG mRNA in either cell culture system. Furthermore, cultures were incubated in low-potassium (0.7 mM) media for 24-72 h to decrease intracellular pH while maintaining normal extracellular pH. LLC-PK(1)-FBPase(+) cells again responded with increased rates of ammonia production and increased levels of the 4.5-kb PDG and PEPCK mRNAs, suggesting that an intracellular acidosis is the initiator of this adaptive response. Because all of the observed responses closely mimic those characterized in vivo, the LLC-PK(1)-FBPase(+) cells represent a valuable tissue culture model to study the molecular mechanisms that regulate renal gene expression in response to changes in acid-base balance.

Acid-Base Equilibrium↗

PubMed2000

Specificity and functional analysis of the pH-responsive element within renal glutaminase mRNA.

The specificity and the functional significance of the binding of a specific cytosolic protein to a direct repeat of an eight-base AU sequence within the 3'-nontranslated region of the glutaminase (GA) mRNA were characterized. Competition experiments established that the protein that binds to this sequence is not an AUUUA binding protein. When expressed in LLC-PK(1)-F(+) cells, the half-life of a beta-globin reporter construct, betaG-phosphoenolpyruvate carboxykinase, was only slightly affected (1.3-fold) by growth in acidic (pH 6.9, 10 mM HCO(-)(3)) vs. normal (pH 7.4, 25 mM HCO(-)(3)) medium. However, insertion of short segments of GA mRNA containing the direct repeat or a single eight-base AU sequence was sufficient to impart a fivefold pH-responsive stabilization to the chimeric mRNA. Furthermore, site-directed mutation of the direct repeat of the 8-base AU sequence in a betaG-GA mRNA, which contains 956 bases of the 3'-nontranslated region of the GA mRNA, completely abolished the pH-responsive stabilization of the wild-type betaG-GA mRNA. Thus either the direct repeat or a single eight-base AU sequence is both sufficient and necessary to create a functional pH-response element.

Acidosis↗

PubMed2000

Mapping and functional analysis of an instability element in phosphoenolpyruvate carboxykinase mRNA.

Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of renal gluconeogenesis. The 3'-nontranslated region of the PEPCK mRNA contains an instability element that facilitates its rapid turnover and contributes to the regulation of PEPCK gene expression. Such processes are mediated by specific protein-binding elements. Thus RNA gel shift analysis was used to identify proteins in rat renal cortical cytosolic extracts that bind to the 3'-nontranslated region of the PEPCK mRNA. Deletion constructs were then used to map the binding interactions to two adjacent RNA segments (PEPCK-6 and PEPCK-7). However, competition experiments established that only the binding to PEPCK-7 was specific. Functional studies were performed by cloning similar segments in a luciferase reporter construct, pLuc/Zeo. This analysis indicated that both PEPCK-6 and PEPCK-7 segments were necessary to produce a decrease in luciferase activity equivalent to that observed with the full-length 3'-nontranslated region. Thus the PEPCK-7 segment binds a specific protein that may recruit one or more proteins to form a complex that mediates the rapid decay of the PEPCK mRNA.

3' Untranslated Regions↗

PubMed1998

PMA and staurosporine affect expression of the PCK gene in LLC-PK1-F+ cells.

The addition of phorbol 12-myristate 13-acetate (PMA) to renal LLC-PK1-F+ cells caused a rapid decrease in the level of phosphoenolpyruvate carboxykinase (PCK) mRNA and reversed the stimulatory effects of exposure to acidic medium (pH 6.9, 10 mM HCO-3) or cAMP. In contrast, prolonged treatment with PMA increased the levels of PCK mRNA. The two effects correlated with the membrane translocation and downregulation of the alpha-isozyme of protein kinase C and were blocked by pretreatment with specific inhibitors of protein kinase C. The rapid decrease in PCK mRNA caused by PMA occurred with a half-life (t1/2 = 1 h) that is significantly faster than that measured during recovery from acid medium or following inhibition of transcription (t1/2 = 4 h). The effect of PMA was reversed by staurosporine, which apparently acts by inhibiting a signaling pathway other than protein kinase C. Staurosporine had no effect on the half-life of the PCK mRNA, but it stimulated the activity of a chloramphenicol acetyltransferase gene that was driven by the initial 490 base pairs of the PCK promoter and transiently transfected into LLC-PK1-F+ cells. This effect was additive to that of cAMP, and neither stimulation was reversed by PMA. The stimulatory effect of staurosporine was mapped to the cAMP response element (CRE-1) and P3(II) element of the PCK promoter. The data indicate that, in LLC-PK1-F+ cells, activation of protein kinase C decreases the stability of the PCK mRNA, whereas transcription of the PCK gene may be suppressed by a kinase that is inhibited by staurosporine.

Animals↗

PubMed1997

Identification of an mRNA-binding protein and the specific elements that may mediate the pH-responsive induction of renal glutaminase mRNA.

Various segments of the 3'-nontranslated region of the renal glutaminase (GA) mRNA were tested for their ability to enhance turnover and pH responsiveness. The combined effects were retained in the 340-base R-2 segment. However, the combined R-1 and R-3 fragments also imparted a partial destabilization and pH responsiveness to a chimeric beta-globin mRNA. RNA electrophoretic mobility shift assays indicated that cytosolic extracts of rat renal cortex contain a protein that binds to the R-2 and R-3 RNAs. The binding observed with the R-2 RNA was mapped to a direct repeat of an 8-base AU sequence. This binding was effectively competed with an excess of the same RNA, but not by adjacent or unrelated RNAs. UV cross-linking experiments identified a 48-kDa protein that binds to the AU repeats of the R-2 RNA. The apparent binding of this protein was greatly reduced in renal cytosolic extracts prepared from acutely acidotic rats. Two related RNA sequences in the R-3 segment also exhibited specific binding. However, the latter binding was more effectively competed by R-2 RNA than by itself, indicating that the homologous sites may be weaker binding sites for the same 48-kDa protein. Thus, a single protein may bind specifically to multiple instability elements within the 3'-nontranslated region of the GA mRNA and mediate its pH-responsive stabilization.

Animals↗

PubMed1997

Use of thermostable and Escherichia coli RNase H in RNA mapping studies.

A recently introduced thermostable RNase H was tested to determine its effectiveness in RNase H mapping reactions. Procedures are described which should have general use with both the thermostable and the Escherichia coli RNase H enzymes. Using the thermostable RNase H at higher temperatures extends the range of oligodeoxyribonucleotide/RNA combinations that yield satisfactory results. Northern blot analyses of total RNA was used to demonstrate that native RNAs can be analyzed by oligodeoxyribonucleotide directed RNase H digestion with minimal sample processing as long as care is taken to maintain thermal stringency both during reaction assembly and termination. Increased thermal stringency allows for higher DNA concentrations to ensure complete site-specific digestion of target RNAs or to permit simultaneous cleavage with multiple oligodeoxyribonucleotides. Partial digests can also be controlled by manipulating oligodeoxyribonucleotide concentrations. In addition, the thermostable RNase H was shown to be active at magnesium ion concentrations as low as 0.1 mM. This allows for optimization of Mg2+ effects on overall sample integrity and DNA/RNA interactions over at least a 20-fold range (2.0-0.1 mM).

Animals↗

PubMed1996

The 3'-nontranslated region of rat renal glutaminase mRNA contains a pH-responsive stability element.

Rat kidney expresses two forms of glutaminase (GA) mRNA which probably result from the use of alternative polyadenylation signals. The two mRNAs are increased coordinately in response to metabolic acidosis via a mechanism that apparently does not involve transcriptional or translational regulation. A 956-bp fragment that contains the 3'-nontranslated sequence of the smaller GA cDNA was cloned into an expression vector (p beta G) that encodes a chimeric beta-globin growth hormone mRNA. Both the parent and the derived construct (p beta G-GA) were transfected into LLC-PK1-F+ cells. Stable transfectants express sixfold lower levels of beta G-GA mRNA than that of the parent beta G mRNA. However, only the beta G-GA mRNA is increased 2.5-fold by growth in acidic medium (pH 6.9, 10 mM HCO3-). The apparent half-life of the beta G mRNA (> 24 h) is unaffected by the pH of the growth media. In contrast, the apparent half-life of the beta G-GA mRNA is increased from 4.5 h to approximately 24 h when cells are transferred to acidic medium for 8 h. The observed pH response is not reproduced when the beta G-GA construct is stably transfected into COS-7 cells or when a beta-globin-phosphoenolpyruvate carboxykinase chimeric gene is expressed in LLC-PK1-F+ cells. Thus the 3'-nontranslated region of the GA mRNA contains a pH-responsive stability element.

Animals↗

PubMed1996

Promoter elements that mediate the pH response of PCK mRNA in LLC-PK1-F+ cells.

The onset of metabolic acidosis causes an increased transcription of the renal phosphoenolpyruvate carboxykinase (PCK) gene. When transgenic mice carrying a bovine growth hormone (bGH) gene driven by the -460 to +73 segment of the PCK promoter were made chronically acidotic, the bGH mRNA was increased twofold after 4 days. Confluent and well-differentiated cultures of LLC-PK1-F+ cells exhibit a 2.5-fold increase in PCK mRNA when transferred to acidic media (pH 6.9, 10 mM HCO3-) for 16 h. Confluent cultures transfected with PCK-490 CAT exhibit an increase (3.5-fold) in chloramphenicol acetyltransferase (CAT) activity when shifted to acidic medium for 48 h. Mutation or deletion of the P2 element causes a four- to fivefold decrease in basal CAT activity but does not affect the pH response. In contrast, mutations of the P3(II) element or the CRE-1 cAMP-response element have little effect on basal activity but cause a 50% decrease in the pH response. Other deletions or mutations have little effect on either activity. Thus changes in the activity or levels of the protein(s) in the renal proximal tubule that binds to the P3(II) and CRE-1 elements may mediate increased transcription of the PCK gene during metabolic acidosis.

Animals↗

PubMed1996

cAMP activation of phosphoenolpyruvate carboxykinase transcription in renal LLC-PK1-F+ cells.

Phosphoenolpyruvate carboxykinase (PCK) is a key regulatory enzyme in renal ammoniagenesis and gluconeogenesis. LLC-PK1-F+ cells are porcine renal proximal tubule-like cells that express significant levels of the cytosolic PCK. Treatment of subconfluent LLC-PK1-F+ cells with 0.1 mM 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) for 8 h causes a 21-fold increase in PCK mRNA. This response is very rapid and is not inhibited by 0.5 mM cycloheximide, indicating that ongoing protein synthesis is not required. Similarly, cells transfected with PCK(-490)CAT exhibit an 8- to 10-fold increase in chloramphenicol acetyltransferase (CAT) activity when treated with cAMP for 24 h. The addition of okadaic acid, a protein phosphatase inhibitor, both stimulated the CAT activity and potentiated the cAMP effect by twofold, suggesting that phosphorylation may contribute to the transcriptional activation. Assays using a series of PCK-CAT constructs containing specific deletions or block mutations established that the CRE-1 the P3(II) elements are required for the cAMP response. Cotransfection experiments using dominant negative expression vectors indicated that a CCAAT enhancer binding protein (C/EBP) transcription factor, and not CREB, mediates cAMP activation of transcription in LLC-PK1-F+ cells.

Animals↗

PubMed1995

In vitro characterization of the mitochondrial processing and the potential function of the 68-kDa subunit of renal glutaminase.

Rat renal mitochondrial glutaminase (GA) is initially synthesized in primary cultures of proximal tubule cells as a 74-kDa precursor and is processed via a 72-kDa intermediate to generate a heterotetrameric enzyme which contains three 66-kDa subunits and one 68-kDa subunit (Perera, S. Y., Chen, T. C., and Curthoys, N. P. (1990) J. Biol. Chem. 265, 17764-17770). The two mature subunits may be derived by either of two possible mechanisms: 1) alternative proteolytic processing or 2) initial synthesis of the 66-kDa subunit followed by its covalent modification to generate the 68-kDa subunit. An in vitro system was utilized to further characterize this unique processing pathway and to investigate the potential function of the 68-kDa subunit. In vitro transcription and translation of the GA cDNA yields a single 74-kDa precursor. Upon incubation with isolated rat liver mitochondria, the precursor is translocated into the mitochondria and processed via a 72-kDa intermediate to yield a 3:1 ratio of the 66- and 68-kDa subunits, respectively. The kinetics of the in vitro processing reaction also closely approximate the kinetics observed in cultured cells. Mitochondrial processing is blocked by o-phenanthroline, an inhibitor of the matrix processing peptidase (MPP). The 72-amino acid presequence of the 66-kDa subunit contains a large proportion of basic amino acids. Two-dimensional gel electrophoresis of mature GA established that the 68-kDa subunit is slightly more basic than the 66-kDa subunit. In addition, incubation of the 74-kDa precursor with purified MPP yields equimolar amounts of the two mature peptides. A cDNA construct, p delta GA, was created which lacks the nucleotides that encode the amino acid residues 32 through 72 of GA. When transcribed and translated in vitro, p delta GA yields a 70-kDa precursor. This precursor is processed by mitochondria to a single mature subunit with a M of 66 kDa. This observation suggests that the 68-kDa subunit is not produced by covalent modification of the 66-kDa subunit and further supports the conclusion that the two mature subunits of GA are produced by alternative processing reactions which can be catalyzed by MPP. However, the yield of products obtained in intact mitochondria may be determined by some unidentified accessory factor. Submitochondrial fractionation of imported GA and delta GA precursors suggest that the 68-kDa subunit may function to retain the mature GA within the mitochondrial matrix.

Animals↗

PubMed1995

Role of the N-terminal 118 amino acids in the processing of the rat renal mitochondrial glutaminase precursor.

Rat renal mitochondrial glutaminase (GA) is synthesized as a 74-kDa cytosolic precursor that is translocated into mitochondria and processed via a 72-kDa intermediate to yield a 3:1 ratio of mature 66- and 68-kDa subunits, respectively. The 66-kDa subunit is derived by removal of a 72-amino-acid presequence. The structural determinants necessary for translocation and proteolytic processing were further delineated by characterizing the processing of different chimeric constructs formed by fusing various segments of the N-terminal sequence of the GA precursor to chloramphenicol acetyl transferase (CAT). GA1-118 CAT is translocated and processed in isolated rat liver mitochondria or cleaved by purified mitochondrial processing peptidase (MPP) to yield an intermediate peptide and two mature subunits that are analogous to the products of processing of the GA precursor. The two reactions also occur with kinetics which are similar to those observed for processing of the GA precursor. Thus, all of the information required for the translocation and synthesis of the mature subunits of GA reside in the N-terminal 118 amino acids of the GA precursor. In contrast, GA1-72 CAT, a construct that contains the GA presequence fused to CAT, is apparently translocated and processed less efficiently. It yields only two peptides that are analogous to the intermediate and 68 kDa forms of GA. In addition, GA1-31 CAT associates with mitochondria but is not proteolytically processed and GA1-31,73-118 CAT is slowly translocated and processed to a single peptide that is analogous to the 66 kDa form of GA. The latter results suggest that the MPP cleavage reactions which yield the GA intermediate and the 66-kDa subunit depend primarily on information that is present C-terminal to the respective sites of cleavage.

Amino Acid Sequence↗