Joseph M. Tager

Rapid separation of particulate components and soluble cytoplasm of isolated rat-liver cells.

A method is described for the rapid separation of mitochondria (plus other particulate components) from the soluble cytoplasm of isolated rat-liver cells. The cells were incubated briefly with a low concentration of digitonin. After rapid centrifugation, the pellet contained more than 90% of the total adenylate kinase and glutamate dehydrogenase activities and the supernatant at least 80% of the lactate dehydrogenase activity. About 60% of total adenine nucleotides in hepatocytes were found in the soluble cytoplasm. The ATP/ADP ratio in the particulate fraction 80 s after exposure to digitonin of hepatocytes metabolizing alanine was 2.0-2.4, and that in the soluble cytoplasm 6-19. In the presence of atractyloside, these values were 3.5-4.4 and 1.3-2.2, respectively.

Peroxisomal disorders: a newly recognised group of genetic diseases

A new group of genetic diseases, in which peroxisomal functions are impaired, has recently been recognised. Although peroxisomes are subcellular organelles that are universally found in animal cells (except mature erythrocytes), their function in mammalian metabolism remained unclear until recently. Now, however, it is evident that peroxisomes play an essential role in several metabolic processes and that dysfunction of these organelles can have serious clinical consequences. Genetic diseases involving peroxisomes can be divided into two classes: firstly, those in which only a single peroxisomal function is impaired (acatalasaemia, X-linked adrenoleukodystrophy, the adult form of Refsum disease); secondly, those in which there is an impairment of more than one peroxisomal function (cerebro-hepato-renal (Zellweger) syndrome, the infantile form of Refsum disease, the neonatal form of adrenoleukodystrophy, the rhizomelic type of chondrodysplasia punctata and hyperpipecolic acidaemia). Whether genetic diseases like intrahepatic bile duct hypoplasia with elevation of trihydroxycoprostanoic acid and cerebrotendinous xanthomatosis can also be classified as peroxisomal disorders remains to be established. The aim of this paper is to summarise the rapid developments that have occurred during the last 3 years with regard to our knowledge of peroxisomal disorders and to describe briefly the clinical, morphological and biochemical characteristics of this new group of genetic disorders.

Control of mitochondrial respiration

The control theory of Kacser and Burns [in: Rate Control of Biological Processes (Davies, D.D. ed) pp. 65–104, Cambridge University Press, London, 1973] and Heinrich and Rapoport ([Eur. J. Biochem. (1974) 42, 97‐105] has been used to quantify the amount of control exerted by different steps on mitochondrial oxidative phosphorylation in rat‐liver mitochondria. Inhibitors were used to manipulate the amount of active enzyme. The control strength of the adenine nucleotide translocator was measured by carrying out titrations with carboxyatractyloside. In state 4, the control strength of the translocator was found to be zero. As the rate of respiration was increased by adding hexokinase, the control strength of the translocator increased to a maximum value of ∼30% at ∼80% of state 3 respiration. In state 3, control of respiration is distributed between a number of steps, including the adenine nucleotide translocator, the dicarboxylate carrier and cytochrome c oxidase. The measured values for the distribution of control agree very well with those calculated with the aid of a model for mitochondrial oxidative phosphorylation developed by Bohnensack [Biochim. Biophys. Acta (1982) 680, 271–280].

Activity of peroxisomal enzymes and intracellular distribution of catalase in Zellweger syndrome

The activity of peroxisomal enzymes was studied in human liver and cultured human skin fibroblasts in relation to the finding (Goldfischer, S. et al. (1973) Science 182, 62-64) that morphologically distinct peroxisomes are not detectable in patients with the cerebro-hepato-renal (Zellweger) syndrome. In homogenates of liver from the patients, dihydroxyacetone phosphate acyltransferase, a membrane-bound peroxisomal enzyme, is deficient (Schutgens, R.B.H., et al. (1984) Biochem. Biophys. Res. Commun. 120, 179-184). In contrast, there is no deficiency of the soluble peroxisomal matrix enzymes catalase, L-alpha-hydroxyacid oxidase and E-aminoacid oxidase. Catalase is also not deficient in homogenates of cultured skin fibroblasts from the patients. The results of digitonin titration experiments showed that in control fibroblasts at least 70% of the catalase activity is present in subcellular particles distinct from mitochondria or lysosomes. In contrast, all of the catalase activity in fibroblasts from Zellweger patients is found in the same compartment as the cytosolic marker enzyme lactate dehydrogenase.

Peroxisomal disorders in neurology

Although peroxisomes were initially believed to play only a minor role in mammalian metabolism, it is now clear that they catalyse essential reactions in a number of different metabolic pathways and thus play an indispensable role in intermediary metabolism. The metabolic pathways in which peroxisomes are involved include the biosynthesis of ether phospholipids and bile acids, the oxidation of very long chain fatty acids, prostaglandins and unsaturated long chain fatty acids and the catabolism of phytanate and (in man) pipecolate and glyoxylate. The importance of peroxisomes in cellular metabolism is stressed by the existence of a group of inherited diseases, the peroxisomal disorders, caused by an impairment in one or more peroxisomal functions. In the last decade our knowledge about peroxisomes and peroxisomal disorders has progressed enormously and has been the subject of several reviews. New developments include the identification of several additional peroxisomal disorders, the discovery of the primary defect in several of these peroxisomal disorders, the recognition of novel peroxisomal functions and the application of complementation analysis to obtain information on the genetic relationship between the different peroxisomal disorders. The peroxisomal disorders recognized at present comprise 12 different diseases, with neurological involvement in 10 of them. These diseases include: (1) those in which peroxisomes are virtually absent leading to a generalized impairment of peroxisomal functions (the cerebro-hepato-renal syndrome of Zellweger, neonatal adrenoleukodystrophy, infantile Refsum disease and hyperpipecolic acidaemia); (2) those in which peroxisomes are present and several peroxisomal functions are impaired (the rhizomelic form of chondrodysplasia punctata, combined peroxisomal beta-oxidation enzyme protein deficiency); and (3) those in which peroxisomes are present and only a single peroxisomal function is impaired (X-linked adrenoleukodystrophy, peroxisomal thiolase deficiency (pseudo-Zellweger syndrome), acyl-CoA oxidase deficiency (pseudo-neonatal adrenoleukodystrophy) and probably, the classic form of Refsum disease.

Genetic heterogeneity in the cerebrohepatorenal (Zellweger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions. A study using complementation analysis.

We have used complementation analysis after somatic cell fusion to investigate the genetic relationships among various genetic diseases in humans in which there is a simultaneous impairment of several peroxisomal functions. The activity of acyl-coenzyme A:dihydroxyacetonephosphate acyltransferase, which is deficient in these diseases, was used as an index of complementation. In some of these diseases peroxisomes are deficient and catalase is present in the cytosol, so that the appearance of particle-bound catalase could be used as an index of complementation. The cell lines studied can be divided into at least five complementation groups. Group 1 is represented by a cell line from a patient with the rhizomelic form of chondrodysplasia punctata. Group 2 consists of cell lines from four patients with the Zellweger syndrome, a patient with the infantile form of Refsum disease and a patient with hyperpipecolic acidemia. Group 3 comprises one cel line from a patient with the Zellweger syndrome, group 4 one cell line from a patient with the neonatal form of adrenoleukodystrophy, and group 5 one cell line from a patient with the Zellweger syndrome. We conclude that at least five genes are required for the assembly of a functional peroxisome.

Direct demonstration that the deficient oxidation of very long chain fatty acids in X-linked adrenoleukodystrophy is due to an impaired ability of peroxisomes to activate very long chain fatty acids

A method was developed to prepare peroxisome-enriched fractions depleted of microsomes and mitochondria from cultured skin fibroblasts. The method consists of differential centrifugation of a postnuclear supernatant followed by density gradient centrifugation on a discontinuous Metrizamide gradient. The activity of hexacosanoyl-CoA synthetase was subsequently measured in postnuclear supernatants and peroxisome-enriched fractions prepared from cultured skin fibroblasts from control subjects and patients with X-linked adrenoleukodystrophy. Whereas the hexacosanoyl-CoA synthetase activity in postnuclear supernatants of X-linked adrenoleukodystrophy fibroblasts was only slightly decreased (77.8 +/- 4.4% of control (n = 15], enzyme activity was found to be much more markedly reduced in peroxisomal fractions isolated from the mutant fibroblasts (19.6 +/- 6.7% of control (n = 5]. This is a direct demonstration that the defect in X-linked adrenoleukodystrophy is at the level of a deficient ability of peroxisomes to activate very long chain fatty acids, as first suggested by Hashmi et al. [Hashmi, M., Stanley, W. and Singh, I. (1986) FEBS Lett. 86, 247-250].

Effect of sulphydryl-blocking reagents on mitochondrial anion-exchange reactions involving phosphate

Chappell and coworkers [l-4] have proposed that the transport of anions across the mitochondrial membrane is brought about by specific carrier systems that mediate exchange-diffusion processes. Two of these carrier systems, or translocators, mediate an exchange between phosphate and hydroxyl and an exchange between phosphate and dicarboxylate, respectively. Fonyo [5] and Tyler [6] have shown that certain sulphydryl-binding reagents specifically interfere with the transport of phosphate across the mitochondrial membrane. The problem arises of whether these inhibitors not only specifically block the exchange diffusion between phosphate and hydroxyl, but also that between dicarboxylate and phosphate. We have recently presented evidence for inhibition of both transport systems by the sulphydryl-binding reagent mersalyl [7] . According to Johnson and Chappell [8] , however, these sulphydryl-blocking reagents inhibit only the phosphate-hydroxyl exchange. We have now found that there is a difference in sensitivity of the phosphate-hydroxyl and phosphatedicarboxylate exchange reactions, depending on the sulphydryl-blocking reagent used. Mersalyl [6] and p-hydroxymercuribenzoate [5] inhibit both of these anion-exchange reactions (see ref. [7] ). On the other hand, N-ethylmaleimide inhibits the phosphatehydroxyl exchange only. These results provide additional evidence for the postulate [l-4] that the phosphate-hydroxyl and the phosphate-dicarboxylate exchange reactions are mediated by distinct translo-

Lipid metabolism in peroxisomes in relation to human disease

Peroxisomes were long believed to play only a minor role in cellular metabolism but it is now clear that they catalyze a number of important functions. The importance of peroxisomes in humans is stressed by the existence of a group of genetic diseases in man in which one or more peroxisomal functions are impaired. Most of the functions known to take place in peroxisomes have to do with lipids. Indeed, peroxisomes are capable of 1. fatty acid beta-oxidation 2. fatty acid alpha-oxidation 3. synthesis of cholesterol and other isoprenoids 4. ether-phospholipid synthesis and 5. biosynthesis of polyunsaturated fatty acids. In Chapters 2-6 we will discuss the functional organization and enzymology of these pathways in detail. Furthermore, attention is paid to the permeability properties of peroxisomes with special emphasis on recent studies which suggest that peroxisomes are closed structures containing specific membrane proteins for transport of metabolites. Finally, the disorders of peroxisomal lipid metabolism will be discussed.

Peroxisomal β-oxidation enzyme proteins in the Zellweger syndrome

Abstract The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities, including an accumulation of very longchain fatty acids. We show by immunoblotting that there is a marked deficiency in livers from patients with the Zellweger syndrome of the peroxisomal β-oxidation enzyme proteins acyl-CoA oxidase, the bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase. Using anti-(acyl-CoA oxidase), increased amounts of cross-reactive material of low M r were seen in the patients. With anti-(oxoacyl-CoA thiolase), high M r cross-reactive material, presumably representing precursor forms of 3-oxoacyl-CoA thiolase, was detected in the patients. Catalase protein was not deficient, in accordance with the finding that catalase activity is not diminished in the patients. Thus in contrast to the situation with catalase functional peroxisomes are required for the stability and normal activity of peroxisomal β-oxidation enzymes.