ABSTRACT
Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by 2-enoyl-CoA hydratase-1 (crotonase). Here we report about the enzymological properties of the Glu144Ala and Glu164Ala variants of rat mitochondrial 2-enoyl-CoA hydratase-1. Size-exclusion chromatography and CD spectroscopy showed that the wild-type protein and mutants have similar oligomerization states and folding. The kcat values of the active site mutants Glu144Ala and Glu164Ala were decreased about 2000-fold, but the Km values were unchanged. For study of the potential intrinsic Delta3-Delta2-enoyl-CoA isomerase activity of mECH-1, a new assay using 2-enoyl-CoA hydratase-2 and (R)-3-hydroxyacyl-CoA dehydrogenase as auxiliary enzymes was introduced. It was demonstrated that rat wild-type mECH-1 is also capable of catalyzing isomerization with the activity ratio (isomerization/hydration) of 1/5000. The kcat values of isomerization in Glu144Ala and Glu164Ala were decreased 10-fold and 1000-fold, respectively. The data are in line with the proposal that Glu164 acts as a protic amino acid residue for both the hydration and the isomerization reaction. The structural factors favoring the hydratase over the isomerase reaction have been addressed by investigating the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. The Gln162 side chain is hydrogen bonded to the Glu164 side chain; nevertheless, these mutants have enzymatic properties similar to that of the wild type, indicating that catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on the interactions with the Gln162 side chain.
Subject(s)
Carbon-Carbon Double Bond Isomerases/chemistry , Carbon-Carbon Double Bond Isomerases/genetics , Enoyl-CoA Hydratase/chemistry , Enoyl-CoA Hydratase/genetics , Mutagenesis, Site-Directed , Alanine/genetics , Amino Acid Sequence , Animals , Binding Sites/genetics , Catalysis , Dodecenoyl-CoA Isomerase , Enoyl-CoA Hydratase/biosynthesis , Enzyme Activation/genetics , Glutamic Acid/genetics , Glutamine/chemistry , Glutamine/genetics , Humans , Hydrogen-Ion Concentration , Leucine/genetics , Mitochondria, Liver/enzymology , Molecular Sequence Data , Rats , Rats, Wistar , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purificationABSTRACT
We have investigated the effects of the truncated trkB receptor isoform T1 (trkB.T1) by transient transfection into mouse N2a neuroblastoma cells. We observed that expression of trkB.T1 leads to a striking change in cell morphology characterized by outgrowth of filopodia and processes. A similar morphological response was also observed in SH-SY5Y human neuroblastoma cells and NIH3T3 fibroblasts transfected with trkB.T1. N2a cells lack endogenous expression of trkB isoforms, but express barely detectable amounts of its ligands, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). The morphological change was ligand-independent, since addition of exogenous BDNF or NT-4 or blockade of endogenous trkB ligands did not influence this response. Filopodia and process outgrowth was significantly suppressed when full-length trkB.TK+ was cotransfected together with trkB.T1 and this inhibitory effect was blocked by tyrosine kinase inhibitor K252a. Transfection of trkB.T1 deletion mutants showed that the morphological response is dependent on the extracellular, but not the intracellular domain of the receptor. Our results suggest a novel ligand-independent role for truncated trkB in the regulation of cellular morphology.
Subject(s)
Neuroblastoma/pathology , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptors, Nerve Growth Factor/biosynthesis , 3T3 Cells , Alternative Splicing , Animals , Brain Neoplasms/diagnosis , Brain-Derived Neurotrophic Factor/pharmacology , Cell Differentiation , Cell Size , Genetic Variation , Humans , Mice , Nerve Growth Factors/pharmacology , Peptide Fragments/biosynthesis , Peptide Fragments/genetics , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Pseudopodia , Receptor Protein-Tyrosine Kinases/genetics , Receptor, Ciliary Neurotrophic Factor , Receptors, Nerve Growth Factor/genetics , Sequence Deletion , TransfectionABSTRACT
AIMS: Variants of renin-angiotensin system genes are shown to be associated with cardiovascular pathology. The association between renin-angiotensin system genes and left ventricular mass was investigated in a population-based case-control study. METHODS AND RESULTS: The association between echocardiographic left ventricular mass and both insertion/deletion polymorphism of the angiotensin-converting enzyme gene and the methionine-threonine variant at position 235 of the angiotensinogen gene was studied in a random cohort of 430 hypertensive and 426 control subjects. No differences in the adjusted left ventricular mass values between the different genotypes were seen among either the hypertensive or the control subjects, whether men or women, or in the subgroups of normotensive or physically active subjects. Gene variation had no statistically significant synergistic effect on left ventricular mass values. In control women, the deletion allele of the angiotensin-converting enzyme gene was associated with an increased risk of left ventricular hypertrophy. However, this finding was based on a small number of women with left ventricular hypertrophy and should be interpreted with caution. CONCLUSION: Variations in renin-angiotensin system genes had no major effect on left ventricular mass in this middle-aged population-based cohort of hypertensives and control subjects.
Subject(s)
Cardiac Volume/genetics , Echocardiography , Genetic Variation , Hypertrophy, Left Ventricular/genetics , Renin-Angiotensin System/genetics , Adult , Alleles , Angiotensinogen/genetics , Cardiac Volume/physiology , Case-Control Studies , Chromosome Deletion , Cohort Studies , Female , Genotype , Humans , Hypertension/genetics , Hypertension/physiopathology , Hypertrophy, Left Ventricular/diagnostic imaging , Male , Middle Aged , Peptidyl-Dipeptidase A/genetics , Polymorphism, Genetic , Risk FactorsABSTRACT
The structure of the hexameric rat mitochondrial enoyl-Coenzyme A (CoA) hydratase, co-crystallised with the inhibitor octanoyl-CoA, has been refined at a resolution of 2.4 A. Enoyl-CoA hydratase catalyses the hydration of 2,3-unsaturated enoyl-CoA thioesters. In the crystal structure only four of the six active sites of the hexamer in the asymmetric unit are occupied with a ligand molecule, showing an unliganded and a liganded active site within the same crystal form. While the protein assembly and fold is identical to the previously solved acetoacetyl-CoA complex, differences are observed close to the fatty acid binding pocket due to the different nature of the ligands. The fatty acid tail of octanoyl-CoA is bound in an extended conformation. This is possible because a high B-factor loop, which separates in the acetoacetyl-CoA complex the binding pocket of the acetoacetyl-CoA fatty acid tail from the intertrimer space, has moved aside to allow binding of the longer octanoyl-CoA moiety. The movement of this loop opens a tunnel which traverses the complete subunit from the solvent space to the intertrimer space. The conformation of the catalytic residues is identical, in both structures as well as in the liganded and the unliganded active sites. In the unliganded active sites a water molecules is bound between the two catalytic glutamate, residues Glu144 and Glu164. After superposition of a liganded active site on an unliganded active site this water molecule is close to the carbon centre that becomes hydroxylated in the hydratase reaction. These findings support the idea that the active site is rigid and that the catalytic residues and the water molecule, as seen in the unliganded active site, are pre-positioned for very efficient catalysis.
Subject(s)
Acyl Coenzyme A/chemistry , Coenzyme A/metabolism , Enoyl-CoA Hydratase/chemistry , Fatty Acids/metabolism , Protein Conformation , Acyl Coenzyme A/metabolism , Amino Acid Sequence , Animals , Binding Sites , Coenzyme A/chemistry , Crystallography, X-Ray , Enoyl-CoA Hydratase/metabolism , Fatty Acids/chemistry , Models, Molecular , Molecular Sequence Data , RatsABSTRACT
To investigate whether the polymorphisms in the angiotensin-converting enzyme and angiotensinogen genes are associated with hypertension, we carried out a case-control study of 508 hypertensive and 523 control subjects randomly selected from the Social Insurance Institution register. The cohorts were well characterized and matched for age and sex. The insertion/ deletion polymorphism of the angiotensin-converting enzyme gene and the methionine-->threonine variant at position 235 of the angiotensinogen gene were determined by the polymerase chain reaction technique. The allele frequencies and genotype distributions of both polymorphisms were similar in hypertensive and control subjects. Systolic and diastolic pressures adjusted for age, body mass index, and alcohol consumption did not differ significantly between the different genotypes of the angiotensin-converting enzyme and angiotensinogen genes. The variation at the angiotensinogen and angiotensin-converting enzyme genes did not have any statistically significant synergistic effect on blood pressure levels. In conclusion, the polymorphisms in the reninangiotensin cascade genes do not confer a significantly increased risk for the development of hypertension in this middle-aged, population-based cohort.
Subject(s)
Angiotensinogen/genetics , Blood Pressure/genetics , Genetic Variation , Peptidyl-Dipeptidase A/genetics , Adult , Cohort Studies , Female , Finland/epidemiology , Genes, ras , Genetics, Population , Genotype , Humans , Hypertension/epidemiology , Hypertension/genetics , Male , Middle Aged , Polymerase Chain Reaction , Prevalence , Random AllocationABSTRACT
OBJECTIVE: Variations in the angiotensin converting enzyme (ACE) gene have been implicated in cardiovascular pathology. Therefore, the association between the intima-media thickness (IMT) of the carotid artery and the insertion/ deletion (I/D) polymorphism of the ACE gene was investigated. SUBJECTS: Three hundred men and 300 women were selected randomly from the middle-aged population living in the town Oulu, Finland, of whom 515 subjects (85.8%) participated. METHODS: The IMT of the carotid arteries was determined by bilateral B-mode ultrasonography. IMT values were adjusted for gender, age, height, plasma low-density lipoprotein cholesterol level, smoking and systolic blood pressure. The I/D polymorphism of the ACE gene was determined by polymerase chain reaction. RESULTS: Among non-smokers, the subjects with the DD genotype had significantly higher carotid IMT than did those with II or ID. The association was found also in combined IMT plaque values. In the total population the association was weaker and it was absent in current smokers. Genotype could explain 1.3-2.7% of the variance of carotid IMT in non-smokers. No association between the amount or size of carotid plaques and genotype was observed. CONCLUSIONS: Variations at the ACE gene locus contribute to the degree of the early changes in carotid atherosclerosis in the population. The gene effect is, however, masked by stronger effects of environmental factors such as smoking. The lack of association between atherosclerotic plaques and genotypes may reflect different mechanisms being involved in plaque development and early arterial wall thickening.
Subject(s)
Arteriosclerosis/genetics , Carotid Artery Diseases/genetics , Peptidyl-Dipeptidase A/genetics , Polymorphism, Genetic , Adult , Arteriosclerosis/enzymology , Carotid Artery Diseases/enzymology , Female , Genetic Variation , Genotype , Humans , Male , Middle Aged , Risk FactorsABSTRACT
In the search for new risk factors for diabetic macroangiopathy the insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme gene was studied in 237 consecutive patients (125 men and 112 women) with non-insulin-dependent diabetes. The female population showed an excess of ischemic electrocardiographic changes or definite myocardial infarctions in the patients homozygous for the deletion [D/D; odds ratio (OR) 2.8; 95% confidence interval (CI) 1.4-5.3] and in the insertion/deletion heterozygotes (I/D; OR 1.8; CI 1.1-3.1) compared with the patients homozygous for the insertion (I/I). In the total series coronary heart disease, cerebrovascular disease, and claudication were more often observed in the patients with I/D (OR 1.5; CI 1.0-2.2) or the D/D genotype patients (OR 1.7; CI 1.1-2.6) than in those with the genotype I/I. The systolic blood pressure was lower in patients with genotype I/I (138 +/- 19 mmHg) than in those with the genotype I/D (149 +/- 22 mmHg) or D/D (150 +/- 21 mmHg; P < 0.02). The prevalence of hypertension and the median urinary albumin excretion rate also tended to be lowest in the I/I genotype patients. Multiple logistic analysis revealed that in women the angiotensin-converting enzyme D/D genotype is independently associated with coronary heart disease. Our findings suggest that variation at the angiotensin-converting enzyme gene locus is one of the factors involved in the predisposition of diabetic patients to the development of arterial disease and hypertension.
Subject(s)
Blood Pressure/genetics , Diabetes Mellitus, Type 2/genetics , Diabetic Angiopathies/genetics , Peptidyl-Dipeptidase A/genetics , Polymorphism, Genetic , Adult , Diabetes Mellitus, Type 2/enzymology , Diabetic Angiopathies/enzymology , Electrocardiography , Female , Humans , Male , Middle Aged , Phenotype , Risk Factors , Sex FactorsABSTRACT
We examined the expression of the neurotrophins (NTFs) and their receptor mRNAs in the rat trigeminal ganglion and the first branchial arch before and at the time of maxillary nerve growth. The maxillary nerve appears first at embryonic day (E)10 and reaches the epithelium of the first branchial arch at E12, as revealed by anti-L1 immunohistochemistry. In situ hybridization demonstrates, that at E10-E11, neurotrophin-3 (NT-3) mRNA is expressed mainly in the mesenchyme, but neurotrophin-4 (NT-4) mRNA in the epithelium of the first branchial arch. NGF and brain-derived neurotrophic factor (BDNF) mRNAs start to be expressed in the distal part of the first brachial arch shortly before its innervation by the maxillary nerve. Trigeminal ganglia strongly express the mRNA of trkA at E10 and thereafter. The expression of mRNAs for low-affinity neurotrophin receptor (LANR), trkB, and trkC in trigeminal ganglia is weak at E10, but increases by E11-E12. NT-3, NT-4, and more prominently BDNF, induce neurite outgrowth from explant cultures of the E10 trigeminal ganglia but no neurites are induced by NGF, despite the expression of trkA. By E12, the neuritogenic potency of NGF also appears. The expression of NT-3 and NT-4 and their receptors in the trigeminal system prior to target field innervation suggests that these NTFs have also other functions than being the target-derived trophic factors.