scholarly journals Reconstitution, morphology and crystallization of a fatty acid β-oxidation multienzyme complex from Pseudomonas fragi

1997 ◽  
Vol 328 (3) ◽  
pp. 815-820 ◽  
Author(s):  
Momoyo ISHIKAWA ◽  
Yuriko MIKAMI ◽  
Jiro USUKURA ◽  
Hiroshi IWASAKI ◽  
Hideo SHINAGAWA ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Gene Product ◽  
Multienzyme Complex ◽  
Β Subunit ◽  
Pseudomonas Fragi ◽  
Α Subunit ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Α And Β Subunits ◽  
Product Forms ◽  
Enoyl Coa Hydratase

The fatty acid β-oxidation multienzyme complex from Pseudomonas fragi, HDT, exhibits predominantly the three enzymic activities of 2-enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The HDT complex is encoded by the faoAB operon, consisting of the faoA and faoB genes that encode two individual constituents, the α-subunit and the β-subunit. We have constructed Escherichia coli overexpression systems for the faoAB gene product (coexpression of the α- and β-subunits), the α-subunit alone and the β-subunit alone, and have purified the three respective products. Gel-filtration analysis revealed that the faoAB gene product forms a heterotetrameric structure, α2β2, identical with the native HDT oligomeric state from P. fragi, whereas the α-subunit and β-subunit individually form dimers. Electron microscopy demonstrated that each protein morphologically adopts the above oligomeric structures. The HDT complex, reconstituted in vitro from the isolated α- and β-subunits, exhibits the three original enzymic activities and yields the same crystal as those from the native enzyme. CD measurements indicated that the α- and β-dimers hardly alter their global conformations upon the formation of the HDT complex. Interestingly, the β-dimer alone does not exhibit 3-oxoacyl-CoA thiolase activity, whereas the α-dimer alone exhibits both the 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities. These results suggest that the contact between the α- and β-subunits is essential for the thiolase activity. We have identified several structurally important proteolytic sites within each subunit, which are protected in the intact heterotetrameric molecule. These findings allow the possible location of the interface between the two subunits, which should be crucial for the exhibition of thiolase activity.

scholarly journals Acquired deficiency of the peroxisomal enzyme enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase is a metabolic vulnerability in hepatoblastoma

2020 ◽  
Author(s):  
Huabo Wang ◽  
Xiaoguang Chen ◽  
Marie Schwalbe ◽  
Joanna E. Gorka ◽  
Jordan A. Mandel ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Intracellular Transport ◽  
Metabolic Reprogramming ◽  
Acid Oxidation ◽  
Small Magnitude ◽  
Peroxisomal Enzyme ◽  
Human Cancers ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Pathway Inhibition ◽  
Enoyl Coa Hydratase

AbstractMetabolic reprogramming provides transformed cells with proliferative and/or survival advantages. However, capitalizing on this therapeutically has been only moderately successful due to the relatively small magnitude of these differences and because cancers may re-program their metabolism to evade metabolic pathway inhibition. Mice lacking the peroxisomal bi-functional enzyme enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) and supplemented with the 12-carbon fatty acid lauric acid (C12) accumulate dodecanedioic acid (DDDA), a toxic C12 metabolite that causes hepatocyte necrosis and acute liver failure. In a murine model of pediatric hepatoblastoma (HB), down-regulation of Ehhadh also occurs in combination with a more general suppression of mitochondrial β- and peroxisomal ω-fatty acid oxidation (FAO) pathways. HB-bearing mice provided with C12 and/or DDDA-supplemented diets survived significantly longer than those on standard diets. The tumors also developed massive necrosis in response to short-term DDDA supplementation. Reduced Ehhadh was noted in murine hepatocellular carcinomas (HCCs) and in substantial subsets of human cancers, including HCCs. Acquired DDDA resistance was not associated with Ehhadh re-expression but was associated with 129 transcript differences ~90% of which were down-regulated in DDDA-resistant tumors and ~two-thirds of which correlated with survival in several human cancers. These transcripts often encoded components of the extracellular matrix suggesting that DDDA resistance arises from its reduced intracellular transport. Our results demonstrate the feasibility of a metabolic intervention that is non-toxic, inexpensive and likely compatible with traditional therapies. C12 and/or DDDA-containing diets could potentially be used to supplement other treatments or as alternative therapeutic choices.

Heterogeneity of human luteinizing hormone. Detection and identification of α- and β-subunits in international reference preparations

1987 ◽  
Vol 114 (4) ◽  
pp. 572-576 ◽  
Author(s):  
L. A. van Ginkel ◽  
J. G. Loeber
Keyword(s):  
Luteinizing Hormone ◽  
Biologically Active ◽  
Β Subunit ◽  
Charge Heterogeneity ◽  
Α Subunit ◽  
Isoelectric Focussing ◽  
International Reference ◽  
Detection And Identification ◽  
Α And Β Subunits ◽  
Two Peaks

Abstract. The charge heterogeneity of three international reference preparations containing biologically active human luteinizing hormone (LH) or its free α-or β-subunits, respectively, was investigated with isoelectric focussing in sucrose density gradients. The immunoreactive profiles throughout the pH-gradient were assessed with radioimmunoassay (RIA) systems specific for intact, i.e. undissociated, LH or free α- or β-subunits. For the preparation of intact LH (MRC 68/40), two peaks were found at pH = 7.69 and 8.05; for the α-subunit preparation (NIBSC 78/554), the peak values were 4.76, 5.04, 5.94, 6.70, 6.96, 7.35, 8.02, 8.72 and 9.32; and for the β-subunit preparation (NIBSC 78/556), the values were 7.60, 8.40, 8.55 and 9.61. These results are in excellent agreement with those obtained for a highly purified LH preparation (NM 14) which was prepared by one of us and on which we have reported earlier. In addition, with the abovementioned techniques, the spontaneous dissociation of MRC 68/40 into subunits upon incubation at 37°C in phosphate buffer was clearly demonstrated by the increased immunoreactivity in the profiles assessed with both RIA-subunit systems. It is concluded that charge heterogeneity of LHi, α- and β-subunits, as observed for different preparations, is confined to a limited population of forms. Differences between preparations are only quantitative. A single preparation, therefore, can be used as a general model for the study of human luteinizing hormone.

scholarly journals Amino acid sequence of the α subunit and computer modelling of the α and β subunits of echicetin from the venom of Echis carinatus (saw-scaled viper)

1997 ◽  
Vol 323 (2) ◽  
pp. 533-537 ◽  
Author(s):  
János POLGÁR ◽  
Edith M. MAGNENAT ◽  
Manuel C. PEITSCH ◽  
Timothy N. C. WELLS ◽  
Mansoor S. A. SAQI ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Computer Modelling ◽  
Platelet Glycoprotein ◽  
Β Subunit ◽  
Α Subunit ◽  
Echis Carinatus ◽  
Α And Β Subunits ◽  
Lectin Protein ◽  
Von Willebrand

Echicetin, a heterodimeric protein from the venom of Echis carinatus, binds to platelet glycoprotein Ib (GPIb) and so inhibits platelet aggregation or agglutination induced by various platelet agonists acting via GPIb. The amino acid sequence of the β subunit of echicetin has been reported and found to belong to the recently identified snake venom subclass of the C-type lectin protein family. Echicetin α and β subunits were purified. N-terminal sequence analysis provided direct evidence that the protein purified was echicetin. The paper presents the complete amino acid sequence of the α subunit and computer models of the α and β subunits. The sequence of α echicetin is highly similar to the α and β chains of various heterodimeric and homodimeric C-type lectins. Neither of the fully reduced and alkylated α or β subunits of echicetin inhibited the platelet agglutination induced by von Willebrand factor–ristocetin or α-thrombin. Earlier reports about the inhibitory activity of reduced and alkylated echicetin β subunit might have been due to partial reduction of the protein.

Interaction of α- and β-subunits in native H-K-ATPase and cultured cells transfected with H-K-ATPase β-subunit

2000 ◽  
Vol 278 (4) ◽  
pp. C727-C738 ◽  
Author(s):  
Curtis T. Okamoto ◽  
Dar C. Chow ◽  
And John G. Forte
Keyword(s):  
Cell Surface ◽  
Cultured Cells ◽  
Mdck Cells ◽  
Live Cells ◽  
Β Subunit ◽  
Stringent Requirement ◽  
Α Subunit ◽  
Α And Β Subunits ◽  
Darby Canine Kidney

The assembly of the β-subunit of the gastric H-K-ATPase (HKβ) with the α-subunit of the H-K-ATPase or the Na-K-ATPase (NaKα) was characterized with two anti-HKβ monoclonal antibodies (MAbs). In fixed gastric oxyntic cells, in H-K-ATPase in vitro, and in Madin-Darby canine kidney (MDCK) cells transfected with HKβ, MAb 2/2E6 was observed to bind to HKβ only when interactions between α- and β-subunits were disrupted by various denaturants. The epitope for MAb 2/2E6 was mapped to the tetrapeptide S226LHY229 of the extracellular domain of HKβ. The epitope for MAb 2G11 was mapped to the eight NH2-terminal amino acids of the cytoplasmic domain of HKβ. In transfected MDCK cells, MAb 2G11 could immunoprecipitate HKβ with α-subunits of the endogenous cell surface NaKα, as well as that from early in the biosynthetic pathway, whereas MAb 2/2E6 immunoprecipitated only a cohort of unassembled endoglycosidase H-sensitive HKβ. In HKβ-transfected LLC-PK1 cells, significant immunofluorescent labeling of HKβ at the cell surface could be detected without postfixation denaturation or in live cells, although a fraction of transfected HKβ could also be coimmunoprecipitated with NaKα. Thus assembly of HKβ with NaKα does not appear to be a stringent requirement for cell surface delivery of HKβ in LLC-PK1 cells but may be required in MDCK cells. In addition, endogenous posttranslational regulatory mechanisms to prevent hybrid α-β heterodimer assembly appear to be compromised in transfected cultured renal epithelial cells. Finally, the extracellular epitope for assembly-sensitive MAb 2/2E6 may represent a region of HKβ that is associated with α-β interaction.

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