Binding of paracetamol metabolites to mouse liver glutathione S-transferases

In mouse liver homogenate with an intact microsomal metabolism covalent binding of [14C]-paracetamol amounted to 1 nmol/mg protein. 65% of the total radioactivity were bound to soluble protein and 35% to microsomes. In the soluble fraction the major radioactivity peak co-chromatographed with glutathione S-transferase activity on Sephacryl S-300. Two different minor labelled fractions with apparent molecular weights of 130 000 and 25 000 daltons were also found. In a second experiment in a reconstituted system of microsomes and supernatant, 86% of the radio-activity was bound to supernatant and 14% by of microsomes. Following ion exchange chromatography of the supernatant on DEAE-Sepharose, the two major radioactivity-containing fractions coincided with GSH-S-transferase activities, but not with selenium-dependent or non-selenium-dependent glutathione peroxidase. The data show that irreversible binding of paracetamol metabolites in mouse liver occurs preferentially to GSH-S-transferases.     

Synthesis in vitro of intrinsic membrane proteins by free, membrane-bound, and Golgi apparatus-associated polyribosomes from rat liver

A fraction of intrinsic membrane proteins was prepared from the major membranous cell components of rat liver by extraction of the membranes with KCl and deoxycholate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the compositions of the intrinsic protein fractions from rough and endoplasmic reticulum, smooth endoplasmic reticulum. Golgi apparatus, plasma membrane, and nuclear envelope were similar to each other but distinct from that of mitochondria. Among endomembranes, differences were in the ratios of protein constituents plus a few protein bands of Golgi apparatus and plasma membranes not found in endoplasmic reticulum or nuclear envelope. The abilities of total rough endoplasmic reticulum, polysomes released from rough endoplasmic reticulum, and free polysomes to incorporate amino acids into the intrinsic protein fraction were tested in vitro. Polysomes bound to endoplasmic reticulum has the greatest capacity to synthesize proteins of this fraction as shown by co-purification of radioactive products and by immunoprecipitation. Although the majority of the radioactive products synthesized by bound polysomes were distinct from those synthesized by free polysomes, certain radioactive products synthesized by free polysomes also co-purified with intrinsic membrane proteins. The results show no absolute segregation between free and bound polysomes in the synthesis of intrinsic membrane proteins. However, the majority of these proteins appear to be synthesized by polysomes bound to the endoplasmic reticulum. Several intrinsic proteins found in plasma membranes do not appear in rough endoplasmic reticulum. To determine where these proteins were synthesized, the ability of other endomembrane components to support in vitro incorporation of [14C]leucine into protein was examined. In contrast to plasma membranes, isolated Golgi apparatus fractions did incorporate [14C]leucine to an extent greater than could be explained by contamination with rough endoplasmic reticulum. Golgi apparatus in situ and isolated from rat liver have polyribosomes associated with a zone of cytoplasm at the Golgi apparatus periphery occupied by tubules and vesicles. The polysomes are not directly attached to membranes as with rough endoplasmic reticulum and may represent a special class of "Golgi apparatus-associated" polysomes. The polysomes, when associated with Golgi apparatus membranes, incorporated amino acids in vitro. The products synthesized in vitro were analyzed by treatment with KCl and deoxycholate and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Certain proteins synthesized by the Golgi apparatus-associated polysomes remained insoluble after the treatment with KCl and deoxycholate. The proteins synthesized by the Golgi apparatus fraction had mobilities similar to proteins in plasma membranes which were absent from endoplasmic reticulum, and which were relatively minor components of Golgi apparatus...     

High-performance liquid chromatography of proteins

Incubation of liver microsomes with GDP [14C] mannose leads to the formation of lipid-linked derivatives of [14C] mannose, a dolichol phosphate monosaccharide and dolichol pyrophosphate oligosaccharides. Standard procedures for separating these two types of compounds from each other were found to be deficient in that fractions thought to contain only dolichol pyrophosphate oligosaccharides are contaminated with dolichol phosphate mannose. This paper presents a column chromatographic procedure which conveniently separates the products of an 8 min labeling experiment into two components; dolichol phosphate [14C]mannose and a [14C]-mannose containing oligosaccharide which is also lipid bound. When this oligosaccharide is released from the lipid by hydrolysis and chromatographed on Sephadex G-50 or G-15 it gives a single peak with an indicated molecular weight of 1100. However, when this released oligosaccharide is chromatographed on concanavalin A Sepharose it is resolved into two peaks suggesting that there may be 2 oligosaccharide of approximately the same size but different structures. After brief periods of labeling with GDP [14C]mannose (5 s) an additional oligosaccharide of 3 to 4 sugar residues can be found in the dolichol pyrophosphate oligosaccharides fraction. Incubation of liver microsomes with UDP [14C]glucose or UDP[14C]galactose produces oligosaccharide components containing 7--8 sugar residues. Labeling of microsomes with UDP[14C]acetylglucosamine gives rise to three different components, including a lipid bound oligosaccharide containing 3- 5 sugar residues.     

HDL3-mediated cholesterol efflux from cultured enterocytes: the role of apoproteins A-I and A-II

High density lipoproteins (HDL) were recently demonstrated in an enterocyte model (CaCo-2 cells) to mediate reverse cholesterol transport by retroendocytosis. The present study was carried out to define the role of the major HDL apoproteins (apo) A-I and apo A-II in this pathway. HDL3 was fractionated by heparin affinity chromatography into the two main fractions containing either apo A-I only (fraction A) or both apo A-I and apo A-II (fraction B). In addition, liposomes were reconstituted from purified apo A-I or apo A-II and dimyristoyl phosphatidylcholine. The cell binding properties and cholesterol efflux potential were studied in the lipoprotein fractions and the liposomes. Both fractions exhibited similar maximal binding capacities of 4427 (A) and 5041 (B) ng/mg cell protein, but their dissociation constants differed (40.5 and 167.7 micrograms/mL, respectively). Fraction A induced cholesterol efflux and stimulated cholesterol synthesis more than did fraction B. Fraction A mobilized both cellular free and esterified cholesterol, whereas fraction B preferentially mobilized cholesteryl esters. Liposomes, containing either apo A-I or apo A-II, showed specific binding, endocytosis and endosomal transport, and were released as intact particles. Apo A-I liposomes also mediated cholesterol efflux. In conclusion, there is evidence that the HDL3 subfractions A and B, as well as reconstituted liposomes containing either apo A-I or apo A-II, were specifically bound and entered a retroendocytosis pathway which was directly linked to cholesterol efflux. Quantitatively, the apo A-I subfraction appeared to play the dominant role in normal enterocytes. The apo A-II content of fraction B was related to the mobilization of cholesteryl esters.     

The lipid composition of plasma membrane subfractions originating from the three major functional domains of the rat hepatocyte cell surface

1. The neutral and phospholipid compositions of three rat liver plasma membrane subfractions originating predominantly from the three major functional domains of the hepatocyte viz the blood sinusoidal, contiguous and bile canalicular fractions, were determined. 2. The sinusoidal and canalicular plasma membrane subfractions, both of which were vesicular, contained a higher lipid to protein weight ratio than the contiguous plasma membrane subfraction that consisted of membrane strips, junctional complexes and some larger vesicles. The three plasma membrane subfractions contained a similar neutral lipid to phospholipid ratio. The highest unesterified cholesterol content was associated with the canalicular plasma membrane subfraction. 3. The phospholipid profiles of the three subfractions were generally similar. However, the canalicular plasma membrane subfraction contained a higher proportion of sphingomyelin than the other subfractions. 4. Correlations between the neutral and phospholipid composition of the subfractions and membrane integrity and function are discussed, especially with respect to a possible role of lipids in governing the resilience of the canalicular plasma membrane to the action of bile salts.     

Extensive exchange of rat liver microsomal phospholipids

Liver microsomal fractions were prepared from rats injected with a single dose of choline [14C]methylchloride or with single or multiple doses of 32Pi. Exchangeability of microsomal phospholipids was determined by incubation with an excess of mitochondria and phospholipid exchange proteins derived from beef heart, beef liver or rat liver. Labeled phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were found to act as a single pool and were 85--95% exchangeable in 1--2h. High latencies of mannose-6-phosphate phosphohydrolase activities and impermeability of microsomes to EDTA proved that phospholipid exchange proteins did not have access to the intracisternal space. If microsomal membranes are largely composed of phospholipid bilayers, the experiments suggest that one or more of the phospholipid classes in microsomal membranes undergo rapid translocation between the inner and outer portions of the bilayer.     

Tissue- and subcellular-distribution of the binding site of [3H]9- methyl-7-bromoeudistomin D, a potent caffeine-like Ca2+ releaser, in rabbits

Tissue and subcellular distribution of the binding site of 3H-labelled 9-methyl-7-bromoeudistomin D ([3H]MBED), a powerful caffeine-like Ca2+ releaser, were investigated in rabbits. The order of specific activities of total homogenates was liver > brain > other tissues. All binding was completely suppressed by 10 mM caffeine, indicating that all [3H]MBED binding sites are modulated by caffeine. [3H]MBED binding sites distributed mainly in membrane fractions rather than soluble fractions in most tissues. In lung and liver, [3H]MBED binding was enriched in microsomes. [3H]MBED may be useful as a probe to investigate the actions of caffeine at the molecular level not only in muscles but also in a variety of tissues including liver, kidney and lung.     

Pharmacology and subcellular distribution of [3H]rilmenidine binding sites in rat brain

We have previously reported that in rat brain membranes, [3H]rilmenidine, in addition to labelling alpha2-adrenoceptors and the I2B-subtype of imidazoline receptor binding site (I2B-RBS), may label an additional I-RBS population, distinct from previously classified I1-RBS and I2-RBS. In this study, using crude or fractionated rat brain membranes we examined the possible association of [3H]rilmenidine-labelled I-RBS with the A- and B-isoforms of monoamine oxidase (MAO) by studying the inhibition of [3H]rilmenidine binding by a number of MAO inhibitors; and comparing the maximal binding density (Bmax) and subcellular distribution of [3H]rilmenidine binding sites with that of MAO-A and MAO-B catalytic sites labelled by [3H]RO41-1049 and [3H]RO19-6327 and 12-RBS labelled by [3H]2-BFI. Inhibition of [3H]rilmenidine binding by all MAO inhibitors tested produced very shallow curves (slope 0.29-0.56). Clorgyline and moclobemide (selective MAO-A inhibitors) displayed moderate affinities (60-140 nM), while pargyline (non-selective MAO-inhibitor), RO41-1049 (selective MAO-A inhibitor) and RO19-6327 (selective MAO-B inhibitor) exhibited very low affinities (> 2 microM) for 50-75% of [3H]rilmenidine-labelled I-RBS in crude brain membranes and even lower affinity for the remaining binding. Under identical buffer conditions, the Bmax of [3H]rilmenidine-labelled I-RBS (1.45+/-0.14 pmol/mg protein) was considerably lower than those of MAO-A (13.10+/-0.15 pmol/mg) and MAO-B (10.35+/-0.50 pmol/mg) sites. These results suggest that [3H]rilmenidine does not interact directly with the active catalytic site of either MAO enzyme and could at best only associate with a subpopulation of MAO molecules. Binding studies on five fractions of rat cortex homogenates-nuclear (N), heavy (M) and light (L) mitochondrial, microsomal non-mitochondrial (P), and soluble cytosolic (S) fractions-revealed that 45% of total [3H]rilmenidine binding was present in the P fraction cf. 20 and 23% in the M and L fractions, in contrast to [3H]RO19-6327 and [3H]2-BFI which bound 11-13% in the P fraction and 36-38% and 35-44% in the M and L fractions, respectively. Binding of all ligands in the N fraction was 6-15% of total. These studies reveal that [3H]rilmenidine-labelled I-RBS, unlike the I2-RBS, are not predominantly associated with mitochondrial fractions containing the MAO enzymes (and cytochrome oxidase activity), but appear to be distributed in both the mitochondrial and plasma membrane fractions in rat cerebral cortex.     

Rapid uptake and binding of estradiol-17beta-6-(O-carboxymethyl)oxime:125I-labeled BSA by female rat liver

To investigate potential membrane-mediated responses to estrogen, a membrane-impermeant, radioiodinated, steroid-BSA conjugate--estradiol-17beta-6-(O-carboxymethyl)oxime:125I-labeled BSA (17beta-E-6-125I-BSA)--or related steroid conjugates, or 125I-BSA was injected into female Sprague-Dawley rats, and tissues were collected at varying times postinjection. The liver, adrenal, and spleen displayed the most prominent uptake of 17beta-E-6-125I-BSA, reaching a maximum of 43 times blood levels in sonicated liver samples at 5 min postinjection, but no uptake of 125I-BSA. Isolation of liver membranes by differential centrifugation showed that over 50% of recovered radioactivity was in association with microsomes and plasmalemma (P3 fraction) at 30 sec postinjection. By 60 min postinjection, over 75% of recovered radioactivity was in association with mitochondrial and lysosomal membranes (P2 fraction), and less than 10% remained in the P3 fraction. In vitro competition assays demonstrated two binding sites in liver P3 fractions. The spleen and liver also showed saturable binding in vivo. These data suggest the presence of at least one membrane-binding protein for estrogen in liver, adrenal, and spleen. Initial studies of affinity-purified liver P3 fractions using ligand blots indicated the presence of two binding proteins. These potential membrane estrogen-binding proteins may be involved in a very rapid shuttling of estrogen from the plasmalemma to mitochondria and lysosomes.     

Association of N-ethylmaleimide sensitive fusion (NSF) protein and soluble NSF attachment proteins-alpha and -gamma with glucose transporter-4-containing vesicles in primary rat adipocytes

To investigate the role of N-ethylmaleimide sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAP)-containing fusion complexes in glucose transporter-4 (GLUT4) membrane trafficking, the subcellular distributions of NSF, alpha-SNAP, and gamma-SNAP in primary rat adipocytes were determined. A large fraction of the NSF and SNAPs were associated with intracellular membranes, distributed between the low-density microsomes (LDM) and high-density microsomes. Very little of the NSF and SNAPs were associated with the plasma membrane fraction. This distribution did not change after insulin stimulation. Approximately 75% of the NSF and SNAPs in the LDM fraction were coimmunoprecipitated with 85% of the GLUT4 and 60% of the vesicle associated membrane proteins (VAMPs; synaptobrevins) VAMP-2 and cellubrevin in anti-GLUT4 immunoadsorptions. In contrast to NSF and the SNAPs, the beta-coatomer protein (beta-COP) found in the LDM fraction was excluded from GLUT4 vesicles. When LDM fractions were solubilized with Thesit (octaethylene glycol dodecyl ether) or Triton X-100, approximately 40% of the alpha-SNAP was colocalized with NSF on glycerol gradients in large (approximately 20S), ATP-sensitive complexes. VAMP-2 and cellubrevin are concentrated in the LDM fractions and in GLUT4 vesicles; both were excluded from these complexes. These data suggest that the steady state association of NSF and the SNAPs with GLUT4 vesicles and cell membranes is independent of the formation of fusion complexes.     

The number of oligosaccharides borne by porcine thyroglobulin is variable

Purified porcine thyroglobulin (Tg) was fractionated on a concanavalin A-Sepharose 4B column by a step-wise elution with increasing concentrations of methyl alpha-mannoside (fraction A, 50 mM; B, 100 mM; C, 200 mM; D, 500 mM, and E, 1 M), and its fractional ratio was 12.8:28.6:26.4:19.7:12.4. These five fractions showed the same profile in polyacrylamide gel electrophoresis. The subfractions were analyzed for their relative contents in oligosaccharides of each structure type and for their monosaccharide contents. In fractions B, C, D, and E the former varied between 15-22% for triantennary complex-type, 47-60% for biantennary complex-type, and 22-30% for high mannose-type oligosaccharide. Fraction A showed a higher percentage of triantennary complex-type structures (36%) and a lower percentage of biantennary complex-type structures (17%). The monosaccharide numbers increased from fraction A to E: 85 to 135 mannose residues, 60 to 82 galactose residues, 84 to 115 N-acetyl glucosamine residues, and 22 to 28 sialic acid residues. After analysis of the number of mannose residues contained in the high mannose-type structures, it was possible to calculate the number of oligosaccharides borne by each Tg subfraction. This number was approximately the same for fractions A and B (22.4 and 21.7), then it increased from B to E (21.8 to 32.9). These results account for the separation obtained on the concanavalin A-Sepharose 4B column. Separation of the two first subfractions bearing the same number of oligosaccharides is certainly due to the higher number of high mannose-type structures in B. In conclusion, the studies reported here show that porcine Tg is heterogeneous, and mainly so in terms of total number of N-glycan structures.     

Changes of the expression of protein substrates of Ca2+/calmodulin-dependent protein kinase II in neonate and adult rats

We studied the expression of whole protein substrates of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in the forebrain of neonate and adult rats. Protein substrates were determined by phosphorylation of the soluble and particulate fractions by CaM kinase II with [gamma-33P]ATP. Phosphorylated proteins were analyzed by SDS-PAGE and two-dimensional gel electrophoresis. More than 50 endogenous proteins were found to be phosphorylated by CaM kinase II in both soluble and particulate fractions. The expression of about 15 protein substrates increased in the particulate fraction from neonate to adult rats, and that of several proteins also changed in the soluble fraction. These findings suggest that the expression of protein substrates was regulated during development as well as that of CaM kinase II itself.     

Analysis of Candida albicans adhesion to salivary mucin

Clearance of Candida albicans from the oral cavity is thought to be mediated via specific receptor-ligand interactions between salivary constituents and the fungus. Since surfaces in the oral cavity are normally coated with a saliva-derived pellicle, specific interactions between salivary constituents and C. albicans may also contribute to adhesion of C. albicans to the oral mucosa and dental prostheses. Therefore, the purpose of this study was to identify salivary constituents to which C. albicans is capable of binding. A solid-phase overlay assay was used in which electrophoretically separated rat and human salivary constituents bound to membrane filters were incubated with radiolabelled C. albicans cells. C. albicans adhered to a single salivary component from each host. Correlation of cell-binding activity with specific monoclonal antibody (MAb)-binding activity indicated that the constituent bound by C. albicans in human saliva was low-molecular-weight mucin (MG2) and that in rat saliva was rat submandibular gland (RSMG) mucin. Further studies showed an identical cell hybridization signal and MAb colocalization by using RSMG ductal saliva and an aqueous RSMG extract in the solid-phase overlay assay. Analysis of cell binding to the aqueous extract of RSMG fractionated by anion-exchange chromatography demonstrated that C. albicans binding was restricted to an acidic subfraction of the RSMG extract, which also bound the RSMG mucin-specific MAb. The Candida-binding fraction contained predominantly RSMG mucin glycoprotein and also a noncovalently associated, chloroform-extractable material. Furthermore, we identified two strains of C. albicans which differed severalfold in the ability to bind RSMG mucin in the overlay assay. These results suggest that C. albicans binds to only a specific subfraction of RSMG mucin and that the two C. albicans strains tested differ in the ability to bind RSMG mucin subfractions.     

Ionization, partitioning, and dynamics of tryptophan octyl ester: implications for membrane-bound tryptophan residues

The presence of tryptophan residues as intrinsic fluorophores in most proteins makes them an obvious choice for fluorescence spectroscopic analyses of such proteins. Membrane proteins have been reported to have a significantly higher tryptophan content than soluble proteins. The role of tryptophan residues in the structure and function of membrane proteins has attracted a lot of attention. Tryptophan residues in membrane proteins and peptides are believed to be distributed asymmetrically toward the interfacial region. Tryptophan octyl ester (TOE) is an important model for membrane-bound tryptophan residues. We have characterized this molecule as a fluorescent membrane probe in terms of its ionization, partitioning, and motional characteristics in unilamellar vesicles of dioleoylphosphatidylcholine. The ionization property of this molecule in model membranes has been studied by utilizing its pH-dependent fluorescence characteristics. Analysis of pH-dependent fluorescence intensity and emission maximum shows that deprotonation of the alpha-amino group of TOE occurs with an apparent pKa of approximately 7.5 in the membrane. The fluorescence lifetime of membrane-bound TOE also shows pH dependence. The fluorescence lifetimes of TOE have been interpreted by using the rotamer model for the fluorescence decay of tryptophan. Membrane/water partition coefficients of TOE were measured in both its protonated and deprotonated forms. No appreciable difference was found in its partitioning behavior with ionization. Analysis of fluorescence polarization of TOE as a function of pH showed that there is a decrease in polarization with increasing pH, implying more rotational freedom on deprotonation. This is further supported by pH-dependent red edge excitation shift and the apparent rotational correlation time of membrane-bound TOE. TOE should prove useful in monitoring the organization and dynamics of tryptophan residues incorporated into membranes.     

含铅土壤中铅的浸出行为

采用BCR连续浸提法分析含铅土壤中铅的存在形态,通过静态溶浸与动态淋滤试验研究了土壤中铅的浸出行为,结合浸出毒性分析,探讨了添加活性炭对土壤中铅的稳定性影响。结果表明,供试土壤中可交换态铅可转化为可还原态和可氧化态形式存在,活性炭对不稳定态的铅有较强吸附作用。添加活性炭对降低土壤中铅的浸出有一定效果,并且与活性炭性质和淋溶体系pH有关,醋酸体系浸提剂比硫酸体系对铅的浸出浓度影响更大。     

The yeast Rab escort protein binds intracellular membranes in vivo and in vitro

In both mammals and yeast, intracellular vesicular transport depends on the correct shuttling between membrane and cytosol of the Rab/Ypt small G proteins. Membrane association of these proteins requires prenylation by the Rab geranylgeranyl transferase that recognizes a complex formed by the Rab/Ypt protein and the Rab escort protein (REP). After prenylation the Rab/Ypt protein is delivered to the target membranes by REP. Little is known about the early steps of the Rab-REP complex formation and where this association occurs in the cell. Although prenylation is believed to take place in the cytosol, we show that the yeast Rab escort protein Mrs6 is present in both soluble and particulate fractions of cell extracts. Mrs6p is associated with the heavy microsomal fraction that contains endoplasmic reticulum-Golgi membranes but is absent in the plasma membrane, vacuoles, mitochondria, and microsomal subfraction associated with mitochondria. The solubilization pattern of the particulate pool of Mrs6p implies that this protein is peripherally but tightly associated with membranes via hydrophobic interactions and metal ions. We also report that the C terminus of Mrs6p is important for maintaining the solubility of the protein because its deletion or replacement with the C terminus of RabGDI results in a protein that localizes only to membranes.     

Curare: a preventive of traumatic complications in convulsive shock therapy (including a preliminary report on a synthetic curare-like drug). 1940

Yellow fluorescent lipofuscin deposited in rat kidney was extracted in an aqueous solution and characterized after separation. Centrifugal fractionation of the extract revealed that most of the yellow fluorescence was detected in the 105,000 x g-supernatant, and little in nuclei, cell debris, mitochondria, lysosomes, microsomes and plasma membrane. The yellow fluorescence in the supernatant was fractionated by gel filtration through Sephadex columns into 5 yellow fluorescent fractions A, B (B1, B2 and B3) and C showing the same fluorescence spectra with excitation maximum/emission maximum at 400/620 nm. The components in fraction A were converted into the smaller molecular-weight components in fraction B on treatment with 4 M urea or protease, suggesting that they were proteinaceous. The smallest molecular-weight fluorescent components in fraction C were adherent to solid cellulose materials. The fluorescent components in all the fractions were soluble in water and insoluble in chloroform-methanol, indicating that they were not lipidic materials. The fluorophores in these fractions were kept stable on borohydride treatment, but readily converted into non-fluorescent components on heavy-metal ion treatment. The characteristics of the yellow fluorescence in these fractions were quite different from those of bluish lipofuscin-like fluorophores that may be generated in tissues during lipid peroxidation.     

Phosphoinositide 3-kinase in rat liver nuclei

Biochemical and immunochemical data from the present investigation reveal the existence of a p85/p110 phosphoinositide 3-kinase (PI 3-kinase) in rat liver nuclei. 32P-Labeling of membrane phosphoinositides by incubating intact nuclei with [gamma-32P]ATP results in the formation of [32P]phosphatidyl-inositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3], accompanied by small quantities of [32P]phosphatidylinositol 3-phosphate [PtdIns(3)P]. Studies with subnuclear fractions indicate that the PI 3-kinase is not confined to nuclear membranes. The nuclear soluble fraction also contains PI 3-kinase and an array of inositide-metabolizing enzymes, including phospholipase C (PLC), phosphoinositide phosphatase, and diacylglycerol (DAG) kinase. As a result, exposure of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to the nuclear extract in the presence of [gamma-32P]ATP generates a series of 32P-labeled D-3 phosphoinositides and phosphatidic acid (PA) in an interdependent manner. On the basis of the immunological reactivity and kinetic behavior, the nuclear PI 3-kinase is analogous, if not identical, to PI 3-kinase alpha, and constitutes about 5% of the total PI 3-kinase in the cell. Moreover, we test the premise that nuclear PI 3-kinase may, in part, be regulated through the control of substrate availability by PtdIns(4,5)P2-binding proteins. Effect of CapG, a nuclear actin-regulatory protein, on PI 3-kinase activity is examined in view of its unique Ca2+-dependent PtdIns(4, 5)P2-binding capability. In vitro data show that the CapG-mediated inhibition of nuclear PI 3-kinase is prompted by PKC phosphorylation of CapG and elevated [Ca2+]. This CapG-dependent regulation provides a plausible link between nuclear PLC and PI 3-kinase pathways for cross-communications. Taken together, these findings provide definite data concerning the presence of an autonomous PI 3-kinase cycle in rat liver nuclei. The nuclear location of PI 3-kinase may lead to a better understanding regarding its functional role in transducing signals from the plasma membrane to the nucleus in response to diverse physiological stimuli.     

Relation of renal hemodynamics to metabolism of angiotensin II by the canine kidney

Incubation of brain cortex slices in the presence of glucose resulted in the permeation of about 65% of [14C] mescaline into slices. Of this, about one-third radioactivity was bound with nuclei, mitochondria, microsomes, and ribosomes. Dialysis of subcellular fractions did not markedly reduce the amounts of radioactivity bound to the fractions. The permeation into slices and the binding of mescaline to subcellular fractions were fairly time-dependent, but were inhibited by the presence of potassium cyanide, or by the absence of glucose and by heating to 80 degrees C for 1 min.     

Comparative effects of cadmium, zinc, and lead in vitro on pulmonary, adrenal, and hepatic microsomal metabolism in the guinea pig

The in vitro effects of Cd, Zn, and Pb on pulmonary, adrenal, and hepatic microsomal enzyme activities in guinea pigs were compared. Cd and Zn produced concentration-dependent (20-200 microM) decreases in benzphetamine demethylase and biphenyl hydroxylase activities in adrenal, liver, and lung. Pb had no significant effect on either enzyme in any of the tissues studied. Adrenal and pulmonary enzymes were more sensitive to the effects of Cd and Zn than were hepatic enzymes. Benzo[a]pyrene hydroxylase and ethoxycoumarin deethylase activities were decreased by Zn, Cd, and Pb in adrenal, liver, and lung microsomes. The inhibitory effect on benzo[a]pyrene and ethoxycoumarin metabolism were far greater than those on benzphetamine or biphenyl metabolism. The relative potencies of the metals as inhibitors of xenobiotic metabolism were Zn greater than Cd greater than Pb. Cd and Zn also inhibited steroid 21-hydroxylase activity in adrenal microsomes, but Pb had no effect on steroid metabolism. In addition, microsomal epoxide hydratase activity in adrenal, liver, and lung was inhibited by Cd but not by Zn or Pb. The results demonstrate that adrenal and pulmonary microsomal enzymes, like those in liver, are inhibited by various metals. Inhibition of mixed-function oxidases by metals in vitro is apparently not related to changes in cytochrome P-450 levels or substrate binding to cytochrome P-450. In addition, the actions of Cd, Zn, and Pb in each tissue are highly dependent on the substrates employed.