48 research outputs found
REACTION-MECHANISM OF THE RECONSTITUTED TRICARBOXYLATE CARRIER FROM RAT-LIVER MITOCHONDRIA
No full textBISACCIA F, DEPALMA A, Dierks T, KRAMER R, PALMIERI F. REACTION-MECHANISM OF THE RECONSTITUTED TRICARBOXYLATE CARRIER FROM RAT-LIVER MITOCHONDRIA. BIOCHIMICA ET BIOPHYSICA ACTA. 1993;1142(1-2):139-145.Transport of citrate and malate by the tricarboxylate carrier from rat liver mitochondria has been studied in a reconstituted system. Homologous citrate/citrate antiport and heterologous (electroneutral) citrate/malate antiport was kinetically analyzed. The maximal rates of the two exchange modes did not vary significantly within pH 7.0 to 7.8 which is the optimum pH-range for transport activity. On the other hand, the apparent transport affinity varied considerably within this range. Calculations on the basis of the different pK values for citrate and malate indicate that only H-citrate 2 -and malate 2 -are accepted as transport species by the tricarboxylate carrier. A complete set of half-saturation constants was established for citrate and malate on both the external and the internal side of the membrane. Both the K(m) and V(max) for citrate and malate were independent of the nature of the countersubstrate at the other side of the membrane. Bisubstrate initial velocity analyses of the exchange reaction resulted in a kinetic pattern which is consistent with a sequential antiport mechanism. This type of mechanism implies formation of a ternary complex of the carrier with two substrate molecules before the transport reaction occurs. Thus the tricarboxylate carrier falls into the functional family of mitochondrial carrier proteins showing sequential transport mechanisms
KINETIC DISCRIMINATION OF 2 SUBSTRATE BINDING-SITES OF THE RECONSTITUTED DICARBOXYLATE CARRIER FROM RAT-LIVER MITOCHONDRIA
No full textINDIVERI C, Dierks T, KRAMER R, PALMIERI F. KINETIC DISCRIMINATION OF 2 SUBSTRATE BINDING-SITES OF THE RECONSTITUTED DICARBOXYLATE CARRIER FROM RAT-LIVER MITOCHONDRIA. BIOCHIMICA ET BIOPHYSICA ACTA. 1989;977(2):194-199
KINETIC CHARACTERIZATION OF THE RECONSTITUTED DICARBOXYLATE CARRIER FROM MITOCHONDRIA - A 4-BINDING-SITE SEQUENTIAL TRANSPORT-SYSTEM
No full textINDIVERI C, PREZIOSO G, Dierks T, KRAMER R, PALMIERI F. KINETIC CHARACTERIZATION OF THE RECONSTITUTED DICARBOXYLATE CARRIER FROM MITOCHONDRIA - A 4-BINDING-SITE SEQUENTIAL TRANSPORT-SYSTEM. BIOCHIMICA ET BIOPHYSICA ACTA. 1993;1143(3):310-318.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 carrier's apparent V(max) 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
THE MITOCHONDRIAL CARNITINE CARRIER - CHARACTERIZATION OF SH-GROUPS RELEVANT FOR ITS TRANSPORT FUNCTION
No full textINDIVERI C, TONAZZI A, Dierks T, KRAMER R, PALMIERI F. THE MITOCHONDRIAL CARNITINE CARRIER - CHARACTERIZATION OF SH-GROUPS RELEVANT FOR ITS TRANSPORT FUNCTION. BIOCHIMICA ET BIOPHYSICA ACTA. 1992;1140(1):53-58.The transport function of the purified and reconstituted carnitine carrier from rat liver mitochondria was correlated to modification of its SH-groups by various reagents. The exchange activity and the unidirectional transport, both catalyzed by the carnitine carrier, were effectively inhibited by N-ethylmaleimide and submicromolar concentrations of mercurial reagents, e.g., mersalyl and p-(chloromercuri)benzenesulfonate. When 1 muM HgCl2 or higher concentrations of the above mentioned mercurials were added, another transport mode of the carrier was induced. After this treatment, the reconstituted carnitine carrier catalyzed unidirectional substrate-efflux and -influx with significantly reduced substrate specificity. Control experiments in liposomes without carrier or with inactivated carrier protein proved the dependence of this transport activity on the presence of active carnitine carrier. The mercurial-induced uniport correlated with inhibition of the `physiological' functions of the carrier, i.e., ``change and substrate specific unidirectional transport. The effect of consecutive additions of various reagents including N-ethylmaleimide, mercurials, Cu2+-phenanthroline and diamide on the transport function revealed the presence of at least two different classes of SH-groups. N-Ethylmaleimide blocked the carrier activity by binding to SH-groups of one of these classes. At least one of these SH-groups could be oxidized by the reagents forming S-S bridges. Besides binding to the class of SH-groups to which N-ethylmaleimide binds, mercurials also reacted with SH-groups of the other class. Modification of the latter led to the induction of the efflux-type of carrier activity characterized by loss of substrate specificity
REACTION-MECHANISM OF THE RECONSTITUTED OXOGLUTARATE CARRIER FROM BOVINE HEART-MITOCHONDRIA
No full textINDIVERI C, Dierks T, KRAMER R, PALMIERI F. REACTION-MECHANISM OF THE RECONSTITUTED OXOGLUTARATE CARRIER FROM BOVINE HEART-MITOCHONDRIA. EUROPEAN JOURNAL OF BIOCHEMISTRY. 1991;198(2):339-347.The transport mechanism of the reconstituted oxoglutarate carrier, purified from bovine heart mitochondria, was studied kinetically. A complete set of half-saturation constants (K(m)) was established for the two different substrates oxoglutarate and malate on both the external and the internal sides of the membrane. The internal affinities for oxogulutarate (K(m) 0.17 mM) and malate (K(m) 0.7 mM) were higher than the corresponding external affinities (K(m) 0.3 mM and 1.4 mM, respectively). The exclusive presence of a single transport affinity for each substrate on one side of the membrane indicated a unidirectional insertion of the oxoglutarate carrier into the liposomal membrane. The K(m) values and also the maximum exchange rates (8-11-mu-mol . min-1 . mg protein-1) for oxoglutarate and malate were independent of the nature of the counter substrate on the other side of the membrane. Under these defined conditions we analyzed the antiport mechanism in two-reactant initial velocity studies varying both the internal and external substrate concentrations. From the kinetic patterns obtained, a sequential type of mechanism became evident, implying that one internal and one external substrate molecule from a ternary complex with the carrier before transport occurs. A quantitative analysis of substrate interaction with the unloaded or single-substrate-occupied carrier revealed that rapid-equilibrium random conditions were fulfilled, characterized by a fast and independent binding of internal and external substrate. This kinetic mechanism agrees with previous results obtained in intact mitochondria. Considering also the data available for other mitochondrial carriers, a common kinetic mechanism (sequential type) for this carrier family is suggested
