Evidence for distinct functional molecular sizes of carnitine palmitoyltransferases I and II in rat liver mitochondria.
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ABSTRACT:
SUBMITTER: Zammit VA
PROVIDER: S-EPMC1148872 | biostudies-other | 1988 Mar
REPOSITORIES: biostudies-other
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Project description:The submitochondrial distribution of the overt and latent carnitine palmitoyltransferases (CPT I and II respectively) of rat liver mitochondria were studied. Separation of outer and inner membranes, as well as of a fraction of intermediate density consisting of contact sites between the two membranes, was achieved, as judged by the distribution of marker enzymes. Both CPT I and CPT II were found to be enriched within the contact- site fraction of mitochondria. These data show that the two carnitine acyltransferases are distributed non-uniformly within their respective membranes, and that subpopulations of the two enzymes occur in close proximity within the mitochondrial membrane structure, while retaining their different accessibilities to cytosolic and matrix pools of metabolites. As the number of contact sites is known to vary with changes in the energy status of mitochondria, the possibility that such changes may acutely affect the proportion of CPT I within the distinctive lipid environment of the contact sites, and thus its overall kinetic characteristics, is discussed.
| S-EPMC1219035 | biostudies-other
Project description:While the brain's high energy demands are largely met by glucose, brain is also equipped with the ability to oxidize fatty acids for energy and metabolism. The brain expresses the carnitine palmitoyltransferases (CPTs) that mediate carnitine-dependent entry of long-chain acyl-CoAs into the mitochondrial matrix for ?-oxidation - CPT1a and CPT2 located on the outer and inner mitochondrial membranes, respectively. Their developmental profile, regional distribution and activity as well as cell type expression remain unknown. We determined that brain CPT1a RNA and total protein expression were unchanged throughout post-natal development (PND0, PND7, PND14, PND21 and PND50); however, CPT2 RNA peaked at PND 21 and remained unchanged through PND50 in all regions studied (cortex, hippocampus, midbrain, and cerebellum). Both long-chain acyl CoA dehydrogenase and medium acyl-CoA dehydrogenase showed a similar developmental profile to CPT2. Acylcarnitines, generated as a result of CPT1a activity, significantly increased with age and peaked at PND21 in all brain regions, concurrent with the increased expression of enzymes involved in mitochondrial ?-oxidation. The CPT system is highly enriched in vivo in hippocampus and cerebellum, relative to cortex and midbrain, and is exclusively present in astrocytes and neural progenitor cells, while absent in neurons, microglia, and oligodendrocytes. Using radiolabeled oleate, we demonstrate regional differences in brain fatty acid oxidation that may be blocked by the irreversible CPT1a inhibitor etomoxir. This study contributes to the field of knowledge in brain cell-specific metabolic pathways, which are important for understanding normal brain development and aging, as well as pathophysiology of neurological diseases. Read the Editorial Comment for this article on page 347.
| S-EPMC5927624 | biostudies-literature
Project description:Carnitine palmitoyltransferase (CPT) I is expressed in the intestine of suckling rats; its mRNA increases very rapidly after birth, remains on a plateau until day 18 and decreases until weaning, when basal (adult) values are reached, which remain unchanged thereafter. CPT II mRNA values do not show any appreciable change in this period. CPT I and CPT II are expressed mainly in mucosa and, to a lesser extent, in the muscular part of the intestine. Intestinal expression of CPT I is maximal in duodenum and jejunum, whereas CPT II is expressed in a similar pattern throughout the whole intestine. Dam's milk may influence the intestinal expression of CPT I, since mRNA levels at birth are low but increase after the first lactation. Moreover, rats weaned at either day 18 or 21 decrease their mRNA levels. Apparently, CPT II gene expression is not influenced by the mother's milk. CPT I and CPT II are also expressed in the liver of suckling rats. Hepatic CPT I is maximal at day 3, and levels of CPT II mRNA do not change, in a similar fashion to that in intestine. The profile of expression of CPT I in liver and intestine strongly resembles that previously reported for mitochondrial 3-hydroxy-3-methyl-glutaryl-CoA synthase.
| S-EPMC1136532 | biostudies-other
Project description:Primary carnitine deficiency is caused by a defect in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. It can cause hypoketotic hypoglycemia or cardiomyopathy in children, and sudden death in children and adults. Fibroblasts from affected patients have reduced carnitine transport. We evaluated carnitine transport in fibroblasts from 358 subjects referred for possible carnitine deficiency. Carnitine transport was reduced to 20% or less of normal in fibroblasts of 140 out of 358 subjects. Sequencing of the 10 exons and flanking regions of the SLC22A5 gene in 95 out of 140 subjects identified causative variants in 84% of the alleles. The missense variants identified in our patients and others previously reported (n = 92) were expressed in CHO cells. Carnitine transport was impaired by 73 out of 92 variants expressed. Prediction algorithms (Polyphen-2, SIFT) correctly predicted the functional effects of expressed variants in about 80% of cases. These results indicate that mutations in the coding region of the SLC22A5 gene cannot be identified in about 16% of the alleles causing primary carnitine deficiency. Prediction algorithms failed to determine the functional effects of amino acid substitutions in this transmembrane protein in about 20% of cases. Therefore, functional studies in fibroblasts remain the best strategy to confirm or exclude a diagnosis of primary carnitine deficiency.
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Project description:The carnitine palmitoyltransferase (CPT) family includes CPT 1 and CPT 2 that transport long-chain fatty acids into the mitochondrial compartment for β-oxidation. In this study, three isoforms (CPT 1α, CPT 1β and CPT 2) of the CPT family were cloned from Chinese mitten crab (Eriocheir sinensis) and their complete coding sequences (CDS) were obtained. Sequence analysis revealed deduced amino acid sequences of 915, 775 and 683 amino acids, respectively. Gene expression analysis revealed a broad tissue distribution for all three isoforms, with high CPT 1α and CPT 2 mRNA levels in the hepatopancreas of males and females. In males, CPT 1β was highly expressed in gill, heart, brain ganglia and muscle, while in females, CPT 1β-mRNA levels were relatively high in muscle, hepatopancreas and ovary tissue. The effects of dietary fish oil replacement on the expression of the three CPT isoforms in the hepatopancreas during gonadal development were investigated using five experimental diets formulated with replacement of 0, 25, 50, 75 and 100% fish oil by 1:1 rapeseed oil: soybean oil. The results showed that Diets 2# and 5# yielded higher CPT 1α and CPT 2 mRNA expression in males (P < 0.05), while in females, expression of all three CPT isoforms increased then declined in the hepatopancreas with increasing dietary fish oil replacement. The observed changes in CPT gene expression varied in different isoforms and gender, suggesting the three CPT genes might play different roles in fatty acid β-oxidation in E. sinensis.
| S-EPMC6070261 | biostudies-literature
Project description:1. A continuously recording and sensitive fluorimetric assay is described for carnitine palmitoyltransferase. This assay has been applied to whole or disintegrated mitochondria and to soluble protein fractions. 2. When rat liver mitochondria had been disintegrated by ultrasound, the specific activity of carnitine palmitoyltransferase was 15-20m-units/mg of protein. Only one-fifth of this activity was assayable (with added substrates) before mitochondrial disintegration. 3. It is concluded that there are two carnitine palmitoyltransferase activities in rat liver mitochondria, of which one (type I) is relatively superficial in location and catalyses an acyl-group transfer between added CoA and carnitine, whereas the other (type II) is less superficial and catalyses an acyl-group transfer in unbroken mitochondria between added carnitine and intramitochondrial CoA. The existence of two distinct carnitine palmitoyltransferases was predicted by Fritz & Yue (1963). 4. In unbroken mitochondria, type I transferase is accessible to the inhibitor 2-bromostearoyl-CoA whereas the type II transferase is inaccessible. 5. A major part of the total carnitine palmitoyltransferase activity of rat liver mitochondria is membrane-bound and of type II. 6. These observations, when considered in conjunction with the penetration of mitochondria by CoASH or carnitine, indicate that the type II transferase is attached to the inner mitochondrial membrane.
| S-EPMC1179386 | biostudies-other
Project description:The circulating, endocrine renin-angiotensin system (RAS) is important to circulatory homeostasis, while ubiquitous tissue and cellular RAS play diverse roles, including metabolic regulation. Indeed, inhibition of RAS is associated with improved cellular oxidative capacity. Recently it has been suggested that an intra-mitochondrial RAS directly impacts on metabolism. Here we sought to rigorously explore this hypothesis. Radiolabelled ligand-binding and unbiased proteomic approaches were applied to purified mitochondrial sub-fractions from rat liver, and the impact of AngII on mitochondrial function assessed. Whilst high-affinity AngII binding sites were found in the mitochondria-associated membrane (MAM) fraction, no RAS components could be detected in purified mitochondria. Moreover, AngII had no effect on the function of isolated mitochondria at physiologically relevant concentrations. We thus found no evidence of endogenous mitochondrial AngII production, and conclude that the effects of AngII on cellular energy metabolism are not mediated through its direct binding to mitochondrial targets.
| S-EPMC3747509 | biostudies-literature
Project description:Carnitine palmitoyltransferase II (CPT-II) has a crucial role in the beta-oxidation of long-chain fatty acids in mitochondria. We report here the crystal structure of rat CPT-II at 1.9A resolution. The overall structure shares strong similarity to those of short- and medium-chain carnitine acyltransferases, although detailed structural differences in the active site region have a significant impact on the substrate selectivity of CPT-II. Three aliphatic chains, possibly from a detergent that is used for the crystallization, were found in the structure. Two of them are located in the carnitine and CoA binding sites, respectively. The third aliphatic chain may mimic the long-chain acyl group in the substrate of CPT-II. The binding site for this aliphatic chain does not exist in the short- and medium-chain carnitine acyltransferases, due to conformational differences among the enzymes. A unique insert in CPT-II is positioned on the surface of the enzyme, with a highly hydrophobic surface. It is likely that this surface patch mediates the association of CPT-II with the inner membrane of the mitochondria.
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Project description:Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl coenzyme A (CoA) to form 3-ketodihydrosphingosine (KDS). Eukaryotic SPTs comprise tightly membrane-associated heterodimers belonging to the pyridoxal 5'-phosphate (PLP)-dependent alpha-oxamine synthase family. Sphingomonas paucimobilis, a sphingolipid-containing bacterium, contains an abundant water-soluble homodimeric SPT of the same family (H. Ikushiro et al., J. Biol. Chem. 276:18249-18256, 2001). This enzyme is suitable for the detailed mechanistic studies of SPT, although single crystals appropriate for high-resolution crystallography have not yet been obtained. We have now isolated three novel SPT genes from Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Bdellovibrio stolpii, respectively. Each gene product exhibits an approximately 30% sequence identity to both eukaryotic subunits, and the putative catalytic amino acid residues are conserved. All bacterial SPTs were successfully overproduced in Escherichia coli and purified as water-soluble active homodimers. The spectroscopic properties of the purified SPTs are characteristic of PLP-dependent enzymes. The KDS formation by the bacterial SPTs was confirmed by high-performance liquid chromatography/mass spectrometry. The Sphingobacterium SPTs obeyed normal steady-state ordered Bi-Bi kinetics, while the Bdellovibrio SPT underwent a remarkable substrate inhibition at palmitoyl CoA concentrations higher than 100 microM, as does the eukaryotic enzyme. Immunoelectron microscopy showed that unlike the cytosolic Sphingomonas SPT, S. multivorum and Bdellovibrio SPTs were bound to the inner membrane of cells as peripheral membrane proteins, indicating that these enzymes can be a prokaryotic model mimicking the membrane-associated eukaryotic SPT.
| S-EPMC1951810 | biostudies-literature
Project description:A plethora of novel information has emerged over the past decade regarding protein lipidation. The reversible attachment of palmitic acid to cysteine residues, termed S-palmitoylation, has focused a special attention. This is mainly due to the unique role of this modification in the regulation of protein trafficking and function. A large family of protein acyltransferases (PATs) containing a conserved aspartate-histidine-histidine-cysteine motif use ping-pong kinetic mechanism to catalyze S-palmitoylation of a substrate protein. Here, we discuss the topology of PAT proteins and their cellular localization. We will also give an overview of the mechanism of protein palmitoylation and how it is regulated. New information concerning the recent discovery of depalmitoylating enzymes belonging to the family of α/β-hydrolase domain-containing protein 17 (ABHD17A) is included. Considering the recent advances that have occurred in understanding the mechanisms underlying the interplay between palmitoylation and depalmitoylation, it is clear that we are beginning to understand the fundamental nature of how cellular signal-transduction mediates membrane-level organization in health and disease. Impact statement Protein palmitoylation is one of most important reversible post-translational modifications of protein function in cell-signaling systems. This review gathers the latest information on the molecular mechanism of protein palmitoyl transferase action. It also discusses the issue of substrate specificity of palmitoyl transferases. Another important question is the role of depalmitoylation enzymes. This review should help to formulate questions concerning the regulation of activity of particular PATs as well as of depalmitoylating enzymes (APT).
| S-EPMC5478004 | biostudies-literature