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At least 649 records · Page 36Linked to original sources
PubMed1990

Compartmentalization of low molecular mass GTP-binding proteins among neutrophil secretory granules.

Neutrophils contain several distinct classes of secretory granules that may sequentially fuse with the phagosome after the ingestion of particulates, or that may be differentially exocytosed after cellular activation with soluble stimuli. The exocytosis of neutrophil secretory granules has been shown to be GTP-dependent at a step distal to activation of the transductional G proteins. Inasmuch as ras-related low molecular mass GTP-binding proteins have been shown to play regulatory roles in vesicle sorting in the secretory pathway in yeast, the differential mobilization of neutrophil granules might be regulated by distinct GTP-binding proteins. We therefore explored the distribution and identity of low molecular mass GTP-binding proteins in neutrophil secretory granules and other subcellular fractions. After lysis by nitrogen cavitation, four highly resolved fractions were harvested from discontinuous Percoll gradients: a microsomal fraction enriched for plasma membranes, specific granules, primary granules, and cytosol. At least seven bands of distinct Mr were detected by probing protein blots with [32P]GTP. Microsomes contained a prominent GTP-binding band at 26 kDa and weaker ones at 24 and 22.5 kDa; specific granules contained bands at 26, 24, 22, and 20 kDa; primary granules showed bands at 24 and 23 kDa; cytosol showed strong bands at 23.5 and 19 kDa and a weak band at 26 kDa. Antiserum against ADP-ribosylation factor reacted strongly with the 19-kDa band in cytosol but with none of the membrane fractions. None of these proteins was recognized by antibodies against ras or against Sec4p. Botulinum exoenzyme C3 labeled bands of molecular mass 20 and 21 kDa in cytosol and microsomes that have distinct mobilities from all the blotted [32P]GTP-binding proteins. The highly compartmentalized subcellular distribution of the blotted [32P]GTP-binding proteins in neutrophils is consistent with a regulatory role in the differential mobilization of granule compartments during cellular activation.

Adenosine Diphosphate Ribose↗
PubMed1990

Compartmentation and turnover of the low density lipoprotein receptor in skin fibroblasts.

The low density lipoprotein receptor (LDLR) was immunoprecipitated from [35S]methionine-labeled skin fibroblasts derivatized at 4 or 18 degrees C with an impermeant biotinylating reagent. Separation of derivatized and underivatized receptor from immunoprecipitates by selective binding to streptavidin-agarose allowed assessment of receptor protein cellular compartmentation and rates of intercompartmental transfer. At both 4 and 18 degrees C the amount of LDLR that is derivatized in cells labeled to near steady state saturates after 1-2 h of reaction at, respectively, 47 and 70% of total immunoprecipitable receptor protein. On the basis of temperature titration experiments, protein exposed only to the cell surface reacts at 4 degrees C; raising the temperature of biotinylation to 18 degrees C provides access to an additional pool of receptor protein. Remaining LDLR is derivatized at 37 degrees C. LDLR unreactive at 18 degrees C largely resides in membrane compartment(s) devoid of plasma membrane on the basis of its fractionation on Percoll gradients. While total cellular LDLR and 4 degrees C-derivatized LDLR labeled to steady state turn over in a first order manner (t1/2 = 12-13 h), the specific activity of pulse-labeled, 4 degrees C-accessible protein peaks after 1-2 h of chase and reaches a reduced level by 3 h of chase. These latter results show that the newly synthesized LDLR is transiently enriched at the cell surface prior to achieving equilibrium distribution between the cell surface and intracellular pools.

Biotin↗
PubMedDate not supplied

[Compartmentalization of Ca2+ in subcellular structures of the rat liver in chemical injury of hepatocyte membranes].

Compartmentalization of Ca2+ was studied in homogenates and subcellular fractions (nuclei, mitochondria, microsomes and cytosol fraction) of rat liver tissue under conditions of chemical lesion of hepatocyte membranes caused by CCl4. The studies were carried out within 2, 24, 48, 96 and 168 hrs after the CCl4 administration. The chemical lesion of hepatocyte membranes led to considerable increase in Ca2+ concentration in the subcellular fractions studied, especially in cytosol fraction, where it reached about 424% as compared with the control level. The drastic increase of calcium concentration in cytosol appears to be due to disturbance of both Ca2+ elimination through plasmatic membrane and its mitochondrial sequestration.

Animals↗
PubMed1987

Compartmentation of alpha 2-adrenergic receptors in human erythroleukemia (HEL) cells.

We have identified alpha 2-adrenergic receptors on human erythroleukemia (HEL) cells, a suspension-grown cell line related to human platelets. properties of receptors were assessed in intact cells by binding of the antagonist [3H]yohimbine and by inhibition of cAMP accumulation. [3H]Yohimbine labeled 5900 +/- 2100 receptors/cell with a Kd of 3.6 +/- 0.9 nM (n = 7). alpha 2-Adrenergic receptors were potently coupled to inhibition of adenylate cyclase, with EC50 values for epinephrine, UK-14,304, and p-aminoclonidine in the low nM range. Treatment of cells with pertussis toxin abolished this response. In radioligand binding studies with membrane preparations [3H]yohimbine and [3H]UK-14,304 bound to the same number of sites (71 versus 69 fmol/mg of protein), and epinephrine competed for [3H]yohimbine binding in a biphasic manner. After addition of GTP, no high affinity [3H]UK-14,304 binding was detected, and epinephrine competed for [3H]yohimbine binding with lower affinity at both 4 degrees and 37 degrees. In studies with intact cells, we detected no specific binding of [3H]UK-14,304 at either 37 degrees or 4 degrees. At 37 degrees, epinephrine competed for all [3H]yohimbine binding sites with a low apparent affinity (Ki = 21 microM), whereas at 4 degrees epinephrine (up to 1 mM) was able to compete for only 59 +/- 13% of [3H]yohimbine-binding sites. The potency of epinephrine in competing for [3H]yohimbine sites in intact cells at 4 degrees was greater than at 37 degrees (Ki = 1 microM) and was similar to that observed with membranes in the presence of GTP. We hypothesize that sites not detectable by epinephrine at 4 degrees are sequestered within the cell. Treatment of HEL cells with pertussis toxin reduced the proportion of receptors on the surface from 51 +/- 12% to 23 +/- 7% (n = 3, p less than 0.05) of the total sites. Treatment of HEL cells with epinephrine (100 microM, 1 hr) reduced the cell surface component to 25 +/- 8% (n = 3) of the total sites. This treatment was not accompanied by significant desensitization of the ability of epinephrine to inhibit cAMP accumulation. We conclude that alpha 2-adrenergic receptors exist in more than one compartment in HEL cells and that interaction of receptors with a guanine nucleotide-binding protein or with agonist may regulate this compartmentation. These cells provide a new model system for the study of expression and metabolism of alpha 2-adrenergic receptors.

Adenylyl Cyclases↗
PubMed1987

[Compartmentalization dynamics of the epidermal growth factor in A431 cells].

Dynamics of compartmentalization of epidermal growth factor (EGF) in human carcinoma A431 cells during the first hour after initiation of endocytosis was examined by methods of the organelle fractionation on a 20% Percoll gradient and of the microfluorimetric visualization of endocytosis of rhodamine-labeled EGF (EGF-R). EGF was revealed in small vesicles localized in the peripheral region of cytoplasm in a few minutes after endocytosis initiation. During centrifugation in Percoll these vesicles (endosomes), with an average density of 1.038 g/ml, were seen co-sedimented with Golgi membranes. By one hour after initiation of endocytosis, EGF-R was accumulated in perinuclear zone, in a trans-Golgi region, as numerous big luminous centres that were apparently MB-endosomes and had the same density in Percoll as did small peripheral endosomes. Such centres appeared in several cells already within 5-10 minutes. In A431 cells EGF did not reach lysosomes within 60 minutes, because no accumulation of 125I-EGF was shown in lysosome corresponding regions of Percoll gradient (average density 1.070 g/ml).

Animals↗
PubMed1987

Cause and consequences of dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space in respect to exchange of energy rich phosphates between cytosol and mitochondria.

By means of reconstituted systems consisting in rat heart mitochondria and pyruvate kinase it was shown, that the mitochondrial ATP-production is remarkable increased if ADP is produced by enzymes localized in the mitochondrial intermembrane space as creatine kinase or adenylate kinase. This result and different amount of inhibition by atractyloside or carboxyatractyloside of hexokinase- and creatine kinase stimulated respiration further support the concept of dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space.

Adenine Nucleotides↗
PubMed1987

Effects of starvation and a high-energy diet on rat blood compartmentation of injected radioactive alanine and glucose.

The effects of starvation upon blood compartmentation and differential handling of glucose and alanine have been studied in control and cafeteria-fed rats. The injection of radioactive glucose resulted in higher specific radioactivities in intracellular glucose but lower intracellular amino acid specific radioactivities when compared with the plasma values. The cells/plasma specific radioactivity ratios increased dramatically in cafeteria rats with starvation. The injection of radioactive alanine resulted in higher cell than plasma glucose specific activities, and lower cell amino acid specific activities. All these parameters increased after a 24-hours starvation period. It is concluded that glucose synthetised by the liver is released mainly into the blood intracellular pool, being later liberated into the plasma or directly into the tissues.

Alanine↗
PubMed1985

Ion compartmentalization in frog oocytes as demonstrated by X-ray microanalysis.

The distributions of K, Na, Mg and Ca within frog ovarian and oviductal oocytes were studied by electron probe wavelength dispersive X-ray microanalysis. An important heterogeneity could be found both in nuclear and jelly coated oocytes. The highest K, Mg and, to a lesser extent, Na concentrations were found in the pigmented area of the peripheral cytoplasm. There is a certain correlation between the distribution of K and Mg. The concentration of K (but not of Na) in the nucleus was higher than that in the non-pigmented cytoplasm. The distribution of Ca was rather uniform. The high amounts of K, Na and S determined in the oocyte jelly coat seem to have become accumulated by ion-exchange mechanism. Oocyte pigment granules are believed to be the site of ion compartmentalization and to play a role in regulation of intracellular ionic composition.

Animals↗
PubMed1985

Compartmentation of enzymes: ATP citrate lyase in hepatocytes from fed or fasted rats.

Compared with traditional techniques of tissue homogenization, digitonin fractionation of isolated hepatocytes provides a much more rapid and, in some instances, more accurate determination of enzyme compartmentation. Results with ATP citrate lyase (EC 4.1.3.8) illustrate the information that uniquely can be obtained. Although the enzyme was previously thought to be entirely cytosolic, digitonin fractionation has shown that a portion of total cellular ATP citrate lyase is bound to mitochondria or some other structure, and the amount bound varies with the animal's nutritional state. In hepatocytes from rats that were starved for 2 days, fed NIH stock diet ab libitum, or starved for 2 days and then refed a fat-free diet for 2 days, the noncytosolic activity was, respectively, 52, 21, or 24% of total cellular lyase. However, because starvation/refeeding greatly induces lipogenic enzymes, the amount of bound lyase activity in this dietary state was 10-12 times greater than that in rats that were starved or fed ad libitum. The association of citrate lyase with a subcellular organelle is also influenced by CoA. Addition of 20 microM CoA to the digitonin fractionation medium caused all of the lyase to be released from cells like a cytosolic enzyme. Conversely, when cellular free CoA was decreased by incubating hepatocytes with the hypolipidemic agent 5-(tetradecyloxy)-2-furoic acid, the amount of bound lyase was increased. These results suggest the possibility that the noncytosolic ATP citrate lyase may have a special role in lipogenesis.

ATP Citrate (pro-S)-Lyase↗
PubMedDate not supplied

Plasma membrane compartmentation, related to the mechanism of myxo- and paramyxovirus invasion, to receptor mobility and membrane balance.

According to the model of plasma membrane compartmentation into functional endocytosis-exocytosis units it is suggested that the respective compartments may also take part in the receptor mediated internalization of viruses. The primary effect of myxo- and paramyxovirus invasion would consist in the intercalation of new microzones in the recycling of the host cell membrane. Viral receptor lateral mobility is a result of the zonal migration of the entire plasmalemma, due to the processes of exocytosis and endocytosis, continuously adding and removing, respectively, different membrane microzones.

Cell Compartmentation↗
PubMed1983

[Compartmentalization of metabolism of spatially-delimiting amino acid pools of yeast cells].

The spatially different amino acid pools (i.e. cytoplasmic, vacuolar and mitochondrial) of yeast cells are metabolically compartmentalized. The accumulation of amino acids in these pools occurs at different rates; the highest rates are observed for glutamate and alanine. The former is predominantly accumulated in the cytoplasm, the latter--in the vacuoles. The renewal rates of the amino acid pools are also different. Each of them contains at least two subpools, readily convertible and relatively stable ones. The readily convertible subpools of the cytoplasmic and mitochondrial pools predominantly contain glutamate, aspartate, valine and alanine; that of the vacuolar pool--alanine. The bulk of the readily convertible alanine subpool (67%) is localized in the vacuoles, that of glutamate and aspartate (85 and 68%, respectively)--in the cytoplasm.

Alanine↗
PubMed1984

Cellular compartmentation of Ehrlich ascites tumor cells.

The digitonin method for the study of cellular compartmentation in mitochondrial and cytosolic fractions was applied to Ehrlich ascites tumor cells. The volume of mitochondrial and cytosolic water spaces are calculated to be 1.62 microliter/30 x 10(6) cells respectively, by the technique of 3H2O permeable and (14C)-sucrose impermeable spaces. The validity of the methods was tested by the distribution of cytosolic (lactate dehydrogenase) and mitochondrial (citrate synthase and glutamate dehydrogenase) marker enzymes. As occurs in normal hepatic cells, an asymmetric distribution of ATP and ADP was observed. The ATP/ADP ratio in the cytosolic fraction was 7 times higher than in the mitochondrial fraction.

Adenosine Diphosphate↗
PubMed1982

Unusual compartmentation of precursors for nuclear and mitochondrial DNA in mouse L cells.

Current evidence suggests that distinct mechanisms exist to regulate precursor synthesis for nuclear and mitochondrial DNA replication. We tested this is mouse L cells by asking whether nuclear and mitochondrial DNAs become labeled to equivalent specific activities when provided with an exogenous nucleic acid precursor. Cells were grown in [32P]orthophosphate-containing medium long enough to bring all pools to equivalent specific activities. [6-3H]Uridine was added to the medium as a general pyrimidine precursor. At intervals, cells were harvested and nuclear and mitochondrial DNA was isolated. After enzymatic hydrolysis of each DNA fraction to deoxyribonucleoside 5'-monophosphates, these were separated by high performance liquid chromatography and the 3H/32P ratio in each pyrimidine was determined as an index of the specific activity of DNA pyrimidine residues. The dTMP residues in nuclear and mitochondrial DNA reached roughly equal specific activities and at comparable rates. However, dCMP residues in mitochondrial DNA reached maximal specific activities more rapidly than those in nuclear DNA, and the maximal values attained were nearly twice those seen either with the nuclear DNA dCMP residues or in the dTMP residues from either DNA. This indicates that the pathways leading to dCTP synthesis are organized so that mitochondria can use exogenous precursors more effectively than can the nucleus. The nature of this compartmentation is not clear, but it evidently involves one or more steps beyond the divergence point between pathways to dCTP and dTTP.

Animals↗
PubMed1994

Compartmentalization of certain components of the protein synthesis apparatus in mammalian cells.

A comparative study on the localization of free cytosolic tryptophanyl-tRNA synthetase (TrpRS) and several components of the multi-aminoacyl-tRNA synthetase (ARS) complex (glutamyl-prolyl-tRNA synthetase (GluProRS), arginyl-tRNA synthetase (ArgRS)), and two non-synthetase polypeptides p38 and p43 has been carried out on ultrathin sections of cultured rabbit kidney cells by the immunogold technique using monoclonal antibodies raised against appropriate polypeptides. It has been shown that GluProRS, ArgRS and p38 polypeptide are distributed in the cells similarly to TrpRS and are located mainly in the vicinity of ribosomes. A smaller but significant portion of these proteins has been observed in the nuclei in the diffuse chromatin regions and in the vicinity of interchromatin granules. On the contrary, the main part of p43 protein was found in the cell nuclei; this indicates that this protein may exist in the cell separately from the cytoplasmic multi-ARS complex. Our results argue in favor of compartmentalization of both free ARS (such as TrpRS) and the multi-ARS complex in the vicinity of ribosomes. At the same time, the detection of some ARS in the diffuse chromatin regions in the nucleus implies that these enzymes may exhibit some non-canonical functions in addition to their role in protein synthesis.

Animals↗
PubMed1994

GPI phospholipase C from Trypanosoma brucei causes a GPI-negative phenotype in Leishmania major: I. Implications for GPI-negative mammalian cells; II. Compartmentalization of two GPI biosynthetic pathways.

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide) are glycosylphosphatidylinositol (GPI)-anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associated gp63 could not be detected in immunoblots. gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative protein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmentalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN(1 alpha 6)-myo-inositol-1-phospholipid, is cytosolic. To our knowledge, this represents the first use of a catabolic enzyme, in vivo, to elucidate the topography of biosynthetic pathways. Intriguingly, the phenotype of GPIPLC-expressing L. major, secretion of proteins with GPI addition signals, and depletion of protein-GPI anchor precursors, is similar to that of some protein-GPI mutants in higher eukaryotes. These findings have implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma.

Animals↗
PubMed1994

Compartmentalization, processing and redistribution of the plasma membrane protein CE9 on rodent spermatozoa. Relationship of the annulus to domain boundaries in the plasma membrane of the tail.

Western blotting, immunofluorescence and immunogold electron microscopy were used to examine the compartmentalization, processing and redistribution of the integral plasma membrane protein CE9 on the spermatozoa of rats, mice and hamsters. In each species examined, spermatozoal CE9 was found to undergo endoproteolytic processing followed by a net redistribution from the posterior-tail domain into the anterior-tail domain of the plasma membrane during epididymal maturation. Compared to spermatozoa of the rat and mouse, those of the hamster were found to express a greater proportion of their CE9 within the anterior-tail plasma membrane domain at all stages of maturation. As a consequence, CE9 was judged to be a suitable marker for two different spermatozoal plasma membrane domains: the posterior-tail plasma membrane domain (spermatozoa from the testis and caput epididymidis of the rat and mouse) and the anterior-tail domain (spermatozoa from the cauda epididymidis of the hamster). Immunogold electron microscopy was used to pinpoint the positions of the boundaries of these CE9-containing plasma membrane domains at a high level of resolution. In each case, the position of the CE9 domain boundary was found to be strongly correlated with that of the subplasmalemmal electron-dense ring known as the annulus. The precise spatial relationship between the CE9 domain boundary and the annulus was, however, found to differ significantly among species and/or as a function of maturation.

Animals↗
PubMed1997

Mechanism and ion-dependence of in vitro autoactivation of yeast proteinase A: possible implications for compartmentalized activation in vivo.

Yeast proteinase A is synthesized as a zymogen which transits through the endoplasmic reticulum, the Golgi complex and the endosome to the vacuole. On arrival in the vacuole, activation takes place. It has previously been found that proteinase A can activate autocatalytically; however, the propeptide of proteinase A shows essentially no similarity to other known aspartic proteinase propeptides. To understand why proteinase A activation occurs rapidly in the vacuole but not at all in earlier compartments, we have purified the zymogen and investigated the conditions that trigger autoactivation and the mechanism of autoactivation. Autoactivation was triggered by acidic pH and its rate increased with increasing ionic strength. Kinetic evidence indicates that autoactivation mainly occurs via a bimolecular product-catalysed mechanism in which an active proteinase A molecule activates a zymogen molecule. Both the pH- and ionic-strength-dependence and the predominance of a product-catalysed mechanism are well adapted to the situation in vivo, since slow activation in the absence of active proteinase A helps to prevent activation in prevacuolar compartments, whereas, on delivery to the vacuole, lower pH, higher ionic strength and the presence of already active proteinases ensure rapid activation. Product-catalysed autoactivation may be a general mechanism by which cells ensure autoactivation of intracellular enzymes to be both rapid and compartmentalized.

Aspartic Acid Endopeptidases↗
PubMed1998

Different intracellular compartmentations of cardiac troponins and myosin heavy chains: a causal connection to their different early release after myocardial damage.

We investigated the net myocardial release of creatine kinase isoenzyme MB (CKMB), myoglobin, cardiac troponin T (cTnT), cardiac troponin I (cTnI), and cardiac beta-type myosin heavy chain (beta-MHC) into the coronary circulation after cardioplegic cardiac arrest in humans. Cardiac markers were measured in paired arterial, central venous, and coronary sinus blood in 19 patients undergoing elective coronary artery bypass grafting (CABG) before aortic cross-clamping and 1, 5, 10, and 20 min after aortic declamping. cTnT and cTnI were released into the coronary sinus in parallel to each other and almost simultaneously to myoglobin and CKMB within 20 min of reperfusion. In contrast, no beta-MHC was released in the same patients during the study period. The average soluble cTnT and cTnI pools in right atrial appendages of 11 patients with right atrial and right ventricular pressures within reference values were comparable and were approximately 8% of total myocardial troponin content. The soluble beta-MHC pool was <0.1% in all patients. Our results demonstrate the impact of the different intracellular compartmention of regulatory and contractile proteins on their early release from damaged myocardium.

Adult↗