Complete nucleotide sequence of hepatic 5-aminolaevulinate synthase precursor

Chick embryo liver mitochondrial matrix protein, 5-aminolaevulinate synthase, is synthesised initially as a larger cytosolic precursor. In this report we present the complete nucleotide sequence of a cDNA clone coding for the precursor together with corresponding confirmatory amino acid sequence of peptides derived from purified mature mitochondrial enzyme. The deduced amino acid sequence shows that the precursor consists of mature enzyme of 579 amino acids and an N-terminal extension of 56 amino acids. The latter presequence is highly basic in character as found with other mitochondrial preproteins.     

Small angle X-ray scattering study on bovine porphobilinogen synthase (5-aminolaevulinate dehydratase)

The quaternary structure of the native (zinc) porphobilinogen synthase (5-amino-laevulinate dehydratase) from bovine liver and its lead-substituted derivative is studied in solution by small angle X-ray scattering. In spite of the profound inhibitory effect of lead ions in the enzyme they do not produce a change in the quaternary structure detectable by small angle X-ray scattering. The most important molecular parameters of the native enzyme were found to be: radius of gyration Rg = 4.04 +/- 0.04 nm and maximum dimension Dmax = 12.0 +/- 0.5 nm. The corresponding values for the lead derivative are: Rg = 4.26 +/- 0.1 nm and Dmax = 12.5 +/- 0.5 nm. The quaternary structure of the enzyme in solution is described by a model, which fits the experimental scattering and distance distribution function.     

Electron microscopic studies on guinea pig rib cartilage

Summary The guinea-pig rib cartilage consists of chondrocytes dispersed in an intercellular substance composed of collagen fibrils, often characteristically cross-striated, and polygonal granules. Electron-dense membrane-bounded matrix vesicles are also observed intercellularly, especially in the central, partly calcified zone of the cartilage. With respect to their location in a cross-section of the rib, the chondrocytes differ in size, shape and intracellular fine structure. Thus, three separate types of cells are recognized. Peripheral chondrocytes have a flattened shape and are largely occupied by the nucleus. In the cytoplasm, the granular endoplasmic reticulum is the most extensive organelle. Intermediate chondrocytes are oval or round in shape. The endoplasmic reticulum and the Golgi complex are both prominent. Mitochondria and membrane-bounded cytoplasmic dense bodies are more numerous than in the peripheral cells. The ground cytoplasm often contains a few lipid droplets. In the central chondrocytes, such droplets sometimes fill the entire cytoplasm. Concomitantly, the nucleus is usually completely heterochromatic and the cells are therefore regarded as being metabolically inert.After preparations including ruthenium red staining en bloc, the general stainability of the chondrocytes is decreased. Intracellularly, positive ruthenium red staining of granular material within the Golgi vacuoles are to be observed. Extracellularly, the matrix granules are stained with this polyvalent, cationic dye. Extraction of the cartilage with 4 M guanidine-HCl removes all matrix granules and about 70% of the proteoglycans, measured as hexosamine, from the tissue. It is concluded that the matrix granules contain proteoglycan complexes.Financial support was received from the Swedish Medical Research Council (proj. no. 12X-3355), the Swedish Cancer Society (proj. no. 100-K71-05XK), the King Gustaf V 80th Birthday Fund, the Harald and Greta Jeansson Foundation, the C. B. Nathhorst Foundation, and from the funds of Karolinska Institutet.The skilled technical assistance of Mrs. Eva Lundberg and the secretarial assistance of Mrs. Inger Åhrén are gratefully acknowledged. The authors are indebted to Dr. S. Lohmander for helpful suggestions during the progress of the work.     

Electron microscope studies of glycogen synthase

Summary Glycogen synthase from rabbit muscle was examined with the electron microscope. In preparations of the completely converted glucose-6-phosphate dependent form (GSD) and the independent form (GSI) three structures were observed: toroids, hexagons and stacks of four elements which appear to be aggregates of four toroids. Toroids can be formed from hexagons by radial inward movement of subunits which form the vertices of the hexagons. Analysis of the dimensions of these structures and comparison of the known chemistry of the enzyme to the subunits as inferred from electron microscopy suggests a model for the structure of glycogen synthase. The model allows predictions of types of subunits in the enzyme, their relation to phosphorylatable and -SH sites and the possibilities of control by small effector molecules.     

Measurement of 5-aminolaevulinate synthase activity by gas-liquid chromatography with electron-capture detection.

A specific assay for 5-aminolaevulinate synthase activity is described, with a sensitivity comparable with that of radiochemical assays. It is based on measurement by g.l.c. with electron-capture detection of the pentafluorobenzyl ester of the ethyl acetoacetate pyrrole derivative of 5-aminolaevulinic acid, and of the corresponding compound from 6-amino-5-oxohexanoic acid used as internal standard. Enzyme activity has been measured in homogenates of rat liver, spleen, kidney and brain, and in human lymphocytes.     

Brain 5-aminolaevulinate synthase. Developmental aspects and evidence for regulatory role.

1. Brain 5-aminolaevulinate synthase showed a peak of increased activity in the first few weeks of life, which preceded and accompanied the development of brain cytochromes. 2. In the brain of the adult rat the activity of the enzyme was only 20% of that in the liver (on a per g wet wt. basis), but it was still probably sufficient to maintain the turnover of brain cytochromes. 3. The brain synthase activity could be decreased by treatment of rats with cycloheximide or with large doses of 5-aminolaevulinate, especially when this precursor was given as the methyl ester. 4. Injected haematin and CoCl2 markedly inhibited the synthase activity in the liver but failed to affect the brain enzyme; neither was taken up by the brain in vivo. 5. It is concluded that the brain can itself produce the haem required for the synthesis and turnover of its own haemoproteins and that 5-aminolaevulinate synthase may regulate the pathway in brain as in other tissues. 6. The relevance of the present findings to the pathogenesis of the neurological symptoms of acute porphyria and to the beneficial effect of exogenous haematin in porphyric patients is briefly considered.     

Electron microscopic observations of young rat liver

Summary Electron microscopic observations on normal liver tissue of four-day-old rats reveal the presence of numerous lamellar structures (lamellar bodies). These can be contained within parenchymal cells or in bile canaliculi, Disse's space, and in the lumen of blood sinusoids. Such bodies can also be found in Kupffer and endothelial cells.The lamellar bodies within hepatic cells are generally seen in very intimate relation to glycogen particles and lipide droplets, but in some to agranular endoplasmic reticulum and Golgi membranes as well.On the basis of this intimate relation to intracellular glycogen granules and lipide droplets, it is presumed that lamellar bodies represent a special intermediate stage in carbohydrate and lipide metabolism.Discontinuities in the endothelial layer of intrahepatic sinusoids are described.This work was supported in part by a N.A.T.O. research fellowship of the Consiglio Nazionale delle Ricerche, Roma.Assistant Professor in the Department of Veterinary Anatomy, Histology and Embryology (Dir.: Prof. A. de Girolamo), University of Naples, Naples, Italy.