Studies on peroxisomes. VI. Relationship between the peroxisomal core and urate oxidase.

The peroxisomal core from the liver of rats was purified 450-fold as a marker of urate oxidase [EC 1.7.3.3.] activity. This preparation has a high specific activity of urate oxidase but not of other peroxisomal enzymes: D-amino acid oxidase [EC 1.4.3.3.], L-alpha-hydroxy acid oxidase [EC 1.1.3.15], or catalase [EC 1.11.1.6]. No activity of marker enzymes for other subcellular particles; cytochrome c oxidase [EC1.9.3.1] (mitochondria), acid phosphatase [EC 3.1.3.2] (lysosomes), or glucose-6-phosphatase [EC 3.1.3.9] (microsomes), was detected in this preparation. The core obtained showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the position of the band was found to correspond to a molecular weight 35,000. When the peroxisomal core was subjected to treatment at various pH's with 0.1 M carbonate buffer, urate oxidase was almost completely solubulized at pH 11.0, although approximately 35% of the core protein still remained in the pellet After solubilization of the core at pH 11.0, the specific activity of urate oxidase in the supernatant increased about 1.6 times; the density of the insoluble protein remaining in the pellet was identical with the that of the original core on sucrose density gradient centrifugation.     

Hydroxymethylglutaryl coA reductase (NADPH) in the latex of Hevea brasiliensis

The activity of hydroxymethylglutaryl CoA reductase (NADPH) (EC 1.1.1.34) was studied in the latex of regularly tapped mature trees of Hevea brasiliensis. The reductase activity was found mainly (95% of the total activity) in the pellet fraction (40 000 g) of the centrifuged latex. The enzyme in this fraction had a specific requirement for NADPH as the cofactor and, while not obligatory for activity, was activated by dithiothreitol at the optimum concentration of 2 mM. The pH optimum was found to be 6.6–6.9 in 0.1 M phosphate buffer. Mevalonate and CoA (at 2 mM each) did not affect enzyme activity, while hydroxymethylglutarate (2 mM) was slightly inhibitory. p-Chloromercuribenzoate (1 mM) completely inhibited this enzyme. The reductase activity in the 40 000 g pellet was not easily solubilized either using Triton X-100 or by sonication. The apparent K_m for the washed, membrane-bound enzyme (103 000 g pellet) was 56 μ M (RS-HMG-CoA). Magnesium-ATP (4 mM) inactivated the reductase but this effect was greatly diminished or was absent upon washing the 40 000 g pellet.     

Triterpene Biosynthesis in the Latex of Euphorbia lathyris: Calmodulin Antagonists Are Ineffective in Whole Latex

The calmodulin antagonists chlorpromazine, fluphenazine, trifluoperazine, and 2-(pentachlorophenoxy)ethyl N,N-diethylamine are not inhibitors of acetate incorporation into triterpenols (TOH) and their fatty acid esters (TE) in whole tapped latex from Euphorbia lathyris, although prior work demonstrated that these antagonists are good inhibitors of mevalonate incorporation into TOH and TE in a centrifuged pellet from the latex. Antagonist absorption into the endogenous terpene pool is the primary reason for antagonist ineffectiveness in whole latex; changes in the utilized substrate or chemical deactivation of the antagonists were ruled out as factors. A biosynthetically inactive, latex supernatant fraction containing the endogenous terpene pool was prepared. This fraction blocks antagonist action when added to the latex pellet, and proved to be a useful tool for demonstrating that inhibition of triterpene biosynthesis by a calmodulin antagonist is partially reversible.     

Acrylamide Alters Cytoskeletal Protein Level in Rat Sciatic Nerves

Occupational exposure and experimental intoxication with acrylamide (ACR) produce neuropathy characterized by nerve degeneration. To investigate the mechanism of ACR-induced neuropathy, male adult Wistar rats were given ACR (20, 40 mg/kg i.p. 3 days/week) for 8 weeks. Sciatic nerves were Triton-extracted and centrifuged at a high speed (100,000 × g) to yield pellet and supernatant fractions. The contents of six cytoskeletal proteins (NF-L, NF-M, NF-H, α-tubulin, β-tubulin, and β-actin) in both fractions were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Results showed that the three neurofilament (NF) subunits (NF-L, NF-M, NF-H) in both the pellet and the supernatant fraction decreased significantly (P < 0.01) in the high-dosing group, except for NF-M in the pellet. α-tubulin, β-tubulin, and β-actin increased significantly in the supernatant (P < 0.01), whereas both α-tubulin and β-tubulin decreased significantly in the pellet (P < 0.01). However, β-actin was not altered significantly in the sciatic nerves pellet. These findings suggest that ACR altered the cytoskeletal protein level in sciatic nerve, which may be one of the molecular mechanisms of ACR-induced peripheral neuropathy.     

Solubilization and molecular weight determination of the (Na+ + K+)-ATPase from rectal glands of Squalus acanthias.

The membrane-bound (Na+ + K+)-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) system was treated with the nonionic detergent octaethylene-glycoldodecyl ether, yielding a transparent supernatant after centrifugation. The supernatant was highly active with both ATPase and p-nitrophenylphosphatase, with initial specific activities of 2300 mumol Pi released . mg-1 protein. h-1 and 350 mumol p-nitrophenol released.mg-1 protein.h-1, respectively. The supernatant was purified to 95--100%, with respect to the 96 000 dalton and the 56 000 dalton peptides. The solubilized enzyme was gel filtered in Sepharose 4B-Cl and displayed 2 peaks, both with catalytic activity. The low molecular weight particles eluted at Kav = 0.54, corresponding to a molecular weight of approximately 500 000 daltons and the particles had a specific activity of 2100 mumol Pi.mg-1 protein.h-1. Both peaks contained phospholipid with 60 mol phospholipid bound per 300 000 g protein. The low molecular weight particles had a molecular weight of 276 000 as determined by sedimentation equilibrium analysis.     

Biosynthesis of lipase in the scutellum of maize kernel

In the scutellum of maize kernel after imbibition, lipase activity increased rapidly, concomitant with the decrease in storage triacylglycerols. The enzyme activity peaked at day 6, but remained at the same level from day 6-10 when most of the triacylglycerols had been depleted. By in vitro translation with extracted RNAs followed by immunoprecipitation, and by resolving the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lipase was found to be de novo synthesized in postgermination. The enzyme was synthesized by RNAs extracted from free polyribosomes and not from bound polyribosomes. Both in vitro and in vivo synthesized lipase had the same Mr of 65,000 as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as had the purified authentic enzyme; thus there was no appreciable co- or post-translational processing of the enzyme. Lipase-specific mRNA was present only between day 2-6 after imbibition. At day 6 when lipolysis was most active, more than 60% of the lipase activity was recovered in the lipid body fraction and specifically associated with the organelle membrane. From day 6-10, the lipase activity gradually shifted from the lipid body fraction to other subcellular fractions, including the 10,000 X g pellet, the 120,000 X g pellet, and the 120,000 X g supernatant. Lipase in these subcellular fractions was attributed to represent the enzyme associated with membrane ghosts of the lipid bodies which were fusing with the fragile cell vacuoles; such fusions were observed in situ by electron microscopy.     

Organelle, bead, and microtubule translocations promoted by soluble factors from the squid giant axon

A reconstituted system for examining directed organelle movements along purified microtubules has been developed. Axoplasm from the squid giant axon was separated into soluble supernatant and organelle-enriched fractions. Movement of axoplasmic organelles along MAP-free microtubules occurred consistently only after addition of axoplasmic supernatant and ATP. The velocity of such organelle movement (1.6 micron/sec) was the same as in dissociated axoplasm. The axoplasmic supernatant also supported movement of microtubules along a glass surface and movement of carboxylated latex beads along microtubules at 0.5 micron/sec. The direction of microtubule movement on glass was opposite to that of organelle and bead movement on microtubules. The factors supporting movements of microtubules, beads, and organelles were sensitive to heat, trypsin, AMP-PNP and 100 microM vanadate. All of these movements may be driven by a single, soluble ATPase that binds reversibly to organelles, beads, or glass and generates a translocating force on a microtubule.     

Carbon Disulfide-Induced Changes in Cytoskeleton Protein Content of Rat Cerebral Cortex

To investigate the mechanism of carbon disulfide-induced neuropathy, male wistar rats were administrated by gavage at dosage of 300 or 500 mg/kg carbon disulfide, five times per week for 12 weeks. By the end of the exposure, the animals produced a slight or moderate level of neurological deficits, respectively. Cerebrums of carbon disulfide-intoxicated rats and their age-matched controls were Triton-extracted and centrifuged at a high speed (100,000 × g) to yield a pellet fraction of NF polymer and a corresponding supernatant fraction, which presumably contained mobile monomer. Then, the contents of six cytoskeletal protein (NF-L, NF-M, NF-H, α-tubulin, β-tubulin, and β-actin) in both fractions were determined by immunoblotting. Results showed that the contents of the three neurofilament subunits in the pellet and the supernatant fraction decreased significantly regardless of dose levels (P < 0.01). As for microtubule proteins, in the pellet fraction of cerebrum, the levels of α-tubulin and β-tubulin demonstrated some inconsistent changes. However, in the supernatant fractions, the content of α-tubulin and β-tubulin increased significantly in both two dose groups (P < 0.01). In comparison to neurofilament and tubulin proteins, the content of β-actin changed less markedly, only the supernatant fraction of the high dose group displayed significant increase (P < 0.01), but the others remained unaffected. These findings suggested that the changes of cytoskeleton protein contents in rat cerebrum were associated with the intoxication of carbon disulfide, which might be involved in the development of carbon disulfide neurotoxicity.     

PROPERTIES OF ACID β-d-GALACTOSIDASE ISOLATED FROM I-CELL DISEASE BRAIN AND SPLEEN

Abstract— Acid 4-methylumbelliferyl β- d -galactosidase activity from autopsied I-cell disease brain and spleen tissues was 28% and 35% respectively of normal activity. Acid β- d -gatactosidase (β- d -galactoside galactohydrolase, EC 3.2.1.23) from two I-cell disease brains demonstrated a 5-fold increase over normal for the proportion of enzyme activity which did not adsorb to Concanavalin A-Sepharose 4B, while acid β- d -galactosidase from two I-cell disease spleens demonstrated a 21–35-fold increase in the proportion of unadsorbed enzyme activity. Normal and I-cell disease acid β- d -galactosidase present in crude brain and spleen supernatant fluids and in preparations partially purified on Concanavalin A-Sepharose 4B had similar apparent K _m values with respect to 4-methylumbelliferyl β- d -galactopyranoside and G_M1-ganglioside. Isoelectric focusing profiles of normal and I-cell disease acid β- d -galactosidase from crude brain and spleen-supernatant fluids and partially purified preparations were similar. Neuraminidase treatment and subsequent isoelectric focusing of the partially purified normal and I-cell disease enzyme preparations from brain and spleen revealed increases in the proportion of I-cell β- d -galactosidases found at neutral pH values, suggesting that the electrophoretic variations observed for the I-cell enzymes may not be attributed solely to changes in sialic acid composition.     

INFLUENCE OF COLD EXPOSURE ON THE ACTIVITIES OF SOLUBLE AND MEMBRANE-BOUND DOPAMINE β-HYDROXYLASE IN RAT HEART VESICLES

Abstract— A fraction containing noradrenaline storage vesicles of the sympathetic nerve terminals in the rat heart was obtained by differential centrifugation. In this preparation, 17% of the dopamine β-hydroxylase was present in a soluble form. Cold exposure (3°C) for periods from 5 to 30 min led to an increase in the activity of soluble dopamine β-hydroxylase by about 50%, while the activity of membrane-bound dopamine β-hydroxylase was simultaneously decreased by approx 30%. The nor-adrenaline content of the vesicles rose concomitantly with the increase in the activity of soluble dopamine β-hydroxylase. This rise in noradrenaline content was caused by an enhanced synthesis and not by an alteration in the subcellular distribution. The results are discussed with respect to the fate of dopamine β-hydroxylase during enhanced sympathetic nerve activity.     

Carboxylic ester hydrolases in mitochondria from rat skeletal muscle

Summary A mitochondrial pellet, prepared from rat skeletal muscle, contained a number of carboxylic ester hydrolase isoenzymes. The esterases which split -naphthyl acetate were organophosphate sensitive, whereas two out of three indoxyl acetate hydrolysing enzymes were resistant to both organophosphate and organomercury. The activity of the indoxyl acetate esterases was enhanced by the non-ionic detergents Tween-40 and Lubrol. After freezing, thawing and high speed centrifugation most of the -naphthyl acetate splitting enzymes were found in the supernatant, indicating that the enzymes are loosely bound to mitochondrial membranes.     

The Induction of Endocytosis in Starved Tetrahymena pyriformis

SYNOPSIS. The formation of digestive vacuoles by starved Tetrahymena pyriformis could be induced by mixtures of latex particles and a variety of potentially digestible solutes. Latex particles themselves had little effect in inducing vacuole formation. Protein, polypeptide, and RNA were highly effective inducers, while glutamate, amino acid mixtures, polysacharides, and glucose were moderately effective. Sodium-β-glycerophosphate had a slight effect and sodium acetate was ineffective. The possible stimulus to endocytosis is discussed. The endocytic response to inducers does not appear to be an all-or-none phenomenon and varies with the concentration of inducer. The stimulatory effect for protein-related inducers seems to be produced by a large number of stimulatory molecules acting upon a single cell and the magnitude of the response appears to be related to molecular size.     

Localization of enzymes and alkaloidal metabolites in Papaver latex

In continuing studies on the metabolic activity of Papaver somniferum, latex has been examined for its enzyme and alkaloidal metabolite content. After an initial centrifugation of latex at 1000g, the pellet which contained a heterogeneous population of dense organelles was further resolved on sucrose gradients. Of the enzymes monitored, acid phosphatase and l-3,4-dihydroxyphenylalanine decarboxylase were found to be in the latex 1000g supernatant, whereas catecholase (polyphenolase) was localized in two distinct organelles within the 1000g sediment. The lighter organelles, sedimenting at 30% sucrose, contained a soluble enzyme which was readily released on organelle plasmolysis, whereas the catecholase found within the heavier organelles, sedimenting at 55–60% sucrose, was membrane bound and showed significant activity only in the presence of Triton X-100. These latter organelles also contained the alkaloids, including morphine and thebaine, and were observed to readily accumulate [¹⁴CH₃]morphine. The alkaloid precursor, dopamine, was localized in the same dense vesicle fraction as the alkaloids. The rate of uptake of [7-¹⁴C]dopamine into these fractions at room temperature, however, was markedly lower than that of morphine. Electron microscopic examination of the organelles of various densities revealed that they possessed different morphology. The results are consistent with the concept that both the 1000g and supernatant fractions of the latex are required for alkaloid biosynthesis and that a sub-population of dense organelles found in the 1000g sediment have at least a function as a storage compartment for both alkaloids and their catecholamine precursor.     

The structure of rat liver mitochondria: a reevaluation

A β-N-acetylgalactosaminyltransferases (Ga1NAcT) that catalyzes the synthesis of a triglycosylceramide, GanglioTricer (Ga1NAcβ-Ga1β1-4G1c-cer), from lactosylceramide and UDP-Ga1NAc was isolated from guinea pig bone marrow. The enzyme was present in the supernatant solution obtained after homogenization of guinea pig bone marrow 12,000 × g pellet with 0.32 M sucrose containing 0.6% Triton X-100 and centrifugation at 129,000 × g. The enzyme that catalyzed the transfer of Ga1NAc to a tetraglycosylceramide (Lac-nTet-cer) was found in a membrane-bound fraction. The K_m values were 0.5 mM and 0.7 mM for the lactosylceramide and Lac-nTet-cer, respectively. 97.0% of the terminal [¹⁴C]Ga1NAc was cleaved by the action of pure β-hexosaminidase from [¹⁴C]triglycosylceramide.     

Effect of sedimentation through sucrose solutions on the protein-synthesizing ability of rat liver microsomes

1. Centrifugation of the postmitochondrial supernatant of rat liver through 1.0m-sucrose produces particles that have 85-95% less incorporating ability in a cell-free protein-synthesizing system than either ribosomes or microsomes. 2. The incorporation of [(14)C]phenylalanine into protein by particles prepared by sedimentation through 1.0m-sucrose is stimulated about 20-fold by addition of poly U. 3. The content of rapidly labelled RNA of microsomes is decreased during centrifugation through 1.0m-sucrose. 4. It is suggested that degradation of mRNA occurs during the formation of the pellet in the centrifuge tube as a result of a membrane-bound alkaline ribonuclease, after removal of the ribonuclease inhibitor of the soluble fraction.     

Evaluation of Cathepsins D and G and EC 3.4.24.15 as Candidate β-Secretase Proteases Using Peptide and Amyloid Precursor Protein Substrates

Abstract: No single protease has emerged that possesses all the expected properties for β-secretase, including brain localization, appropriate peptide cleavage specificity, and the ability to cleave amyloid precursor protein exactly at the amino-terminus of β-amyloid peptide. We have isolated and purified a brain-derived activity that cleaves the synthetic peptide substrate SEVKMDAEF between methionine and aspartate residues, as required to generate the amino-terminus of β-amyloid peptide. Its molecular size of 55–60 kDa and inhibitory profile indicate that we have purified the metalloprotease EC 3.4.24.15. We have compared the sequence specificity of EC 3.4.24.15, cathepsin D, and cathepsin G for their ability to cleave the model peptide SEVKMDAEF or related peptides that contain substitutions reported to modulate β-amyloid peptide production. We have also tested the ability of these enzymes to form carboxy-terminal fragments from full-length, membrane-embedded amyloid precursor protein substrate or amyloid precursor protein that contains the Swedish KM → NL mutation. The correct cleavage was tested with an antibody specific for the free amino-terminus of β-amyloid peptide. Our results exclude EC 3.4.24.15 as a candidate β-secretase. Although cathepsin G cleaves the model peptide correctly, it displays poor ability to cleave the Swedish KM → NL peptide and does not generate carboxy-terminal fragments that are immunoreactive with amino-terminal-specific antiserum. Cathepsin D does not cleave the model peptide or show specificity for wild-type amyloid precursor protein; however, it cleaves the Swedish "NL peptide" and "NL precursor" substrates appropriately. Our results suggest that cathepsin D could act as β-secretase in the Swedish type of familial Alzheimer's disease and demonstrate the importance of using full-length substrate to verify the sequence specificity of candidate proteases.     

Ontogenetic changes in adenylate cyclase, cyclic AMP phosphodiesterase and calmodulin in chick ventricular myocardium.

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and hatched (6-day H) chicks. This enzyme form was eluted at approx. 0.27 M-sodium acetate, hydrolysed both cyclic AMP and cyclic GMP, and was sensitive to stimulation by Ca2+-calmodulin, with an apparent Km for calmodulin of approx. 1 nM. In contrast, ventricular supernatant from late-embryonic (18-day E) chicks contained two forms of phosphodiesterase separable on DEAE-Sephacel: the same form as that seen at other ages, plus a cyclic AMP-specific form which was eluted at approx. 0.65 M-sodium acetate and was insensitive to stimulation by Ca2+-calmodulin. The ontogenetic changes in cyclic AMP phosphodiesterase activity in chick ventricular myocardium are consistent with reported ontogenetic changes in the steady-state contents of cyclic AMP in this tissue and suggest that this enzyme may be responsible for the changes that occur in this nucleotide during development of chick myocardium.     

The influence of incubation pH on the activity of rat and human gut flora enzymes

The activities of three bacterial biotransformation enzymes (β-glucuronidase, β-glucosidase, nitrate reductase) were determined in suspensions of rat caecal contents or human faeces over the pH range 6–8. All three enzymes were influenced by pH, as exemplified by β-glucosidase activity which diminished as pH increased. In other instances the rat and human flora showed distinct profiles, with nitrate reductase activity undetectable in human faeces below pH 6–6, whereas the rat caecal flora displayed optimal reduction of nitrate around neutrality. The most pronounced host-species difference was found with β-glucuronidase, which showed maximal activity at pH 6–0 in human faecal bacteria, while the rat caecal flora expressed greatest activity at pH 8–0. All three enzyme activities were associated with that fraction of rat caecal or human faecal material sedimented by centrifugation at 5000 g for 15 min, with little or no metabolism occurring in the 11000 g supernatant fluid. The results demonstrate that pH has a pronounced effect on the enzymic activity of bacterial preparations from rat and human sources.     

Increased content of calmodulin in morris hepatoma 5123 t.c.(h)

The content of calmodulin in the 100,000 × g supernatant and particulate fractions in Morris hepatoma 5123 t.c.(h), assayed by its ability to activate the Ca²⁺-activatable cAMP phosphodiesterase, was significantly higher (about 44%) than that in normal or host liver. Only one peak of calmodulin activity was detected when 100,000 × g supernates (350 mg protein) from sonicated homogenates of normal liver and hepatoma were fractionated on a DEAE cellulose column, eluting at a sodium acetate concentration of 0.65 M. The total calmodulin activity eluted from the hepatoma supernatant was 240% higher than that from nornal liver. The increased content of calmodulin in the hepatoma may contribute to the state of abnormal cell proliferation in this tissue.