Purification and characterization of human liver branched-chain alpha-keto acid dehydrogenase complex

Human liver BCKADH complex was purified. On SDS-polyacrylamide gel electrophoresis, the purified enzyme complex gave three major bands having molecular weights of 51,000, 46,000, and 36,000, and one minor band with a molecular weight of 55,000. The minor band corresponded in molecular weight to lipoamide oxidoreductase which was purified separately. The purified BCKADH represented only approximately 20% of the maximum activity when assayed without addition of exogenous lipoamide oxidoreductase, indicating that lipoamide oxidoreductase component was readily dissociable from the complex. The BCKADH effectively oxidized all of KIV, KIC, and KMV, yielding apparent Km values in the range of 14-17 microM for those alpha-keto acids. Vmax values obtained were 0.86, 0.61, and 0.51 mumole NADH produced/min/mg of protein for KIV, KIC, and KMV, respectively, in the presence of excess amount of lipoamide oxidoreductase. This ratio of Vmax values was practically identical to those of specific activities obtained with respective branched-chain alpha-keto acids at each purification step. The enzyme complex also oxidized pyruvate and alpha-ketoglutarate to a lesser extent. Kinetic experiments gave Km values of 0.98 and 2.9 mM for pyruvate and alpha-ketoglutarate, respectively, with Vmax of 0.43 and 0.08 mumole NADH produced/min/mg of protein. NAD and CoASH were absolutely required for the reaction. Km values for NAD and CoASH were estimated to be 47 and 25 microM, respectively.     

Chest wall and respiratory system mechanics in cats: effects of flow and volume

The effects of inspiratory flow rate and inflation volume on the resistive properties of the chest wall were investigated in six anesthetized paralyzed cats by use of the technique of rapid airway occlusion during constant flow inflation. This allowed measurement of the intrinsic resistance (Rw,min) and overall dynamic inspiratory impedance (Rw,max), which includes the additional pressure losses due to time constant inequalities within the chest wall tissues and/or stress adaptation. These results, together with our previous data pertaining to the lung (Kochi et al., J. Appl. Physiol. 64: 441-450, 1988), allowed us to determine Rmin and Rmax of the total respiratory system (rs). We observed that 1) Rw,max and Rrs,max exhibited marked frequency dependence; 2) Rw,min was independent of flow (V) and inspired volume (delta V), whereas Rrs,min increased linearly with V and decreased with increasing delta V; 3) Rw,max decreased with increasing V, whereas Rrs,max exhibited a minimum value at a flow rate substantially higher than the resting range of V; 4) both Rw,max and Rrs,max increased with increasing delta V. We conclude that during resting breathing, flow resistance of the chest wall and total respiratory system, as conventionally measured, includes a significant component reflecting time constant inequalities and/or stress adaptation phenomena.     

Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver

Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.     

Reduction of the level of the glycine cleavage system in the rat liver resulting from administration of dipropylacetic acid: an experimental approach to hyperglycinemia.

Prolonged administration of dipropylacetic acid, a branched-chain fatty acid, reduced the glycine cleavage activity in the liver of rat to about one-third of the activity in the control rat. The reduction of the activity appeared to be due mainly to reduction of the level of P-protein, a pyridoxal phosphate enzyme, which is responsible for the first step of the glycine cleavage, although dipropylacetic acid was also found to inhibit the activity of P-protein in vitro in a noncompetitive but partially competitive manner with respect to glycine. The rat treated with dipropylacetic acid may provide an experimental approach for the biochemical study of hyperglycinemia which accompanies to metabolic disorders of branchedchain keto acids. In the dipropylacetic acid-treated rat, however, the glycine concentration in the serum was not appreciably elevated and this may be accounted for by the fact that the activities of both the glycine cleavage system and serine dehydratase are considerably higher in the rat liver as compared with those in other animals including human.     

Both induction and activation of the branched-chain 2-oxo acid dehydrogenase complex in primary-cultured rat hepatocytes by clofibrate

Clofibrate administration to rats caused both the activation and induction of the branched-chain 2-oxo acid dehydrogenase complex in the liver; the former phenomenon occurred within the first 6 h after clofibrate administration whereas the latter occurred after 12 h. Essentially the same results were obtained with primary cultures of rat hepatocytes in the presence of 0.5 mM clofibrate, though about three-fourths of the enzyme complex in control cells (without clofibrate addition) was inactivated during a culture for 44 h, with little reduction of the enzyme amount. This was also confirmed by immunotitration analysis with antibodies raised against the purified decarboxylase and transacylase components of the enzyme complex. On the other hand, the activity of dihydrolipoamide dehydrogenase (a constituent of the complex) was little affected by clofibrate administration. The half lives of the decarboxylase and transacylase components in the primary cultures were estimated to be in the range of 22-26 h, and were unchanged in the presence of clofibrate, when determined with the use of cycloheximide and by a pulse-chase experiment. On the contrary, the rates of synthesis of these two enzyme components had increased to about 1.9-fold after 32 h cultivation in the presence of clofibrate. Thus, the increase in the synthesis of both the components resulted in induction of the complex.     

Regulation of hormonal induction of chick liver cytosol-specific phosphoenolpyruvate carboxykinase

Administration of glucagon, epinephrine, or dibutyryl cAMP to chicks induced cytosol-specific phosphoenolpyruvate carboxykinase in liver. In vitro translation assay with poly(A)+RNA indicated that this induction was due to the increase in phosphoenolpyruvate carboxykinase-coding mRNA synthesis which resulted from an increased level of hepatic cAMP. Either hydrocortisone or alpha-adrenergic agonist was ineffective for the induction by itself, but showed a significant effect when administered together with one of the inducing agents given above. In particular, hydrocortisone enhanced the synthesis of phosphoenolpyruvate carboxykinase-specific mRNA without changing the profile of the time courses of the induction and of hepatic cAMP level. Those observations suggest that the fundamental machinery required for induction of cytosol-specific phosphoenolpyruvate carboxykinase in liver is shared in common between rat and chick, and that the absence of appreciable induction of cytosol-specific hepatic phosphoenolpyruvate carboxykinase in starved chicks is due to neither lack nor impairment of the hormone-mediated induction mechanism, but is due to the difference in usage of the genetic information between the two animal species.     

Regulation by induction of branched-chain 2-oxo acid dehydrogenase complex in clofibrate-fed rat liver.

The activity of branched-chain 2-oxo acid dehydrogenase complex increased 3.0-fold in liver of rats fed on 0.1%(w/w) clofibrate. Immunotitration experiments with antibodies against the constituent enzymes of the complex revealed that this increase resulted mainly from the increased amounts of only two(a decarboxylase and a lipoate acyltransferase) of three components of the complex and that the other component(dihydrolipoamide dehydrogenase) remained unchanged in its content, irrespective of clofibrate administration. The increases of both enzyme components were associated with increases in their mRNA levels which were estimated by in vitro translation with poly(A)+ RNA.     

Induction of migration of periodontal ligament cells by selective regulation of integrin subunits

The recruitment of tissue‐resident stem cells is important for wound regeneration. Periodontal ligament cells (PDL cells) are heterogeneous cell populations with stemness features that migrate into wound sites to regenerate periodontal fibres and neighbouring hard tissues. Cell migration is regulated by the local microenvironment, coordinated by growth factors and the extracellular matrix (ECM). Integrin‐mediated cell adhesion to the ECM provides essential signals for migration. We hypothesized that PDL cell migration could be enhanced by selective expression of integrins. The migration of primary cultured PDL cells was induced by platelet‐derived growth factor‐BB (PDGF‐BB). The effects of blocking specific integrins on migration and ECM adhesion were investigated based on the integrin expression profiles observed during migration. Up‐regulation of integrins α3, α5, and fibronectin was identified at distinct localizations in migrating PDL cells. Treatment with anti‐integrin α5 antibodies inhibited PDL cell migration. Treatment with anti‐integrin α3, α3‐blocking peptide, and α3 siRNA significantly enhanced cell migration, comparable to treatment with PDGF‐BB. Furthermore, integrin α3 inhibition preferentially enhanced adhesion to fibronectin via integrin α5. These findings indicate that PDL cell migration is reciprocally regulated by integrin α3‐mediated inhibition and α5‐mediated promotion. Thus, targeting integrin expression is a possible therapeutic strategy for periodontal regeneration.     

A novel mutation in DAX1 gene causing different phenotypes in three siblings with adrenal hypoplasia congenita

Adrenal hypoplasia congenita (AHC) is a rare disease that can be caused by many abnormalities, including an X-linked form. Mutations in the DAX1 gene have been assigned as the genetic cause of AHC. We describe here three siblings with AHC, clinically presented at different ages, two in the neonatal period and one oligosymptomatic during infancy. Molecular analysis was able to detect a novel mutation in exon 1 of the DAX1 gene, consisting of a transition of C to T at position 359, determining a stop codon at position 359 (Q359X). The mutated gene encodes a truncated protein missing a large portion of the ligand-binding domain (C-terminal domain). The recognition of the disease in the index case suggested the diagnosis in the other siblings. Interestingly, the same mutation is presented with different phenotypes, suggesting that first-degree family members of patients with DAX1 mutations should be carefully evaluated routinely.