Functional characterization and mechanism of action of recombinant human kynurenine 3-hydroxylase.

The mitochondrial outer membrane enzyme kynurenine 3-hydroxylase (K3H) is an NADPH-dependent flavin mono-oxygenase involved in the tryptophan pathway, where it catalyzes the hydroxylation of kynurenine. K3H was transiently expressed in COS-1 cells as a glutathione S-transferase (GST) fusion protein, and the pure recombinant protein (rec-K3H) was obtained with a specific activity of about 2000 nmol.min-1.mg-1. Rec-K3H was shown to have an optimum pH at 7.5, to use NADPH more efficiently than NADH, and to contain one molecule of non-covalently bound FAD per molecule of enzyme. The mechanism of the rec-K3H-catalyzed reaction was investigated by overall initial-rate measurements, and a random mechanism in which combination of the enzyme with one substrate does not influence its affinity for the other is proposed. Further kinetic studies revealed that K3H activity was inhibited by both pyridoxal phosphate and Cl-, and that NADPH-catalyzed oxidation occurred even in the absence of kynurenine if 3-hydroxykynurenine was present, suggesting an uncoupling effect of 3-hydroxykynurenine with peroxide formation. This observation could be of clinical interest, as peroxide formation could explain the neurotoxicity of 3-hydroxykynurenine in vivo.

Renal Na/Pi-cotransporters.

Two non-homologous proximal tubular apical Na/Pi-cotransport systems (type I and type II) have been identified thus far by expression cloning. Subsequent studies provided evidence that the type II Na/Pi-cotransporter represents a target for the physiological and pathophysiological regulation of proximal reabsorption of phosphate. The exact role of the type I Na/Pi-cotransporter in proximal Pi-reabsorption and eventually also in the renal handling of other substrates, such as organic anions, is currently less clear and needs further investigation. Evidence was obtained that acute changes of brush border membrane Na/Pi-cotransport involves endo- and exocytic movement of type II Na/Pi-cotransporters. In particular, we elucidated if and how phosphorylation reactions are involved and defined the intracellular structures of the endo/exocytic apparatus involved. At the level of the gene it will be necessary to elucidate its organization in order to understand the mechanisms involved in chronic regulations of Na/Pi-cotransport related to the type II Na/Pi-cotransporter. Furthermore, for structural investigations of these integral membrane proteins, they have to be isolated in sufficient quantities. Thus far the type II cotransporter (NaPi-2) has been expressed in Sf9 insect cells [20], which may eventually allow a purification of this protein.

Functional characterization of leucine transport induced in Xenopus laevis oocytes injected with mRNA isolated from midguts of lepidopteran larvae (Philosamia cynthia).

The injection of poly(A)+ mRNA prepared from Philosamia cynthia midgut caused time- and dose-dependent increases of leucine transport in Xenopus laevis oocytes, with an increase in leucine uptake 1.5-3 times that of oocytes injected with water. When the NaCl concentration was reduced from 100 to 5 mmol l-1, the difference between mRNA- and water-injected oocytes was greater and a fourfold increase of L-leucine uptake was measured. D-Leucine (10 mmol l-1) completely inhibited the induced uptake of 0.1 mmol l-1 L-leucine. The newly expressed component of L-leucine uptake increased at alkaline pH and was abolished by incubation for 15 min with 15 mmol l-1 phenylglyoxal. The mean Km values, calculated using Na+ activation curves of leucine uptake, were 23.3 +/- 6.1 mmol l-1 in water-injected oocytes and 0.4 +/- 0.2 mmol l-1 for the newly expressed component of leucine uptake in mRNA-injected oocytes. On the basis of these results, we conclude that the increase of L-leucine uptake in mRNA-injected oocytes was due to the expression of a new transport system, which differs from the endogenous ones and shares many features with that found previously in Philosamia cynthia midgut.

Expression cloning of human and rat renal cortex Na/Pi cotransport.

We have isolated two cDNA clones, NaPi-2 and NaPi-3, by screening rat kidney cortex and human kidney cortex cDNA libraries, respectively, for expression of sodium-dependent phosphate transport in Xenopus laevis oocytes. Substrate specificity and a detailed kinetic analysis (Na, Pi, H+ concentrations) suggested that expressed uptake activities relate to proximal tubular brush border membrane Na/Pi cotransport. NaPi-2 cDNA contains 2464 bp encoding a protein of 637 aa; NaPi-3 cDNA contains 2573 bp encoding a protein of 639 aa. NaPi-2- and NaPi-3-deduced protein sequences show high homology to each other but are different from the protein sequence deduced from the previously cloned NaPi-1 cDNA (from rabbit proximal tubules). Hydropathy profile predictions suggest at least eight membrane-spanning regions in NaPi-2/3-related proteins. In vitro translation results in proteins of the expected size and suggests glycosylation. Northern blot analysis shows corresponding mRNA species (approximately 2.7 kb) in kidney cortex of various species but no hybridization with RNAs isolated from a variety of other tissues (including intestinal segments); a hybridization signal (approximately 4.8 kb) was observed only in the lung (human). We conclude that we have structurally identified two closely related proteins most likely involved in human and rat renal brush border Na/Pi cotransport.

Poly(A)+ RNA from rabbit intestinal mucosa induces b0,+ and y+ amino acid transport activities in Xenopus laevis oocytes.

Injection of poly(A)+ RNA (mRNA) isolated from rabbit intestinal mucosa into Xenopus laevis oocytes results in an increase in sodium-independent uptake of L-[3H]leucine, L-[35S]cystine, and L-[3H]arginine. This uptake activity is related to an mRNA species corresponding to the recently isolated rabbit kidney cortex cDNA clone rBAT (related to b0,+ amino acid transporter; Bertran, J., Werner, A., Stange, G., Markovich, D., Moore, M. L., Biber, J., Testar, X., Zorzano, A., Palacin, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 281, 717-723) and to a protein involved in amino acid transport via system y+. This conclusion is based on the following observations: 1) mRNA isolated from mucosa of duodenum, jejunum, and ileum, but not from colon, induces sodium-independent uptake of L-leucine, L-cystine, and L-arginine. 2) In Northern blot analysis, mRNA isolated from mucosa of duodenum, jejunum, and ileum, but not from colon, hybridizes to an rBAT cDNA probe, with signals of 2.2-2.3 kilobases and 3.7-3.9 kilobases. 3) mRNA isolated from mucosa of jejunum induces sodium-independent uptake of L-leucine and L-cysteine which shows an inhibition pattern corresponding to system b0,+; the inhibition pattern of mRNA-induced uptake of L-arginine is compatible with the contribution of system b0,+ and y+. 4) Hybrid depletion with an rBAT antisense oligonucleotide greatly prevents the mRNA-dependent induction of uptake of L-cystine (greater than 90%) and of L-leucine (approximately 75%); it reduces to about 50% the induction of L-arginine uptake. 5) After separation of mRNA on a sucrose density gradient, the fractions resulting in expression of b0,+ transport activity were also those hybridizing with rBAT cDNA; induction of transport activity from these fractions was also sensitive to hybrid depletion. 6) The mRNA-induced component of L-arginine uptake which is resistant to rBAT hybrid depletion is inhibited by L-homoserine, only in the presence of sodium; thus, it is related to a system y(+)-like activity.

Stimulation of system y(+)-like amino acid transport by the heavy chain of human 4F2 surface antigen in Xenopus laevis oocytes.

A kidney cortex cDNA clone (rBAT) has recently been isolated, which upon in vitro transcription and capping complementary RNA (cRNA) and injection into Xenopus laevis oocytes induces a system b0,(+)-like amino acid transport activity. This cDNA encodes a type II membrane glycoprotein that shows significant homology to another type II membrane glycoprotein, the heavy chain of the human and mouse 4F2 surface antigen (4F2hc). Here we demonstrate that injection of human 4F2hc cRNA into oocytes results in the activation of a cation-preferring amino acid transport system that appears to be identical to the y(+)-like transport already present in the oocyte. This is based on the following results: (i) Injection of in vitro transcripts from 4F2hc cDNA (4F2hc cRNA) into oocytes stimulates up to 10-fold the sodium-independent uptake of L-arginine and up to 4.1-fold the sodium-dependent uptake of L-leucine. In contrast, 4F2hc cRNA does not increase the basal sodium-independent uptake of L-leucine. (ii) Basal and 4F2hc cRNA-stimulated sodium-independent uptake of L-arginine is completely inhibited by L-leucine in the presence of sodium. Similarly, the basal and 4F2hc cRNA-stimulated sodium-dependent uptake of L-leucine is entirely inhibited by L-arginine. (iii) The stimulation of sodium-independent uptake of L-arginine and the stimulation of sodium-dependent uptake of L-leucine induced by injection of 4F2hc cRNA are both completely inhibited by dibasic L amino acids and to a lesser extent by D-ornithine. (iv) Both basal and 4F2hc cRNA-stimulated sodium-independent uptake of L-arginine show two additional characteristics of the system y+ transport activity: inhibition of L-arginine uptake by L-homoserine only in the presence of sodium and an increase in the inhibition exerted by L-histidine as the extracellular pH decreased. Our results allow us to propose that an additional family of type II membrane glycoproteins (composed by rBAT and 4F2hc) is involved in amino acid transport, either as specific activators or as components of amino acid transport systems.

Age-related modifications of leucine uptake in brush-border membrane vesicles from rat jejunum.

Leucine uptake in brush-border membrane vesicles purified from rat jejunum is sodium-dependent, sensitive to the membrane electrical potential difference and enhanced by the intravesicular presence of potassium. This last effect is not mediated by the genesis of an electrical potential difference, since potassium activation and electrical potential effects are additive. Sodium-dependent leucine Vmax (1568 +/- 91 pmol/mg per 3 s, is higher in young rats than in adult and old animals. The diffusion component of leucine transport decreases with increasing age. Preloading the vesicles with 100 mM KCl increases leucine Vmax 200% in young animals, 100% in adult and 44% in old animals. The potassium activation is a saturation function of the cation concentration. Leucine uptake in brush border membrane from old animals is less sensitive to the electrical potential difference than in membranes from adult and young animals.

Membrane vesicles in the study of transport processes: a critical analysis of the experimental procedure.

A critical analysis of the use of membrane vesicles in the study of cotransport processes is presented. Transport experiments were simulated according to two different models, stressing those conditions that seemed more relevant in affecting the measurements. In particular, we observed that the experimental Vmax values were underestimated. This underevaluation depended on the incubation time employed to measure the initial uptake rate and on the time necessary to wash the vesicles. Also the temperature and the composition of the washing solution, together with the Q10 of the transport process taken into consideration, had a consistent influence on the uptake. All the above mentioned effects were affected by the vesicle volume: the smaller the volume, the greater the underestimate of the uptake. This theoretical analysis underlines, on the one side, that the experimental data should be interpreted with some caution, on the other, that the examined procedure allows an internal check of its validity by adopting suitable simulations of the experiments. The use of the presented models as a tool for the planning and the critical analysis of the experimental results is suggested.