Biomedical subjects

John C Wallace

Publications and source records attributed to John C Wallace.

At least 19 recordsLinked to original sources

PubMed2007

Precise mapping of an IGF-I-binding site on the IGF-1R.

The IGF-1R [type 1 IGF (insulin-like growth factor) receptor] is activated upon binding to IGF-I and IGF-II leading to cell growth, survival and migration of both normal and cancerous cells. We have characterized the binding interaction between the IGF-1R and its ligands using two high-affinity mouse anti-IGF-1R mAbs (monoclonal antibodies), 7C2 and 9E11. These mAbs both block IGF-I binding to the IGF-1R but have no effect on IGF-II binding. Epitope mapping using chimaeras of the IGF-1R and insulin receptor revealed that the mAbs bind to the CR (cysteine-rich) domain of IGF-1R. The epitope was finely mapped using single point mutations in the IGF-1R. Mutation of Phe241, Phe251 or Phe266 completely abolished 7C2 and 9E11 binding. The three-dimensional structure showed that these residues cluster on the surface of the CR-domain. BIAcore analyses revealed that IGF-I and a chimaeric IGF-II with the IGF-I C-domain competed for the binding of both mAbs with the IGF-1R, whereas neither IGF-II nor a chimaeric IGF-I with the IGF-II C-domain affected antibody binding. We therefore conclude the IGF-I C-domain interacts with the CR (cysteine-rich) domain of the receptor at the cluster of residues Phe241, Phe251 and Phe266. These results allow precise orientation of IGF-I within the IGF-I-IGF-1R complex involving the IGF-I C-domain binding to the IGF-1R CR domain. In addition, mAbs 7C2 and 9E11 inhibited both IGF-I- and IGF-II-induced cancer cell proliferation, migration and IGF-1R down-regulation, demonstrating that targeting the IGF-1R is an effective strategy for inhibition of cancer cell growth.

Animals↗

PubMed2006

Structure, dynamics and heparin binding of the C-terminal domain of insulin-like growth factor-binding protein-2 (IGFBP-2).

Insulin-like growth factor-binding protein-2 (IGFBP-2) is the largest member of a family of six proteins (IGFBP-1 to 6) that bind insulin-like growth factors I and II (IGF-I/II) with high affinity. In addition to regulating IGF actions, IGFBPs have IGF-independent functions. The C-terminal domains of IGFBPs contribute to high-affinity IGF binding, and confer binding specificity and have overlapping but variable interactions with many other molecules. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the solution structure of the C-terminal domain of IGFBP-2 (C-BP-2) and analysed its backbone dynamics based on 15N relaxation parameters. C-BP-2 has a thyroglobulin type 1 fold consisting of an alpha-helix, a three-stranded anti-parallel beta-sheet and three flexible loops. Compared to C-BP-6 and C-BP-1, structural differences that may affect IGF binding and underlie other functional differences were found. C-BP-2 has a longer disordered loop I, and an extended C-terminal tail, which is unstructured and very mobile. The length of the helix is identical with that of C-BP-6 but shorter than that of C-BP-1. Reduced spectral density mapping analysis showed that C-BP-2 possesses significant rapid motion in the loops and termini, and may undergo slower conformational or chemical exchange in the structured core and loop II. An RGD motif is located in a solvent-exposed turn. A pH-dependent heparin-binding site on C-BP-2 has been identified. Protonation of two histidine residues, His271 and His228, seems to be important for this binding, which occurs at slightly acidic pH (6.0) and is more significant at pH 5.5, but is largely suppressed at pH 7.4. Possible preferential binding of IGFBP-2 and its C- domain fragments to glycosaminoglycans in the acidic extracellular matrix (ECM) of tumours may be related to their roles in cancer.

Binding Sites↗

PubMed2006

Computational model for the IGF-II/IGF2r complex that is predictive of mutational and surface plasmon resonance data.

Insulin-like growth factors (IGFs) are key regulators of cell proliferation, differentiation, and transformation, and are thus pivotal in cancer, especially breast, prostate, and colon neoplasm. Their potent mitogenic and anti-apoptotic actions depend primarily on their availability to bind to the signaling IGF cell surface receptors. One mechanism by which IGF-II availability is thought to be modulated is by binding to the nonsignaling IGF-II receptor (IGF2R). This binding is essentially mediated by domain 11 in the multidomain IGF2R extracellular region. The crystal structure of domain 11 of the human IGF-II receptor (IGF2R-d11) has identified a putative IGF-II binding site, and a nuclear magnetic resonance (NMR) solution structure for the IGF-II ligand has also been characterized. These structures have now been used to model in silico the protein-protein interaction between IGF-II and IGF2R-d11 using the program 3D-Dock. Because the IGF-II data comprise an ensemble of 20 structures, all of which satisfy the NMR constraints, the docking procedure was applied to each member of the ensemble. Only those models in which residue Ile1572 of IGF2R-d11, known to be essential for the binding of IGF-II, was at the interface were considered further. These plausible complexes were then critically assessed using an array of analysis techniques including consideration of additional mutagenesis data. One model was strongly supported by these analyses and is discussed here in detail. Furthermore, we demonstrate in vitro experimental support for this model by studying the binding of chimeras of IGF-I and IGF-II to IGF2R fragments.

Binding Sites↗

PubMed2006

Production and characterization of monoclonal antibodies against insulin-like growth factor type 1 receptor.

The type 1 insulin-like growth factor receptor (IGF-1R) has been extensively reported to play an important role in cancer. Activation of the IGF-1R by IGF-I and IGF-II binding to the extracellular domains of the receptor induces mitogenic and anti-apoptotic effects, which are important events in tumor growth and survival. Several cancer cell types overexpress IGF-1R, suggesting a possible use of monoclonal antibodies (MAbs) against IGF-1R as diagnostic reagents. Here, we report the production and characterization of two independent MAbs, namely 7C2 and 9E11, generated by immunizing mice with the soluble extracellular part of this receptor (amino acids 1-906). Both MAbs bind to membrane bound IGF-1R and do not cross-react with insulin receptor isoforms, IR-A and IR-B expressed on IGF-1R() cells. MAbs 7C2 and 9E11 stained the IGF- 1R on frozen or paraffin-embedded tissue sections or frozen cells. The MAbs 7C2 and 9E11 immunoprecipitated the IGF-1R from P6 cell lysates (cells overexpressing human IGF-1R) and could detect non-reduced intact IGF-1R on immunoblots. However, the MAbs were not able to detect reduced and denatured receptor alpha and beta chains. Sequencing of the heavy- and light-chain variable regions revealed that the 7C2 and 9E11 CDR amino acid sequences are different but result in antibodies with similar properties. MAbs 7C2 and 9E11 are therefore potentially useful diagnostic tools and could be of therapeutic use for humans in the future.

Amino Acid Sequence↗

PubMed2005

Differential activation of insulin receptor isoforms by insulin-like growth factors is determined by the C domain.

The actions of IGF-I and IGF-II are thought to be largely due to their activation of the IGF-I receptor. However, IGF-II can also bind with high affinity to, and effectively activate, an isoform of the insulin receptor (IR-A) that lacks a sequence at the carboxyl-terminal end of the extracellular alpha subunit due to the alternative splicing of exon 11. This isoform is poorly activated by IGF-I. Here, we show that IGF-II, but not IGF-I, induces potent autophosphorylation of residues Y1158, Y1162, and Y1163 in the activation loop of the kinase domain and tyrosine 960 in the juxtamembrane region of both IR-A and IR-B (exon 11+) isoforms. We have also found, by using IGF chimeras, that the C domain of IGF-II completely accounts for the ability of IGF-II to stimulate IR autophosphorylation compared with IGF-I. We further show that the C domains are responsible for the differential abilities of IGF-II and IGF-I to activate phosphorylation of insulin receptor substrate-1 and Akt, as well as their ability to induce migration and cell survival via the IR-A. Finally, we show for the first time that IGF signaling through the IR-A can protect cells from butyrate-induced apoptosis. In summary, our studies define the structural determinants that allow potent IGF-II signaling and regulation of cellular functions through the IR-A and provide novel insights into IGF signaling via the IR.

3T3 Cells↗

PubMed2005

Transactivation of CXCR4 by the insulin-like growth factor-1 receptor (IGF-1R) in human MDA-MB-231 breast cancer epithelial cells.

In the multimolecular environment in tissues and organs, cross-talk between growth factor and G protein-coupled receptors is likely to play an important role in both normal and pathological responses. In this report, we demonstrate transactivation of the chemokine receptor CXCR4 by the growth factor insulin-like growth factor (IGF)-1 is required for IGF-1-induced cell migration in metastatic MDA-MB-231 cells. The induction of chemotaxis in MDA-MB-231 cells by IGF-1 was inhibited by pretreatment of the cells with pertussis toxin (PTX) and by RNAi-mediated knockdown of CXCR4. Transactivation of the CXCR4 pathway by IGF-1 occurred independently of CXCL12, the chemokine ligand of CXCR4. Neither CXCR4 knockdown nor PTX had any effect on the ability of IGF-1 to activate IGF-1R, suggesting that CXCR4 and G proteins are activated subsequent to, or independently of, phosphorylation of IGF-1R by IGF-1. Coprecipitation studies revealed the presence of a constitutive complex containing IGF-1R, CXCR4, and the G protein subunits, G(i)alpha2 and Gbeta, and stimulation of MDA-MB-231 cells with IGF-1 led to the release of G(i)alpha2 and Gbeta from CXCR4. Based on our findings, we propose that CXCR4 constitutively forms a complex with IGF-1R in MDA-MB-231 cells, and that this interaction allows IGF-1 to activate migrational signaling pathways through CXCR4, G(i)alpha2 and Gbeta.

Animals↗

PubMed2005

The peroxisome proliferator-activated receptor-gamma regulates murine pyruvate carboxylase gene expression in vivo and in vitro.

Pyruvate carboxylase (PC) plays a crucial role in various metabolic pathways, including gluconeogenesis, lipogenesis, and glucose-induced insulin secretion. Here we showed for the first time that the PC gene is transcriptionally regulated by peroxisome proliferator-activated receptor-gamma (PPARgamma) in vitro and in vivo in white and brown adipose tissue. PC mRNA and protein are markedly increased during differentiation of 3T3-L1 cells and HIB-1B, in parallel with the expression of the adipogenic transcription factors, CCAAT-enhancer binding protein alpha, PPARgamma1, and PPARgamma2. Tumor necrosis factor-alpha, a cytokine that blocks differentiation of 3T3-L1 cells, suppressed PC expression. Co-transfection studies in 3T3-L1 preadipocytes or HEK293T cells with a 2.3-kb promoter fragment of mouse PC gene linked to a luciferase reporter construct and with plasmids overexpressing retinoid X receptor alpha/PPARgamma1 or retinoid X receptor alpha/PPARgamma2 showed a 6-8-fold increase above the basal promoter activity. Furthermore, treatment of these transfected cells with the PPARgamma agonist doubled the promoter activity. Mutation of the putative PPAR-response element-(-386/-374) of this 2.3-kb PC promoter fragment abolished the PPARgamma response. Gel shift and chromatin immunoprecipitation assays demonstrated that endogenous PPARgamma binds to this functional PPAR-response element of the PC promoter. Mice with targeted disruption of the PPARgamma2 gene displayed approximately 50-60% reduction of PC mRNA and protein in white adipose tissue. Similarly, in brown adipose tissue of PPARgamma2-deficient mice subjected to cold exposure, PC mRNA was 40% lower than that of wild type mice. Impaired in vitro differentiation of white adipocytes of PPARgamma2 knock-out mice was also associated with a marked reduction of PC mRNA. Our findings identified PC as a PPARgamma-regulated gene and suggested a role for PPARgamma regulating intermediary metabolism.

3T3-L1 Cells↗

PubMed2005

Involvement of specific proteins (Sp1/Sp3) and nuclear factor Y in basal transcription of the distal promoter of the rat pyruvate carboxylase gene in beta-cells.

Pyruvate carboxylase plays diverse roles in different biosynthetic pathways, including glucose-induced insulin secretion in pancreatic beta-cells. We have localized the control region of the P2 promoter by generating a series of 5'-nested deletion constructs, and both 25- and 9-bp internal deletion constructs, as well as by performing site-directed mutagenesis. Transient transfections of these constructs into INS-1 cells identified a CCAAT box and a GC box that are located at -65/-61 and -48/-41, respectively, as the important determinants. Disruption of the GC box resulted in a 4-fold reduction of the reporter activity, while disruption of the proximal CCAAT box (-65/-61) but not the distal CCAAT box (-95/-91) increased the reporter activity by 3-fold. Simultaneous disruptions of both the GC box and the CCAAT box reduced the reporter activity to a level that was close to that of the single GC box mutation. Electrophoretic mobility shift assays (EMSAs) and supershift EMSAs using nuclear extract from INS-1 cells demonstrated that Sp1 and Sp3 bind a GC box while the nuclear factor Y was shown to bind the proximal but not the distal CCAAT box.

Animals↗

PubMed2004

Structural and functional characteristics of the Val44Met insulin-like growth factor I missense mutation: correlation with effects on growth and development.

We have previously described the phenotype resulting from a missense mutation in the IGF-I gene, which leads to expression of IGF-I with a methionine instead of a valine at position 44 (Val44Met IGF-I). This mutation caused severe growth and mental retardation as well as deafness evident at birth and growth retardation in childhood, but is relatively well tolerated in adulthood. We have conducted a biochemical and structural analysis of Val44Met IGF-I to provide a molecular basis for the phenotype observed. Val44Met IGF-I exhibits a 90-fold decrease in type 1 IGF receptor (IGF-1R) binding compared with wild-type human IGF-I and only poorly stimulates autophosphorylation of the IGF-1R. The ability of Val44Met IGF-I to signal via the extracellular signal-regulated kinase 1/2 and Akt/protein kinase B pathways and to stimulate DNA synthesis is correspondingly poorer. Binding or activation of both insulin receptor isoforms is not detectable even at micromolar concentrations. However, Val44Met IGF-I binds IGF-binding protein-2 (IGFBP-2), IGFBP-3, and IGFBP-6 with equal affinity to IGF-I, suggesting the maintenance of overall structure, particularly in the IGFBP binding domain. Structural analysis by nuclear magnetic resonance confirms retention of near-native structure with only local side-chain disruptions despite the significant loss of function. To our knowledge, our results provide the first structural study of a naturally occurring mutant human IGF-I associated with growth and developmental abnormalities and identifies Val44 as an essential residue involved in the IGF-IGF-1R interaction.

Binding Sites↗

PubMed2004

Binding site for the C-domain of insulin-like growth factor (IGF) binding protein-6 on IGF-II; implications for inhibition of IGF actions.

Insulin-like growth factors (IGFs) are important mediators of growth and IGF-binding proteins (IGFBPs) 1-6 regulate IGF actions. As IGFBP C-terminal domains contribute to high-affinity IGF binding, we have defined the binding site for the C-domain of IGFBP-6 on IGF-II using NMR. This site lies adjacent to and between the binding sites for the IGFBP N-domain and IGF-I receptor (IGFIR), which have previously been found on opposite sides of the IGF molecule. The C-domain is therefore likely to interfere with IGF binding to the IGFIR, providing a structural basis for the potent inhibitory effects of intact IGFBPs on IGF actions.

Binding Sites↗

PubMed2004

Structural determinants for high-affinity binding of insulin-like growth factor II to insulin receptor (IR)-A, the exon 11 minus isoform of the IR.

The insulin receptor (IR) lacking the alternatively spliced exon 11 (IR-A) is preferentially expressed in fetal and cancer cells. The IR-A has been identified as a high-affinity receptor for insulin and IGF-II but not IGF-I, which it binds with substantially lower affinity. Several cancer cell types that express the IR-A also overexpress IGF-II, suggesting a possible autocrine proliferative loop. To determine the regions of IGF-I and IGF-II responsible for this differential affinity, chimeras were made where the C and D domains were exchanged between IGF-I and IGF-II either singly or together. The abilities of these chimeras to bind to, and activate, the IR-A were investigated. We also investigated the ability of these chimeras to bind and activate the IR exon 11+ isoform (IR-B) and as a positive control, the IGF-I receptor (IGF-1R). We show that the C domain and, to a lesser extent, the D domains represent the principal determinants of the binding differences between IGF-I and IGF-II to IR-A. The C and D domains of IGF-II promote higher affinity binding to the IR-A than the equivalent domains of IGF-I, resulting in an affinity close to that of insulin for the IR-A. The C and D domains also regulate the IR-B binding specificity of the IGFs in a similar manner, although the level of binding for all IGF ligands to IR-B is lower than to IR-A. In contrast, the C and D domains of IGF-I allow higher affinity binding to the IGF-1R than the analogous domains of IGF-II. Activation of IGF-1R by the chimeras reflected their binding affinities whereas the phosphorylation of the two IR isoforms was more complex.

3T3 Cells↗

PubMed2004

Expression of human pyruvate carboxylase in insect cells using the baculovirus system.

Constructs containing cDNA encoding human pyruvate carboxylase (PC) with and without a hexahistidine (6x His) tag at the N-terminal of the mature enzyme have been cloned under the control of the polyhedrin promoter. These two constructs were co-transfected with the baculovirus genome into Sf9 cells to produce recombinant baculoviruses harbouring human PC cDNA. The expression of human PC under the control of the polyhedrin promoter was found to be at its highest level at 4 days post-infection. The expressed material accounted for up to 70% of total cellular protein with 5% of this expressed material being found in the soluble fraction. The recombinant human 6x His-PC isolated with a purity of approximately 50% using a Ni-NTA agarose column was found to have the specific activity of 7U/mg, which was similar to that produced from a 293T stable line [Biochem. Biophys. Res. Commun. 266 (1999) 512]. This is the first report of a heterologous expression system for recombinant human PC.

Amino Acid Sequence↗

PubMed2004

Kinetic characterization of yeast pyruvate carboxylase isozyme Pyc1 and the Pyc1 mutant, C249A.

The yeast Pyc1 isoform of pyruvate carboxylase has been further characterized and shown to differ from the Pyc2 isoform in its K(a) for K(+) activation. Pyc1 differs from chicken liver pyruvate carboxylase in the lack of effect of acetyl-CoA on ADP phosphorylation by carbamoyl phosphate, which may be a result of differences in the loci of action of the effector between the two enzymes. Solvent D(2)O isotope effects have been measured with Pyc1 on the full pyruvate carboxylation reaction, the ATPase reaction in the absence of pyruvate, and the carbamoyl phosphate-ADP phosphorylation reaction for the first time for pyruvate carboxylase. Proton inventories indicate that the measured isotope effects are due to a single proton transfer step in the reaction. The inverse isotope effects observed in all reactions suggest that the proton transfer step converts the enzyme from an inactive to an active form. Kinetic measurements on the C249A mutant enzyme suggest that C249 is involved in the binding and action of enzyme activators K(+) and acetyl-CoA. C249 is not involved in ATP binding as was observed for the corresponding residue in the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase, nor is it directly responsible for the measured inverse (D)(k(cat)/K(m)) isotope effects. The size of the inverse isotope effects indicates that they may result from formation of a low-barrier hydrogen bond. Modification of the wild type and C249A mutant with o-phthalaldehyde suggests that C249 is involved in isoindole formation but that the modification of this residue is not directly responsible for the accompanying major loss of enzyme activity.

Acetyl Coenzyme A↗

PubMed2004

Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance.

Host-defence peptides secreted from the skin glands of Australian frogs and toads, are, with a few notable exceptions, different from those produced by anurans elsewhere. This review summarizes the current knowledge of the following classes of peptide isolated and characterized from Australian anurans: neuropeptides (including smooth muscle active peptides, and peptides that inhibit the production of nitric oxide from neuronal nitric oxide synthase), antimicrobial and anticancer active peptides, antifungal peptides and antimalarial peptides. Other topics covered include sex pheromones of anurans, and the application of peptide profiling to (i). recognize particular populations of anurans of the same species and to differentiate between species, and (ii). investigate evolutionary aspects of peptide formation.

Animals↗

PubMed2003

Isolating substrates for an engineered alpha-lytic protease by phage display.

Panning of a substrate phage library with an alpha-lytic protease mutant showed that substrate phage display can be used to isolate sequences with improved protease sensitivity even for proteases of relatively broad specificity. Two panning experiments were performed with an engineered alpha-lytic protease mutant known to have a preference for cleavage after His or Met residues. Both experiments led to the isolation of protease-sensitive phage containing linker sequences in which His and Met residues were enriched compared with the initial library. Despite the relatively hydrophobic substrate binding site of the enzyme, the predominant protease-sensitive sequence isolated from the second library panning had the sequence Asp-Ser-Thr-Met. Kinetic studies showed that this sequence was cleaved up to 4.5-fold faster than rationally designed positive controls. Protease-resistant phage particles were also selected and characterized, with the finding that Gly and Pro appeared frequently at the putative P4 positions, whereas Asp dominated the putative P1 position.

Amino Acid Motifs↗

PubMed2003

The biotin enzyme family: conserved structural motifs and domain rearrangements.

The biotin carboxylase family is comprised of a group of enzymes that utilize a covalently bound prosthetic group, biotin, as a cofactor. These enzymes, which include acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, geranoyl-CoA carboxylase, oxaloacetate decarboxylase, methylmalonyl-CoA decarboxylase, transcarboxylase and urea amidolyase, are found in diverse biosynthetic pathways in both pro-karyotes and eukaryotes. The reactions catalyzed by most members of this group of enzymes share two common features: (1) carboxylation of biotin, apparently via the formation of a carboxyphosphate intermediate, followed by (2) transcarboxylation of CO(2) from biotin to specific acceptor molecules to yield different products. Structural determinations by NMR and X-ray crystallography, complemented by mutagenesis studies, have identified some motifs that are structurally or catalytically important. Analysis of the amino acid sequences of a number of biotin carboxylases not only shows remarkable similarities within certain domains but also that there appears to have been domain rearrangements between groups of carboxylases. Acyl-coenzyme A derivatives, which bind either as substrates or as allosteric regulators of the biotin carboxylases, do not appear to share any of the CoA binding motifs that have been identified in other CoA-SH/acyl-CoA binding proteins. Further comparisons of biotin-dependent carboxylases with other groups of enzymes in the protein data bank reveal that this family of biotin enzymes has strong similarities in specific domains to a number of ATP-utilizing enzymes and to the lipoyl-containing enzymes. These structural homologies are so extensive as to be highly suggestive of evolutionary relationships between biotin carboxylases and these other enzymes.

Acetyl-CoA Carboxylase↗

PubMed2002

Molecular cloning and domain structure of chicken pyruvate carboxylase.

Pyruvate carboxylase (PC) [EC 6.4.1.1] is a biotin-dependent carboxylase that catalyses the conversion of pyruvate to oxaloacetate. Here we have determined the complete nucleotide sequence encoding chicken PC (cPC) by screening a liver cDNA library, by RT-PCR of poly(A)(+) RNA, and by PCR of genomic DNA. The full-length transcript contains an open reading frame of 3537 nucleotides, including the stop codon, encoding a polypeptide of 1178 amino acids with M(r) of 127,262. The amino acid sequence of cPC shows approximately 77% identity to mammalian PC. Limited proteolysis of pure cPC with chymotrypsin yields a major stable 75 kDa C-terminal peptide, including the biotinyl domain and a minor, unstable 39 kDa N-terminal peptide. Northern analysis of poly(A)(+) RNA isolated from chicken liver has shown that cPC's mRNA is approximately 5 kb in length, including a very long 3'-untranslated region.

Amino Acid Sequence↗

PubMed2002

Kinetic characterization of yeast pyruvate carboxylase isozyme pyc1.

Yeast (Saccharomyces cerevisiae) is unusual in being the only organism thus far identified as having two genes for pyruvate carboxylase. The expression of the two isozymes Pyc1 and Pyc2 appears to be differentially regulated, and since both are expressed cytoplasmically, this suggests that they have different properties. To the present, little has been done to characterize these isozymes, and almost all of the published kinetic information on yeast pyruvate carboxylase comes from measurements of enzyme prepared from bakers' yeast which is likely to be a mixture of both isozymes. Here we have measured basic kinetic parameters for Pyc1 and found that the K(a) of this isozyme for acetyl CoA is in the order of 8-10-fold higher than previously recorded, suggesting that Pyc1 and Pyc2 may be differentially regulated by this effector. Pyc1 is highly dependent on the presence of acetyl CoA for activity and in this respect is similar to chicken liver pyruvate carboxylase. However, unlike the chicken liver enzyme, the quaternary structure of the enzyme is quite stable in the absence of acetyl CoA, and the major locus of action of this effector appears to lie outside of the stimulation of the biotin carboxylation reaction.

Acetyl Coenzyme A↗