HETEROGENEOUS DISTRIBUTION OF ENZYMES IN SUBMICROSOMAL MEMBRANE FRAGMENTS

Microsomal membranes are postulated to contain either a homogeneous arrangement of individual enzymes or groupings of functionally related enzymes. In the present study we attempt to distinguish between these hypotheses in subfractions of rough microsomes from rat liver. After sonication, the individual vesicles that make up the rough-membrane fraction average less than 1/100 of their previous mass. The vesicles in the sonicated suspension are fractionated roughly according to size on a continuous sucrose gradient. Enzyme activity or concentration in fractions of the gradient is expressed on a phospholipid basis. Fractions containing primarily small vesicles differ from those containing larger vesicles in a manner suggesting a certain degree of separation of NADH-linked from NADPH-linked enzymes. NADH-ferricyanide reductase, NADH-cytochrome c reductase and cytochrome b₅ are most concentrated within the large vesicles in the lowest third of the gradient. In contrast, NADPH-cytochrome c reductase and cytochrome P-450 are found in highest concentration in the small vesicles that make up the upper third of the gradient. The results suggest a nonrandom distribution of these two enzyme groups in the membranes of the endoplasmic reticulum.     

An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b₅ reductase and cytochrome b₅.     

Lateral enzyme topology in the rough endoplasmic reticulum of rat liver

Summary Rough microsomes were subfractionated on the basis of different properties in order to investigate the nature and extent of the enzyme heterogeneity of these vesicles. A discontinuous gradient, containing monovalent cations allowed the separation of a ribosome-poor membrane fraction which was enriched in electron transport enzymes and relatively poor in phosphatases. Zonal centrifugation on a stabilizing gradient separated 3 fractions characterized by enrichment of electron transport enzymes, glucose-6-phosphatase and adenosinetriphosphatase, respectively. An essentially similar pattern was seen when ribosomes were removed with EDTA and the denuded vesicles subfractionated on a sucrose gradient. Rough microsomes from phenobarbitaltreated rats exhibited the same pattern both qualitatively and quantitatively. It appears that electron transport enzymes and two types of phosphatases are heterogeneously distributed among rough microsomal vesicles.This work has been supported by grants from the Swedish Medical Research Council. The authors wish to thank Mrs. Ulla-Britta Torndal for her valuable technical assistance     

Cyclic nucleotide phosphodiesterase. Partial purification and characterization of a high affinity enzyme activity from human lung tissue.

Part of the soluble cyclic nucleotide phosphodiesterase activity of crude human lung tissue can be attributed to a thermosensitive (37 degrees) enzyme with a high apparent affinity for both adenosine 3':5'-monophosphate (cyclic AMP) and guanosine 3':5'-monophosphate (cyclic GMP). The enzyme can be partially purified by DEAE-Sephadex chromatography. In the presence of 0.1 mM EDTA or ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), it is eluted from the column immediately before a cyclic GMP-specific phosphodiesterase, but in the presence of 0.2 mM Ca2+, the elution follows that of the cyclic GMP-specific enzyme. The two forms of the nonspecific phosphodiesterase activity are referred to as DEAD-Sephadex Fractions Ia and Ic, respectively. Their apparent molecular weights, recorded at gel filtration, vary with different preparations from 230,000 to 150,000. Occasionally, corresponding recordings for main peaks of activity also cluster round the values 120,000, 105,000, and 78,000. The enzymatic properties of Fractions Ia and Ic closely resemble each other. The enzyme activity is blocked by EDTA, partially inhibited in the presence of 1,10-phenanthroline, but only slightly affected by EGTA. The inhibitory effect of EDTA can be overcome by Mg2+ and Mn2+ and that of 1,10-phenanthroline, in part, by Zn2+; this cation in itself is inhibitory at millimolar concentrations. With submicromolar substrate concentrations, the activity of either fraction obeys linear kinetics displaying an apparent Km of approximately 0.4 micron for both substrates. Reciprocal inhibition experiments suggest that hydrolysis of both cyclic AMP and cyclic GMP is performed by the same active site. Examination of the activity using extended substrate concentration ranges indicates nonlinear kinetics; Hill plots of such data also show nonlinear curvature. The activity is inhibited by micromolar concentrations of inosine 3':5'-monophosphate (cyclic IMP), 3-isobutyl-1-methylxanthine, papervine, and some antiallergic agents. Theophylline and disodium cromoglycate are less potent inhibitors. Inhibition of activity by Lubrol PX follows a biphasic dose response curve. The activity of Fraction Ia can be enhanced 2- to 3-fold by a Ca2+-dependent activator prepared from lung tissue, whose action is counteracted by chlorpromazine, and by lysophosphatidylcholine. It is initially enhanced but subsequently decreased at exposure to trypsin. Fraction Ic is less prone to activation by these agents. The results indicate that the present activity represents an enzyme form that differs from three previously described phosphodiesterases of human lung tissue. It is apparently related to, but also shows distinct differences from the Ca2+-dependent enzyme(s) of brain and heart tissue.     

Heme Amplifies the Innate Immune Response to Microbial Molecules through Spleen Tyrosine Kinase (Syk)-dependent Reactive Oxygen Species Generation

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions, hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor κB (NF-κB) and MAPKs and the generation of reactive oxygen species were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of spleen tyrosine kinase (Syk) and was abrogated in dendritic cells deficient in Syk. Moreover, inhibition of Syk and the downstream molecules PKC and PI3K reduced the reactive oxygen species generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.     

Preparation and characterization of total,rough and smooth microsomes from the lung of control and methylcholanthrene-treated rats

Optimal conditions for the preparation of relatively pure microsomes and microsomal subfractions from rat lung have been determined. The most important of these conditions is homogenization of a 20% (w/v) suspension of lung tissue in 0.44 M sucrose/1% (w/v) bovine serum albumin with four up-and-down strokes at 440 rev./min in a Potter-Elvehjem homogenizer. The 10 000 × g supernatant prepared from this homogenate can be centrifuged at 105 000 × g to obtain total microsomes or subfractionated into rough and smooth microsomes on a Cs⁺-containing discontinuous sucrose gradient. The total, rough and smooth microsomes have been characterized in terms of their chemical composition, enzymatic activity, and morphology. These preparations should prove useful in studies of various enzymes in lung (e.g. benzpyrene monooxygenase, epoxide hydrase, enzymes of phospholipid and ascorbic acid synthesis) and in subfractionations designed to reveal heterogeneites in the lateral plane of the lung endoplasmic reticulum.     

Partial purification and characterization of adenosine- and guanosine-3',5'-monophosphate phosphodiesterases from human lung tissue.

Crude extracts of human lung tissue were examined for cyclic adenosine- and guanosine-3',5'-monophosphate (cAMP and cGMP) phosphodiesterase activities. Nonlinear reciprocal plots were observed for each substrate. DEAE-Sephadex chromatography of the extracts revealed four main fractions of activity, which were further purified by Sephadex gel filtration. The phosphodiesterase activity of the resulting individual fractions was partially characterized with respect to substrate specificity, kinetic parameters, apparent molecular weight (gel filtration), thermal stability at 30 and 37 degrees C, effect of the cyclic nucleotide not utilized as substrate, and the possible influence of Ca2+-dependent protein activator. The results indicate that the tissue contains phosphodiesterases with strict specificity and a high apparent affinity for each of the two cyclic nucleotides (the Km values determined were approximately 0.3-0.4 muM). The high affinity cAMP phosphodiesterase activity was enriched in two of the purified fractions; both activities probably represent fragments of the native high affinity cAMP specific enzyme. A third purified phosphodiesterase showed mixed substrate specificity. The Km value recorded for hydrolysis of either substrate with this enzyme was approximately 25 muM. A fourth, irregularly occurring, phosphodiesterase activity also showed mixed substrate specificity. The Km value registered for hydrolysis of either substrate with this fraction was approximately 0.4 muM. There was no evidence for a Ca2+-dependent specific activation by a boiled lung tissue supernatant of any of the purified enzymes.     

HETEROGENEITY OF ROUGH-SURFACED LIVER MICROSOMAL MEMBRANES OF ADULT,PHENOBARBITAL-TREATED,AND NEWBORN RATS

Rough microsomes from the livers of adult, phenobarbital-treated, and newborn rats were subfractionated on a continuous sucrose gradient. Among the subfractions a marked heterogeneity in the distribution patterns of some enzyme activities appears. The isopycnic density of the various fractions in aqueous sucrose ranges from 1.17 to 1.25. The sedimentation coefficients (s₀) in 0.25 M sucrose lie between 0.4 x 10³ S and 1.2 x 10³ S. In adult animals, the NADH- and NADPH-cytochrome c reductase as well as the G6Pase activities are much higher in the slower sedimenting fractions than in the pellet. The increase in the level of G6Pase induced by fasting as well as the phenobarbital-induced changes are most prominent in the slowly sedimenting fractions. Three injections of phenobarbital have no effect on the specific NADPH-cytochrome c reductase activity in the pellet, but cause a significant increase of this enzyme activity in the light fractions. In the newborn animal, the NADH-ferricyanide reductase and NADPH-cytochrome c reductase activities are highest in the light fractions. On the other hand, the amount of cytochrome b ₅ is evenly distributed in all cases. Short-term incorporation of leucine-¹⁴C and glycerol-³H in vivo after phenobarbital treatment shows contrasting results, as the former is increased and the latter is decreased in the slowly sedimenting fractions. Leucine-¹⁴C incorporation into isolated, total membrane proteins is greater in both phenobarbital-treated and newborn animals than in untreated adults. The data support a multistep model for membrane biogenesis and indicate dynamic and individual behavior of the different parts of the rough-surfaced endoplasmic reticulum.     

Enzyme purification by genetically attached polycysteine and polyphenylalanine affinity tails

Two novel affinity tails, polycysteine and polyphenylalanine, have been genetically attached to galactokinase (EC 2.7.1.6) and beta-galactosidase (EC 3.2.1.23) in order to facilitate their purification. A chemically synthesized DNA linker encoding four cysteine residues was thus fused in frame with the galactokinase gene. The gene product, cysteine galactokinase, was significantly retarded on a column of thiopropyl-Sepharose. Using pulse elution, cysteine galactokinase was eluted at 10 mM DTT. Under the condition used, native galactokinase did not bind to thiopropyl-Sepharose. Homopolymer tailing was employed to prepare a phenylalanine-modified beta-galactosidase. One of the obtained genetic transformants coding for a beta-galactosidase carrying 11 phenylalanine residues at the N-terminus of the enzyme was isolated. With the aid of hydrophobic interaction chromatography the modified enzyme could be purified to homogeneity on fast protein liquid chromatography using a phenyl-Superose column.