G. Prezioso

Kinetic characterization of the reconstituted carnitine carrier from rat liver mitochondria

The carnitine carrier was purified from rat liver mitochondria and reconstituted into liposomes by removing the detergent from mixed micelles by Amberlite. Optimal transport activity was obtained with 1 microgram/ml and 12.5 mg/ml of protein and phospholipid concentration, respectively, with a Triton X-100/phospholipid ratio of 1.8 and with 16 passages through the same Amberlite column. The activity of the carrier was influenced by the phospholipid composition of the liposomes, being increased in the presence of cardiolipin and decreased in the presence of phosphatidylinositol. In the reconstituted system the incorporated carnitine carrier catalyzed a carnitine/carnitine exchange which followed a first-order reaction. The maximum transport rate of external [3H]carnitine was 1.7 mmol/min per g protein at 25 degrees C and was independent of the type of countersubstrate. The half-saturation constant (Km) for carnitine was 0.51 mM. The affinity of the carrier for acylcarnitines was in the microM range and depended on the carbon chain length. The activation energy of the carnitine/carnitine exchange was 133 kJ/mol. The carrier function was independent of the pH in the range between 6 and 8 and was inhibited at pH below 6.

A simple and rapid method for the purification of the mitochondrial porin from mammalian tissues.

A new, simple and rapid procedure for the purification of high amounts of mitochondrial porins from different tissues of mammalia is described. The method consists in a single step hydroxyapatite/celite chromatography of Triton X-100 solubilized mitochondrial membranes. For optimal purification several factors are critical such as the absence of salts, a low protein/detergent ratio and an exact hydroxyapatite/celite ratio of 2:1.

KINETIC CHARACTERIZATION OF THE RECONSTITUTED TRICARBOXYLATE CARRIER FROM RAT LIVER MITOCHONDRIA

The tricarboxylate carrier from rat liver mitochondria was purified by chromatography on hydroxyapatite/celite and reconstituted in phospholipid vesicles by removing the detergent using hydrophobic chromatography on Amberlite. Optimal transport activity was obtained by using a Triton X-114/phospholipid ratio of 0.8, 6% cardiolipin and 24 passages through a single Amberlite column. In the reconstituted system the incorporated tricarboxylate carrier catalyzed a first-order reaction of citrate/citrate or citrate/malate exchange. The activation energy of the exchange reaction was 70.1 kJ/mol. The rate of the exchange had a pH optimum between 7 and 8. The half-saturation constant was 0.13 mM for citrate and 0.76 mM for malate. All these properties were similar to those described for the tricarboxylate transport system in intact mitochondria. In proteoliposomes the maximum exchange rate at 25 degrees C reached 2000 mumols/min per g protein. This value was independent of the type of substrate present at the external or internal space of the liposomes (citrate or malate).

Kinetics of succinate uptake by rat-liver mitochondria

The permeation of anions into mitochondria has been kinetically studied so far only in a qualitative manner. No quantitative values on the rates and related kinetic parameters are available, although such data are of main importance for an understanding of these carrier-catalyzed transport processes. Only for the adenine nucleotide translocation a detailed kinetic study is available by applying a number of special techniques. Particularly short times of permeation have been measured by stopping the permeation on addition of inhibitor, such as atractyloside for the adenine nucleotide translocation .[ 1,2] . By applying the same principle, the kinetics of succinate permeation are followed as described in the present communication with a resolution of less than 1 sec. Recently, Kraayenhof et al. [3] also studied the kinetics of succinate uptake into the mitochondria with a technique based on centrifugal filtration (cf. also [3a]). Their method, however, has a dead time of 4 set, which is too long to resolve kinetics above 5”. The uptake of succinate into mitochondria has been the subject of previous studies of this laboratory and of other groups [4131. It was known that the rate of respiration with succinate is controlled by the uptake of succinate to the mitochondria. Furthermore, it was revealed that the degree of accumulation of succinate is dependent primarily on the pH difference across the mitochondrial membrane [ 121.

Kinetic characterization of the reconstituted dicarboxylate carrier from mitochondria: a four-binding-site sequential transport system

The mitochondrial antiport carriers form a protein family with respect to their structure and function. The kinetic antiport mechanism, being of the sequential type, shows that the dicarboxylate carrier also belongs to this family. This was demonstrated by bireactant initial velocity studies of the purified and reconstituted carrier protein. The transport affinity of the carrier for the internal substrate was largely independent of the external substrate concentration and vice versa, whereas the carriers apparent Vmax rose with increasing saturation of internal and external binding sites. Thus, the carrier forms a catalytic ternary complex with one internal and one external substrate molecule. As compared to other mitochondrial antiport carriers, however, the situation with the dicarboxylate carrier is more complex. On each membrane side of the protein two separate binding sites exist, one specific for phosphate (or its analogue phenyl phosphate), the other specific for dicarboxylate (or butyl malonate), that can be occupied by the respective substrates without mutual interference. This became evident from the non-competitive interaction of these substrates (or analogues) with the carrier. The two external, but not the two internal binding sites could be saturated simultaneously with phosphate and malate, thereby causing inhibition of transport. All four binding sites must be associated with the same translocation pathway through the carrier protein, since the sequential antiport mechanism held true for the phosphate/malate heteroexchange as well as for the malate/malate or phosphate/phosphate homoexchange.

myo-Inositol transport in rat intestinal brush border membrane vesicles, and its inhibition by d-glucose

The uptake of myo-inositol into rat intestinal brush border membrane vesicles (BBMV) has been investigated. It is demonstrated that myo-inositol is transported into the vesicles by a secondary active process, specifically using the sodium gradient as the driving force. In the absence of sodium gradient, the transport reaction is still sodium dependent, and rheogenic, indicating that a myo-inositol/sodium cotransport is likely to occur. A kinetic analysis shows an hyperbolic saturation process with a Km of 0.16 +/- 0.02 mM with respect to myo-inositol and Vmax of 68.5 +/- 21.2 pmol/min per mg protein. The transport is inhibited by D-glucose, phloridzin and few other sugars. The mechanism of D-glucose inhibition appears to be of the mixed type. Finally, the myo-inositol transport is trans-activated by myo-inositol itself, but not by D-glucose. It is concluded that myo-inositol is transported into rat intestine BBMV by a specific transport system, which is also able to bind D-glucose, but not efficiently transport it across the membrane.

Inhibition of the mitochondrial tricarboxylate carrier by arginine-specific reagents

The effect of arginine‐specific reagents on the activity of the partially purified and reconstituted tricarboxylate carrier of the inner mitochondrial membrane has been studied. It has been found that 1,2‐cyclohexanedione, 2,3‐butanedione, phenylglyoxal and phenylglyoxal derivatives inhibit the reconstituted citrate/citrate exchange activity. The inhibitory potency of the phenylglyoxal derivatives increases with increasing hydrophilic character of the molecule. Citrate protects the tricarboxylate carrier against inactivation caused by the arginine‐specific reagents. Other tricarboxylates, which are not substrates of the carrier, have no protective effect. The results indicate that at least one essential arginine residue is located at the substrate‐binding site of the tricarboxylate carrier and that the vicinity of the essential arginine(s) has a hydrophilic character.

Photoaffinity labeling of the mitochondrial phosphate carrier by 4-azido-2-nitrophenyl phosphate

The effect of 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive analogue of phosphate, on the phosphate carrier of pig-heart mitochondria has been investigated. In the dark, ANPP inhibits the transport of phosphate in a competitive manner with a Ki of 3.2 mM. Upon photoirradiation with visible light, [32P]ANPP binds covalently to the phosphate carrier and the inhibition becomes irreversible. Both the inhibition of phosphate transport and the incorporation of [32P]ANPP into the phosphate carrier depend on the concentration of the inhibitor and the pH of the medium. Incubation of the mitochondria with phosphate during illumination in the presence of ANPP protects the carrier against inactivation and decreases the amount of radioactivity which is found to be associated with the purified protein. By extrapolation it is calculated that at 100% inactivation of the phosphate carrier 0.35 mol of reagent are bound per mol of 33 kDa carrier protein. It is concluded that ANPP can be used for photoaffinity labeling of the mitochondrial phosphate carrier at the substrate-binding site.

Transport of citrate in submitochondrial particles

The efflux of citrate from mitochondria provides precursors for biosynthetic processes such as fatty acid synthesis. It also provides a mean for transporting reducing equivalents from the matrix to the cytoplasm. There is evidence, in RLM, that the transport of citrate is mediated by a specific transport system, known as the tricarboxylate carrier, which catalyzes an exchange between citrate, malate and PEP [ I]. The exchange is inhibited by 1,2,3_benzenetricarboxylate, and less specifically by piodobenzylmalonate [ 1,2]. The tricarboxylate carrier is virtually absent in mitochondria from heart [3], where the fatty acid synthesis is very low. In this report it is shown that SMP from RLM retain the transport system for citrate, but with a decreased activity. Part of this work has been communicated [4].

Citrate uniport by the mitochondrial tricarboxylate carrier: A basis for a new hypothesis for the transport mechanism

The tricarboxylate transport system located in the inner mitochondrial membrane was studied as an isolated protein reconstituted in proteoliposomes. The effects on the transport of citrate by various reagents, specific for different aminoacid residues, were analyzed. In the group of SH reagents, it was found that N-ethylmaleimide is an irreversible inhibitor of the citrate–citrate exchange, while HgCl2 and the mercurial mersalyl cause a rapid unidirectional efflux of citrate from liposomes. It was demonstrated that NEM and mercurials act on different SH groups. Dithioerythritol is not able to reverse the effect of mersalyl unless another reagent, pyridoxalphosphate, is present. Pyridoxalphosphate itself, a reagent specific for NH2 residues, is an effective inhibitor of citrate exchange transport, as measured in both influx and efflux, but it has no effect on the mercurial-induced efflux. The same behavior was observed with diethylpyrocarbonate, a reagent specific for histidine and tyrosine residues. Interestingly, a slow basic efflux of internal citrate, in the absence of countersubstrate, was observed in proteoliposomes. Because it is inhibited by the same reagents acting on the exchange process, it is deduced that it is catalyzed by the tricarboxylate carrier. The ability of the carrier to perform a uniport of the substrate suggests the presence of a single substrate binding site on the carrier protein. A preliminary kinetic approach indicates that such a transport model is compatible with this theory.