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PubMed2004

Simian retrovirus serogroup 2 constitutive transport element recognizes the ribosomal L10-like protein and translocon gamma subunit-like protein in a yeast three-hybrid assay.

The simian retrovirus (SRV) serogroup 2 genome contains a constitutive transport element (CTE) within its 3' intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA. In a previous report [Virology 264 (1999) 37], CTE RNA-protein complexes were detected using UV-crosslinking/sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To identify these CTE-interacting cellular proteins, we utilized yeast three-hybrid interaction approaches using the complete IR as bait, modified to eliminate transcriptional termination signals recognized by RNA polymerase III, and identified several interactive clones from a Hela cell cDNA activation domain (AD) library. UV-crosslinking of RNA-protein complexes, using Hela cell extracts and the modified IR bait, were conducted prior to library screening, to verify appropriate interaction of CTE RNA-protein complexes. Over one million recombinants were screened, and our yeast hybrid results indicate that the CTE interacts with several molecules involved in cellular translational and translocation machinery, including the ribosomal L10-like protein and the tranlocon protein gamma subunit-like protein. UV-crosslinking/immunoblot assays have verified the interaction of the CTE region with molecules immunologically reactive to antibodies recognizing the ribosomal L10-like protein.

Active Transport, Cell Nucleus↗
PubMed2000

Sam68, RNA helicase A and Tap cooperate in the post-transcriptional regulation of human immunodeficiency virus and type D retroviral mRNA.

Unlike cellular mRNA, retroviral mRNA bypasses the tight coupling of the splicing and nuclear export steps to allow the export of intron-containing viral RNA transcripts to the cytoplasm. Two distinct nuclear export pathways for retroviral mRNA have been described: a CRM-1 dependent pathway mediated by the HIV-1 Rev protein and the Rev Response Element (RRE), and a CRM-1 independent pathway mediated by the Constitutive Transport Element (CTE) of type D retroviruses. Two CTE-binding proteins, RNA helicase A (RHA) and Tap, have been implicated in the nuclear export of CTE-containing RNA. Recently, we reported that expression of RRE-containing RNA could also be mediated by a cellular protein, Sam68, independently of Rev. Here we report evidence that Sam68, RHA and Tap cooperate in the nuclear export of both CTE- and RRE-containing RNA. RHA binds to Sam68 and to Tap both in vivo and in vitro. Over-expression of Sam68 activates both RRE- and CTE-regulated reporter gene expression in human cells and in quail cells in the presence of human Tap. This activation was competitively inhibited by the nuclear transport domain (NTD) of RHA and a transdominant negative mutant of Tap. Conversely, the activation of CTE by Tap in quail cells was inhibited by a transdominant mutant of Sam68 and NTD. We propose that both HIV and type D retroviruses may access the same constitutive RNA nuclear export pathway involving RHA, Tap and Sam68, even though HIV also utilizes the Rev protein for more efficient nuclear export. it is likely that this constitutive export pathway is also used by cellular mRNA, but at a different interface with the splicing process.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Trail making test in chronic toxic encephalopathy: performance and discriminative potential.

The objective of this study was to examine the influence of chronic toxic encephalopathy (CTE) on Trail Making Test (TMT) performance, with special focus on the discriminative potential of this test. We assessed TMT performance in patients diagnosed with CTE, patients with similar symptoms but no diagnosis, and healthy participants. Inferior performance was seen in CTE, and increasing age had a negative effect on TMT performance only for the CTE group. This effect was most pronounced in TMT-B. However, the ability of the TMT to identify CTE was low, whereas all healthy participants were identified as healthy. Thus, the sensitivity of TMT alone was low, but it succeeded in correctly classifying normal subjects. The pattern of results indicates that normal TMT performance may be seen in individuals with mild to moderate brain syndromes, such as CTE, whereas poor performance should not be expected in healthy individuals.

Adult↗
PubMed1998

Audiological disturbances caused by long-term exposure to industrial solvents. Relation to the diagnosis of toxic encephalopathy.

Sixty workers, consecutively admitted due to suspicion of solvent-induced chronic toxic encephalopathy (CTE), were investigated with pure-tone audiometry, determination of speech recognition of monosyllabic words and distorted speech and cortical response audiometry (CRA). Eighteen workers not exposed to occupational solvents and noise were also investigated. The scores in the distorted speech test were significantly lower and the CRA latencies were significantly longer in the solvent group than in the control group. There was no difference between the groups in the pure-tone and monosyllabic speech recognition tests. In the solvent group, 19 subjects had one or several pathological audiological test results (values exceeding the mean result of the control group by 2 SD). Independently of the audiological examination all the workers in the solvent group underwent the traditional clinical assessment of CTE, which is based on symptoms, history of exposure, clinical neurological examination and a neuropsychological investigation. They were classified in three groups--CTE, incipient CTE and non-CTE. There was no correlation between these groups and the audiological test results. A previous report on vestibular pathology in the same group of subjects and the present investigation on hearing deficits suggest that long-term exposure to solvents causes disturbances of the central pathways in the otovestibular system. Hitherto, no attention has been paid to these disturbances in the definition of the CTE syndrome.

Adult↗
PubMed1998

DNA immunization of mice against SIVmac239 Gag and Env using Rev-independent expression plasmids.

Simian immunodeficiency virus (SIV) structural gene expression, including gag and env, strictly depends on the interaction of the viral posttranscriptional regulator Rev with its target RNA, the Rev-responsive element (RRE). A small RNA element, termed the constitutive transport element (CTE), located in the 3' portion of simian retrovirus 1 (SRV-1) mRNA, can efficiently substitute for the human immunodeficiency virus (HIV) Rev-RRE interaction, and thus render HIV expression and replication Rev independent. We tested the ability of the SRV-1 CTE to drive the expression of SIVmac239 env and gag from subgenomic constructs designed for possible use in vaccine trials. In vitro expression studies showed that when the SRV-1 sequence is coupled to the SIV gag and env mRNAs, it functions in an orientation-dependent fashion, and leads to strong expression of SIV Gag and Env in human and monkey cell lines; levels of CTE-mediated protein expression were similar to those obtained with a functional Rev-RRE system. On the other hand, in murine fibroblast-like cells, SIV Gag and Env were expressed from constructs at relatively high levels even in the absence of Rev-RRE; nevertheless, their expression was increased by the presence of the SRV-1 CTE. As reported previously for HIV, the murine cell lines appeared to be defective for Rev-RRE activity, and required overexpression of Rev to induce a Rev response. Intramuscular injection of the gag-CTE and env-CTE constructs in BALB/c mice resulted in the expression of the corresponding mRNAs, and the production of anti-Gag and anti-Env antibodies, thus suggesting that these vectors might be used for genetic immunization approaches.

Animals↗
PubMed2001

Thrombophilic factors in chronic thromboembolic pulmonary hypertension.

Chronic thromboembolic pulmonary hypertension (CTE-PH) is an infrequent cause of pulmonary hypertension that develops in 0.1-0.2% of patients who survive after an acute venous thromboembolic event. According to the largest series so far reported, 15-30% of patients with diagnosis of CTE-PH have an underlying congenital or acquired hypercoagulable state. To determine the prevalence of thrombophilic factors in our population, we analyzed 24 patients admitted to our institution between November 1992 and March 2000 fulfilling criteria for CTE-PH. Eighteen patients disclosed abnormal results in the screening for thrombophilia. The presence of antiphospholipid antibodies (lupus anticoagulant and/or anticardiolipin antibodies) was the abnormality most frequently found (12 out of 24 patients). We found hyperhomocysteinaemia in 7/14, true protein S deficiency in 1/10, protein C deficiency in 1/13, activated protein C resistance in 1/22, antithrombin III deficiency in 1/24, and prothrombin gene G20210A mutation in 1/18 patients. Factor V Leiden was normal in all 18 patients studied. Five patients (20.8%) disclosed more than one thrombophilic abnormality. In conclusion, contrary to the largest series of patients with CTE-PH so far reported, we found that 75% of patients with CTE-PH presented at least one thrombophilic risk factor, being antiphospholipid antibodies in 50% of the cases. We recommend a thorough screening for thrombophilia in all patients with diagnosis of CTE-PH.

Activated Protein C Resistance↗
PubMed1999

The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences.

The constitutive transport element (CTE) encoded by simian type D retroviruses directs unspliced viral RNAs into a nuclear export pathway that is congruent with the pathway used by cellular mRNAs. Here, we show that quail cells are refractory to CTE function but become highly permissive upon expression of the human Tap protein, a candidate CTE cofactor. Tap contains a novel sequence-specific RNA binding domain that is sufficient for CTE binding but inadequate to support CTE function. Using microinjection assays, we have defined two NLSs and one NES in Tap. Mutational inactivation of the Tap NES, which lies outside the RNA-binding domain, not only blocks Tap function but also generates dominant-negative forms of Tap. Whereas replacement of the Tap NES with the well-defined Rev NES rescues the ability of Tap to support CTE function, this substitution also confers sensitivity to agents that block the activity of Crm1, the Rev NES cofactor. Together, these data validate Tap as the first human sequence-specific nuclear mRNA export factor and identify a novel type of NES that can support nuclear mRNA export but does not act via Crm1.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1985

In vivo biological effect of allogeneic cultured thymic epithelium on thymus-dependent immunity in athymic nude rats.

We have extended our previous study of induction of T-lymphocyte immunocompetence in athymic nude rats by cultured epithelium (CTE) of syngeneic origin to that by CTE of allogeneic origin. Immune responsiveness (IgG-class antibody and delayed-type hypersensitivity) after ovalbumin immunization is detectable by 4-6 weeks after transplantation. However, the antibody appears at a slower rate when compared with heterozygous immunocompetent littermates. Seven weeks after transplantation phytohaemagglutinin responsiveness of spleen cells is detectable, and in T-dependent areas of lymphoid organs lymphocytes with helper and non-helper T-cell phenotype are present, but at lower levels than those in heterozygous immunocompetent littermates. Levels comparable to that of immunocompetent rats are reached about 20 weeks after transplantation. Since CTE contains thymocytes, control experiments consisted of transplantation with high numbers of allogeneic freshly isolated thymocytes in athymic nude rats. These animals showed IgG-class antibody formation after ovalbumin immunization, but at lower levels than CTE-treated rats, and were almost negative in T-cell immunocompetence assessed in the other assays. We conclude that CTE of allogeneic origin induces T-cell immunocompetence in athymic nude rats to the level of heterozygous immunocompetent littermates. This study adds to the rationale of CTE transplantation applied in treatment of thymic dysfunction.

Animals↗
PubMed1996

The posttranscriptional control element of the simian retrovirus type 1 forms an extensive RNA secondary structure necessary for its function.

It was previously shown that a 240-nucleotide (nt) RNA element (cis-acting transactivation element [CTE]) located between the env gene and the 3' long terminal repeat of simian retrovirus type 1 (SRV-1) can functionally replace posttranscriptional activation directed by Rev and the Rev-responsive element (RRE) when inserted into a Rev- and RRE-deficient molecular clone of human immunodeficiency virus type 1, resulting in efficient virus replication. Here, we analyze the molecular and structural requirements for function of this RNA element. Deletion mutagenesis demonstrated that the core element spans 173 nt. SRV-2 and Mason-Pfizer monkey virus have highly homologous elements, which function similarly when inserted into the Rev/RRE-deficient human immunodeficiency virus type 1. Computer prediction indicated that the core CTEs of all three viruses have similar extensive secondary structures. Mutagenesis of the SRV-1 CTE revealed that both sequence and secondary structure are essential for function. Nuclease probing of the SRV-1 CTE further supported the genetic analysis and confirmed the predicted structural features of the RNA element. Sequence analysis of the 240-nt SRV-1 CTE, after continuous long-term propagation of the Rev-independent viruses, revealed that the genetically defined core element remained unchanged, while regions outside the core CTE underwent deletions or duplications. These data further support our in vitro mutagenesis data and demonstrate the importance of the sequence and structure of the SRV-1 CTE for appropriate function.

Base Sequence↗
PubMed1999

Identifying the cause of unilateral hypoperfusion in patients suspected to have chronic pulmonary thromboembolism: diagnostic accuracy of helical CT and conventional angiography.

PURPOSE: To determine the prevalence of unilateral hypoperfusion in patients suspected to have chronic thromboembolism (CTE), to identify the most common cause of hypoperfusion, and to compare the accuracy of helical computed tomographic (CT) angiography with that of conventional angiography in helping to determine the cause. MATERIALS AND METHODS: Radionuclide lung scan reports showed asymmetric hypoperfusion in 47 of 410 consecutive patients referred because of suspected CTE. Twenty-seven patients had unilateral or predominantly unilateral perfusion abnormalities. Each pulmonary angiogram and CT angiogram in these patients was interpreted independently by two readers blinded to clinical information and surgical outcome. Surgical confirmation of the diagnosis was available in 39 of the 47 patients with asymmetric hypoperfusion. RESULTS: Unilateral (n = 11) or predominantly unilateral hypoperfusion (n = 16) was found in 6.6% (27 of 410 patients) of patients referred, and CTE was the most common cause. The accuracies of CT angiogram readers (reader 1, 83%; reader 2, 89%) were greater than those of conventional angiogram readers (reader 1, 73%; reader 2, 65%) for distinguishing CTE from other causes. CONCLUSION: Unilateral hypoperfusion occurred in 6.6% of our study population, most frequently because of CTE. CT angiography is an excellent diagnostic alternative to conventional angiography for distinguishing patients with CTE from those with other causes.

Adolescent↗
PubMedDate not supplied

Psychological treatment of patients with chronic toxic encephalopathy: lessons from studies of chronic fatigue and whiplash.

BACKGROUND: Chronic toxic encephalopathy (CTE), which can result from long-term exposure to organic solvents, is characterized by problems of attention and memory, fatigue and affective symptoms. There is little experience with (neuro)psychological treatment in this patient group. We reviewed treatment outcome studies of CTE and comparable syndromes, namely, chronic whiplash-associated disorder (WAD) and chronic fatigue syndrome (CFS), with a view to providing recommendations for the psychological treatment of patients with CTE. METHODS: PubMed and PsychLIT were systematically searched and reference lists of retrieved articles were studied. The articles were classified according to study design and level of evidence. RESULTS: The studies of CFS provided high-level evidence for the effectiveness of cognitive-behavior therapy (CBT) in challenging dysfunctional cognitions regarding the effectiveness of rest and in stimulating graded activity. The studies of WAD were methodologically weaker, and most evaluated a combination of CBT and graded activity training. There was some evidence that changing fatigue- or pain-related behaviors may result in cognitive improvement. Two uncontrolled studies of CTE evaluated cognitive rehabilitation techniques but yielded inconsistent findings. CONCLUSIONS: CBT techniques focusing on changing illness attributions and on stimulating graded activity might be useful for patients with CTE, diminishing fatigue-related problems of concentration and memory. Future studies should evaluate whether cognitive deficits of CTE patients as a result of neurotoxic effects of exposure should be treated by cognitive rehabilitation.

Chronic Disease↗
PubMed2000

Acyl-CoA thioesterases belong to a novel gene family of peroxisome proliferator-regulated enzymes involved in lipid metabolism.

Acyl-CoA thioesterases hydrolyze acyl-CoAs to the corresponding free fatty acid plus coenzyme A. The activity is strongly induced in rat and mouse liver after feeding the animals peroxisome proliferators (PPs). To elucidate the role of these enzymes in lipid metabolism, the authors have cloned the cDNAs corresponding to the inducible cytosolic and mitochondrial type I enzymes (CTE-I and MTE-I), and studied tissue expression and nutritional regulation of expression of the mRNAs in mice. The constitutive expression of both mRNAs was low in liver, with CTE-I expressed mainly in kidney and brown adipose tissue, and MTE-I expressed in brown adipose tissue and heart. As expected, the expression in liver of both the CTE-I and MTE-I mRNAs were strongly induced (> 50-fold) by treatment with clofibrate. A similar level of induction was observed by fasting and a time-course study showed that the CTE-I and MTE-I mRNAs were increased already at 6 h after removal of the diet. Refeeding normal chow diet to mice fasted for 24 h normalized the mRNA levels with a T1/2 of about 3-4 h. Feeding mice a fat-free diet further decreased the expression, possibly indicating repression of expression. The strong expression of MTE-I and CTE-I in the heart was increased about 10-fold by fasting. To further characterize these highly regulated enzymes, the authors have cloned the corresponding genes and promoter regions. The structures of the two genes were found to be very similar, consisting of three exons and two introns. Exon-intron borders conform to general consensus sequences, and, especially, the first exon appears to be highly conserved. The promoter regions of both the CTE-I and MTE-I genes contain putative PP response elements, suggesting an involvement of PP-activated receptors in the regulation of these genes.

Amino Acid Sequence↗
PubMed2000

Involvement of the peroxisome proliferator-activated receptor alpha in regulating long-chain acyl-CoA thioesterases.

Long-chain acyl-CoA thioesterases catalyze the hydrolysis of acyl-CoAs to the corresponding free fatty acid and CoA. We recently cloned four members of a novel multi-gene family of peroxisome proliferator-induced genes encoding cytosolic (CTE-I), mitochondrial (MTE-I), and peroxisomal (PTE-Ia and PTE-Ib) acyl-CoA thioesterases (Hunt et al. 1999. J. Biol. Chem. 274: 34317-34326). As the peroxisome proliferator-activated receptor alpha (PPARalpha) plays a central role in regulating genes involved in lipid metabolism, we examined the involvement of this receptor in regulation of the thioesterases, particularly CTE-I and MTE-I. Northern blot analysis shows that the induction of these thioesterases by clofibrate is mediated through a strictly PPARalpha-dependent mechanism. All four acyl-CoA thioesterases are induced at mRNA level by fasting and using PPARalpha-null mice, it is evident that the increase in CTE-I due to fasting is mainly independent of the PPARalpha in liver and heart. The CTE-I gene responds rapidly to fasting, with induction of mRNA and protein evident after 6 h. This fasting effect is rapidly reversible, with CTE-I mRNA returning almost to control levels after 3 h refeeding, and being further repressed to 20% of control after 9 h refeeding. Although CTE-I mRNA shows a low basal expression in liver, it can be suppressed 90% by feeding a fat-free diet. These data demonstrate that the nutritional regulation of the thioesterases involves the PPARalpha and other signaling pathways responsible for activation and repression. Putative physiological functions for the acyl-CoA thioesterases are discussed.

Animals↗
PubMed2001

[Solvent-induced chronic encephalopathy; the 'solvent team' project].

Long-term exposure to organic solvents can result in damage of the central nervous system. The WHO recognises the following stages: organic affective syndrome, mild chronic toxic encephalopathy (CTE), and severe CTE. There is no golden standard for the diagnosis of CTE. Mild CTE is characterised by fatigue, mood disturbances, memory and attention disorders. In the Netherlands, a so-called 'solvent team' consisting of an occupational physician, a neurologist, a neuropsychologist and an occupational hygienist, assesses patients suspected of having CTE. The diagnostic procedure consists of three steps: (a) interview and blood tests, (b) the computer-based Neurobehavioral Evaluation System, and (c) assessment of an exposure index, neuropsychological investigation, and clinical neurological examination. In the period 1997-1999 approximately 250 patients were assessed yearly in the Netherlands; resulting in 50 diagnoses of mild CTE a year. The real incidence is most likely higher at present, but will decline due to diminished exposure to organic solvents.

Chronic Disease↗
PubMedDate not supplied

[Comparative evaluation of the cytotoxic effect of hydrogen peroxide and tumor necrosis factor alpha on nonischemic and ischemic endothelial cells].

We studied cytotoxic effects (CTE) induced in confluent cultures of human umbilical vein endothelial cells (HUVEC) by initiators of free-radical reactions (FRR): H2O2 (10(-6)-10(-9) M), recombinant human tumor necrosis factor-[symbol; see text] (TNF-alpha, 0.05-100 ng/ml), and a combination of TNF-alpha with low-density lipoproteins (LDL, 100 microgram/ml). HUVEC were incubated with these substances for 6 or 24 h in parallel tests performed under aerobic (CO2-incubator) and ischemic conditions (a mixture of 95% N2 + 5% CO2 in RPMI-1640 medium containing no substrate additives, growth factor or protein). HUVEC viability was determined by counting cells adherent to the bottom of wells after 24 h of reincubation under aerobic conditions in the growth medium (Plating Efficiency Index). The data showed that: 1) CTE of these compounds were dose-dependent (H2O2 and TNF-alpha) and time-dependent (TNF-alpha); 2) CTE of FRR initiators and CTE of ischemia were synergistic, that is, their combination produced a greater decrease HUVEC viability than any substance examined or ischemia alone; 3) CTE of TNF-alpha observed in experiments in substrate-deficient, protein-free medium was considerably stronger than in the growth medium; 4) a combination of TNF-a and LDL caused a stronger CTE on HUVEC than either factor alone, and this synergism was more pronounced during incubation under ischemic conditions. Thus, the data indicate that FRR initiators and TNF-alpha + LDL particularly increase the severity of ischemic injuries of EC and therefore they can be factors which in hypercholesterolemic patiens predispose vascular wall to atherosclerosis.

Cells, Cultured↗
PubMed1999

Nucleocytoplasmic export of type D simian retrovirus genomic RNA: identification of important genetic subregions and interacting cellular proteins.

The simian retrovirus (SRV) genome contains a constitutive transport element (CTE) within its 3' intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA. The serogroup 2 SRV CTE is predicted to form a stable stem-loop structure containing two major internal loops exhibiting 180 degrees inverse symmetry, with loop face sequences A, A', B, and B' and additional minor internal and terminal loops. To begin the identification of potential CTE-interacting proteins and to assess structural requirements for protein interaction, we conducted RNA mobility shift assays using IR fragments that obliterated this region's known stable stem-loop structure. Using immunoblotting assays, we have determined that RNA helicase A, implicated in the nuclear export of unspliced SRV genomic RNA, does not appear to interact directly with either the complete serogroup 2 SRV 3' IR or the subregion RNAs and that formation of RNA-protein complexes is conferred by interaction with other novel proteins. UV crosslinking of RNA-protein complexes, coupled with RNase T1/A digestion, indicates that a novel protein of 120 kDa molecular weight interacts with the complete CTE or with individual subregion RNAs. Transfection analyses indicate that SRV recombinants containing A, A', B, or B' sequences forming the faces for two open loops undergo RNA export; only the complete sense CTE recombinant or a second recombinant containing two subregions in sense orientation that reconstitute the 3' two-thirds of the 3' IR, and contain only A' and B that form the faces for two terminal loops, are capable of SRV RNA export. These experiments indicate that secondary structural determinants of the 3' IR and multiple protein interactions may be important factors in the nuclear export of unspliced SRV RNA.

Animals↗
PubMed1999

Cloning and regulation of peroxisome proliferator-induced acyl-CoA thioesterases from mouse liver.

1.1. Acyl-CoA thioesterases hydrolyze acyl-CoAs to the corresponding free fatty acid plus CoASH. The activity is strongly induced in rat and mouse liver after feeding the animals peroxisome proliferators. To elucidate the role of these enzymes in lipid metabolism, we have cloned the cDNAs corresponding to the inducible cytosolic and mitochondrial type I enzymes (CTE-I and MTE-I) and studied tissue expression and nutritional regulation of expression of the mRNAs in mice. The constitutive expression of both mRNAs was low in liver, with CTE-I being expressed mainly in kidney and brown adipose tissue and MTE-I expressed in brown adipose tissue and heart. As expected, the expression in liver of both the CTE-I and MTE-I mRNAs was strongly induced (> 50-fold) by treatment with clofibrate. A similar level of induction was observed by fasting and a time-course study showed that both mRNAs were increased already at 6 hours after removal of the diet. Refeeding normal chow diet to mice fasted for 24 hours normalized the mRNA levels with a T1/2 of about 3-4 hours. Feeding mice a fat-free diet further decreased the expression, possibly indicating repression of expression. The strong expression of MTE-I and CTE-I in the heart was increased about 10-fold by fasting. To further characterize these highly regulated enzymes, we have cloned the corresponding genes and promoter regions. The structures of the two genes were found to be very similar, consisting of three exons and two introns. Exon-intron borders conform to general consensus sequences and especially the first exon appears to be highly conserved. The promoter regions of both the CTE-I and MTE-I genes contain putative peroxisome proliferator response elements (PPREs), suggesting an involvement of peroxisome proliferator-activated receptors in the regulation of these genes.

Animals↗
PubMed1996

Functional studies of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunits A and B expressed in Escherichia coli: formation of highly active A4 and B4 homotetramers and evidence that aggregation of the B4 complex is mediated by the B subunit carboxy terminus.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (phosphorylating, E.C. 1.2.1.13) (GAPDH) of higher plants exists as an A2B2 heterotetramer that catalyses the reductive step of the Calvin cycle. In dark chloroplasts the enzyme exhibits a molecular mass of 600 kDa, whereas in illuminated chloroplasts the molecular mass is altered in favor of the more active 150 kDa form. We have expressed in Escherichia coli proteins corresponding to the mature A and B subunits of spinach chloroplast GAPDH (GapA and GapB, respectively) in addition to a derivative of the B subunit lacking the GapB-specific C-terminal extension (CTE). One mg of each of the three proteins so expressed was purified to electrophoretic homogeneity with conventional methods. Spinach GapA purified from E. coli is shown to be a highly active homotetramer (50-70 U/mg) which does not associate under aggregating conditions in vitro to high-molecular-mass (HMM) forms of ca. 600 kDa. Since B4 forms of the enzyme have not been described from any source, we were surprised to find that spinach GapB purified from E. coli was active (15-35 U/mg). Spinach GapB lacking the CTE purified from E. coli is more highly active (130 U/mg) than GapB with the CTE. Under aggregating conditions, GapB lacking the CTE is a tetramer that does not associate to HMM forms whereas GapB with the CTE occurs exclusively as an aggregated HMM form. The data indicate that intertetramer association of chloroplast GAPDH in vitro occurs through GapB-mediated protein-protein interaction.

Amino Acid Sequence↗