Jan Kopecký

Uncoupling protein in embryonic brown adipose tissue — existence of nonthermogenic and thermogenic mitochondria

Embryonic development of mouse and rat brown adipose tissue was characterized by electron microscopy and by quantifying the mitochondrial oxidative, phosphorylating and thermogenic capacities immunochemically, using antibodies against cytochrome oxidase, F1-ATPase and uncoupling protein, respectively. Mitochondria and cytochrome oxidase were detected from the 15-16th day of pregnancy and their amounts continuously increased toward birth. F1-ATPase was also found on the 15th day but it reached a maximum level already on the 19th day when the uncoupling protein appeared and rapidly increased during further maturation of brown adipose tissue. It thus appears that mitochondria in early prenatal brown adipose tissue lack completely uncoupling protein and are nonthermogenic. They transform into typical thermogenic mitochondria abruptly only 2 days before birth.

Tissue Metabolism and Plasma Levels of Thyroid Hormones in Critically Ill Very Premature Infants

Thyroid status was characterized in very preterm infants (gestational age≤32 wk; n = 61) from birth through d 14, and in infants who died within 16 d after delivery (n = 10), where it was also correlated with metabolism of iodothyronines in peripheral tissues (brain, liver, kidney, skeletal muscle, and adipose tissue). At 3 d of life, mean plasma levels of thyroxine, triiodothyronine, and TSH started to decrease, being lower in the critically ill compared with healthy premature neonates. Activities of the three iodothyronine deiodinases enzymes (type I, II, and III, respectively) were detected in all postmortem tissue samples, except for absence of the type II activity in kidney. All activities were the highest in liver and differed in other tissues. Lack of correlation between the type I activity in liver(and kidney), and plasma levels of thyroid hormones suggested that the thyroid was the primary source of circulating triiodothyronine. On the other hand, namely in brain, correlations between activity of the deiodinases and plasma hormone levels were found which suggested a complex control by thyroid hormones of their own metabolism. High activity of type III in liver, adipose tissue, and skeletal muscle demonstrated a role of these tissues in thyroid hormones degradation. Results support the view that peripheral tissues of very preterm infants are engaged in local generation of triiodothyronine, and inactivation of thyroid hormones, but do not represent a major source of circulating triiodothyronine.

Molecular mechanism of uncoupling in brown adipose tissue mitochondria: the non-identity of proton and chloride conducting pathways

Specific permeability properties of the inner membrane of brown adipose tissue mitochondria were analysed with the aid of simultaneous pH measurements outside mitochondria and of mitochondria swelling. It was shown that valinomycin‐induced potassium diffusion potential drives a parallel passive uptake of chlorides and extrusion of proton. Electrogenic H+‐extrusion was independent on anion transport, no competition was found between the two processes and the former process exerted a lower sensitivity to the inhibitory effect of GDP. The existence of two distinct, independent pathways for translocation of protons and halide anions across the membrane is suggested.

Evaluation of the specific dicyclohexylcarbodiimide binding sites in brown adipose tissue mitochondria

1. The content of the membrane sector of the ATPase complex (Fo) in brown adipose tissue mitochondria was determined by means of specific [14C]-DCCD binding. 2. The specific DCCD binding to the F0 protein was distinguished from the nonspecific binding to the other membrane proteins and phospholipids by: (a) Scatchard plot analysis of the equilibrium binding data, (b) SDS-polyacrylamide gel electrophoresis of the 14C-labelled membrane proteins, (c) partial purification of the chloroform-methanol extractable DCCD-binding protein. It was found that the specific DCCD binding was present in three polypeptides of a relative molecular weight of 9000, 16 000 and 32 000. In brown adipose tissue mitochondria the specific binding was 10-times lower than in heart or liver mitochondria. The binding to the other membrane proteins and to phospholipids was quite similar in all mitochondrial preparations studied. 3. The decreased quantity of the specific binding sites in brown adipose tissue mitochondria demonstrated that the reduction of F0 parallels the reduction of the F1-ATPase and revealed that in these mitochondrial membranes the ratio between the respiratory chain enzymes and the ATPase complex is 10- to 20- times higher than in heart or liver mitochondria.

Structure and function of the membrane-integral components of the mitochondrial H+-ATPase

ConclusionsBased on our present knowledge about the composition of mitochondrial F0, it is evident that its mode of interaction with F1 is more complex in comparison with bacteria and chloroplasts. As far as the H+-channel is concerned, no definite conclusion about the involvement of other subunits besides the DCCD-binding protein can be drawn at present. This holds for mitochondria as well as for chloroplasts and bacteria. Experimental evidence is accumulating in favor of the oligomeric and asymmetrical arrangement of the H+-channel. Extraction of its few polar amino acid residues by specific agents reveals the fundamental functional importance of these residues in the path of protons across the membrane. In particular, the use of DCCD was of primary importance for elucidation of the structural features underlying the protonophoric activity. It may be hoped that application of similar new approaches in combination with studies of the intact phosphorylating assembly will help us to clarify the molecular mechanism of ATP synthesis.

Differentiation of dicyclohexylcarbodiimide reactive sites of the ATPase complex in bovine heart mitochondria

1. In isolated bovine heart mitochondria, the 14C-labelled dicyclohexylcarbodiimide (DCCD) induced inhibition of the ATPase activity is accompanied by labelling of three polypeptides of Mx 9000, 16 000 and 33 000. Of these, only the 9000 polypeptide reacts with [14C]DCCD proportionally to the inhibitory effect, being saturated when the enzyme is maximally inhibited. 2. The 9000 and 16 000 polypeptides are extracted by neutral chloroform/methanol (2 : 1 v/v) while the 33 000 polypeptide remains in the non-extractable residue. No disaggregation of the polypeptides takes place during the extraction. 3. In the ATPase complex immunoprecipitated with antibody against F1, the 9000 and 16 000 polypeptides are present, but the 33 000 polypeptide is absent. 4. The results obtained indicate that the 33 000 polypeptide is not a component of the ATPase complex. As far as F0 is concerned, two types of the binding sites for DCCD were demonstrated, corresponding to the 9000 and 16 000 polypeptides. Their existence is explained by a non-random arrangement among individual monomers of the DCCD-binding protein.

The binding of dicyclohexylcarbodiimide to uncoupling protein in brown adipose tissue mitochondria

The generation of heat in brown adipose tissue has been related to the uncoupling of respiration in their mitochondria (reviews [ 1,2]). The uncoupling in these mitochondria is achieved by a high ion conductance of the mitochondrial membrane and is specifically controlled by exogenous purine nucleotides [3-71. The binding site for these nucleotides was identified as a 32 000 Mr protein that is present in considerable amount only in brown adipose tissue mitochondria [S111. It is proposed that this protein (so called GDP-binding or uncoupling protein) is directly responsible for the physiological uncoupling of brown adipose tissue mitochondria. Here, we report that N,N’-dicyclohexylcarbodiimide a well-established inhibitor or proton-pumping activity of several membrane enzymes [12-161, binds rather specifically to the uncoupling protein in brown adipose tissue mitochondria. In addition, it inhibits the high chloride permeability of brown adipose tissue mitochondrial membrane. We propose that the 32 000 Mr uncoupling protein may function in mitochondrial membrane of brown adipose tissue similarly to other proton-translocating DCCD binding proteins.

Is the mitochondrial dicyclohexylcarbodiimide-reactive protein of Mr 33 000 identical with the phosphate transport protein?

Dicyclohexylcarbodiimide acts at low concentrations as a specific inhibitor of H’-ATPase due to the covalent binding to a hydrophobic subunit of F, [1,2]. In mammalian mitochondria, however, an additional protein of different M, (33 000) is also labelled at H’-ATPase-inhibitor concentrations of [‘4C]DCCD f2-41. In contrast to the DC~D-boding protein of F, it is neither extracted by chloroformmethanol (2: 1, v/v>, nor is it detected in the isolated H’-ATPase complex [3,4]. In different types of mitochondria the 33 000 DCCD-reactive protein is present in a near stoichiometrical quantity with respect to F,, even though mitochondria differ in the natural content of H’-ATPase up to 10 times [5]. Here, we describe the separation of the 33 000 DCCD-reactive protein from the DCCD-binding subunit of F, and its purification. Evidence is presented for its identity with the TV-ethylmaleimide (NEM)-sensitive phosphate~proton symporter of the mitochondrial membrane.

Stoicheiometry of dicyclohexylcarbodiimide-ATPase interaction in mitochondria

1. The oligomeric dicyclohexylcarbodiimide (DCCD)-binding protein of mitochondrial ATPase was studied using (a) the relationship between (14C] DCCD binding and inhibition of ATPase activities and (b) the analysis of the kinetics of inhibition. 2. The [14C]DCCD binding to bovine heart mitochondria is linearly proportional to the inhibition of ATP hydrolysis up to a 50% decrease of the original activity resulting in 0.6 mol DCCD bound covalently to the specific inhibitory site (Houstĕk, J., Svoboda, P., Kopecký, J., Kuzela, S. and Drahota, Z. (1981) Biochim. Biophys. Acta 634, 331-339) per mol of the fully inhibited enzyme. 3. Kinetics of the inhibition of both the ATPase activity (heart and liver mitochondria) and ADP-stimulated respiration (liver) reveal that 1 mol DCCD per mol ATPase eliminates both the synthetic and the hydrolytic activities. It is inferred that the activity-binding correlation underestimates that number of DCCD-reactive sites. 4. The second-order rate constant of the DCCD-ATPase interaction (k) is inversely related to the concentration of membranes, indicating that DCCD reaches the inhibitory site by concentrating in the hydrophobic (phospholipid) environment. 5. At a given concentration of liver mitochondria, comparable k values are obtained both for the inhibition of ATP hydrolysis (k = 5.35.10(2)M-1.min-1) and ADP-stimulated respiration (k = 5.67.10(2)M-1.min-1). 6. It is concluded that both the synthetic and the hydrolytic functions if ATPase are inhibited via a common single DCCD-reactive site. This site is represented by one of the several polypeptide chains forming the oligomer of the DCCD-binding protein. The inhibitor-ATPase interaction does not exhibit cooperativity, indicating that the preferential reactivity towards DCCD is an inherent property of the inhibitory site.

Reaction of oxiranecarbonitrile with L-cysteine methyl ester

Abstract The title reaction yields (3R,5R) and (3R,5S) isomers of methyl 5-cyanotetrahydro-1,4-2H-thiazine-3-carboxylate, together with methyl 2-acetylthiazole-4-carboxylate as a minor by-product. The stereochemistry of the tetrahydrothiazine derivatives is discussed.