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PubMed1979

Subcellular distributions of parathyroid hormone, hormonal precursors, and parathyroid secretory protein.

Distributions of parathyroid hormone (PTH), proparathyroid hormone (ProPTH), preproparathyroid hormone (PreProPTH), and parathyroid secretory protein (PSP) were analyzed in subcellular fractions prepared from homogenates of bovine parathyroid glands. Slices of bovine parathyroid glands were incubated with radiolabeled amino acids for 3--30 min to selectively label newly synthesized proteins. Subcellular fractions were prepared from homogenates of the gland slices by differential centrifugation. Newly synthesized labeled hormonal polypeptides in the fractions were analyzed by electrophoresis on polyacrylamide gels, and total amounts of PTH and ProPTH (previously formed and newly synthesized) were determined by immunoassay. Ninety percent of total immunoreactive, 70--80% of newly synthesized PTH, ProPTH, and PreProPTH, and 50% of PSP were found in sedimentable particulate fractions. The low speed (800 X g) pellet, which consisted predominantly of cell debris and nuclei with adherent remnants of cytoplasm, contained 30--50% of the ProPTH and PTH. The intermediate speed (10,000 X g) pellet, which contained granules, was relatively enriched in PTH. Most particulate-associated hormone could be solubilized by treatment with deoxycholate (DOC) 98% and 97% of radiolabeled and 93% and 83% of immunoreactive ProPTH and PTH, respectively, in particulates sedimenting at 10,000 and 105,000 X g were rendered DOC-soluble. Approximately 50% of the PTH and ProPTH in the particulates resisted digestion by combined trypsin and chymotrypsin, whereas PreProPTH was completely susceptible to proteolysis. Up to 50% of the radiolabeled PTH and ProPTH added exogenously to parathyroid gland slices before homogenization became associated with the particulate fractions, and 70--80% or radiolabeled PreProPTH added to the subcellular fractions readily associated with the sedimentable material. The results indicate that in homogenates of parathyroid glands, PTH, ProPTH, PreProPTH, and PSP are associated with particulate structures. Furthermore, up to 50% of the association of ProPTH, PTH, and PSP with particulate fractions seems to be nonsepcific and occurs during the disruption of the tissues. The remaining 50% or more of hormonal protein is presumably sequestered within membrane-limited structures, such as microsomal vesicles. The complete susceptibility in particulate fractions of newly synthesized PreProPTH, but not of ProPTH, to limited proteolysis indicates that the two precursors are located in different subcellular compartments and suggests that PreProPTH is converted to ProPTH before its entry into the intracisternal space of the endoplasmic reticulum. Alternatively, the PreProPTH identified in parathyroid gland slices may represent polypeptide chains synthesized in the cell sol on polyribosomes that are not attached to endoplasmic reticulum but are adsorbed nonspecifically to the particulate fraction of the cell during the process of tissue homogenization.

Animals↗

PubMed1998

Effects of insulin on subcellular localization of hexokinase II in human skeletal muscle in vivo.

The phosphorylation of glucose to glucose-6-phosphate, catalyzed by hexokinase, is the first committed step in glucose uptake into skeletal muscle. Two isoforms of hexokinase, HKI and HKII, are expressed in human skeletal muscle, but only HKII expression is regulated by insulin. HKII messenger RNA, protein, and activity are increased after 4 h of insulin infusion; however, glucose uptake is stimulated much more rapidly, occurring within minutes. Studies in rat muscle suggest that changes in the subcellular distribution of HKII may be an important regulatory factor for glucose uptake. The present studies were undertaken to determine if insulin causes an acute redistribution of HKII activity in human skeletal muscle in vivo. Muscle biopsies (vastus lateralis muscle) were performed before and at the end of 30 min insulin infusion, performed using the euglycemic clamp technique. Muscle biopsies were subfractionated into soluble and particulate fractions to determine if insulin acutely changes the subcellular distribution of HKII. Insulin decreased HKII activity in the soluble fraction from 2.20 +/- 0.31 to 1.40 +/- 0.18 pmoles/(min[chempt]micrograms) and increased HKII activity in the particulate fraction from 3.02 +/- 0.46 to 3.45 +/- 0.46 pmoles/(min[chempt]micrograms) (P < 0.01 for both). These changes in HKII activity were correlated with changes in HKII protein, as determined by immunoblot analysis (r = 0.53, P = 0.05). Insulin had no effect on the subcellular distribution of HKII activity, which was primarily restricted to the soluble fraction. These studies are consistent with the conclusion that, in vivo in human skeletal muscle, insulin changes the subcellular distribution of HKII within 30 min.

Adult↗

PubMed2003

Role of the LXXLL-motif and activation function 2 domain in subcellular localization of Dax-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1).

Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (Dax-1, NR0B1) is an orphan nuclear receptor that represses transcription by Ad4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1, NR5A1). Observations on human diseases and the phenotypes of mice, in which the corresponding genes have been disrupted, have elucidated essential roles of these two nuclear receptors in differentiation of steroidogenic tissues. However, little is known about how the functions of these factors are regulated. Here we have examined their subcellular localization and have clarified the molecular mechanisms regulating subcellular localization of Dax-1. Prompted by the finding that nuclear localization of Dax-1 correlates with the presence of Ad4BP/SF-1 in the early stages of pituitary development, we have tested the possibility that interaction between the two factors is essential for the nuclear localization of Dax-1. In vitro studies with cultured cells demonstrated that an interaction involving the LXXLL motifs in the N-terminal repeat region of Dax-1 plays a key role in its subcellular localization. In addition, we found that a mutant form of DAX-1 (L466R), from a patient with adrenal hypoplasia congenita, was defective in nuclear localization in spite of having an intact N terminus. Taken together, the results reveal that the subcellular localization of Dax-1 is influenced by the presence of Ad4BP/SF-1, and that two regions of Dax-1 have important roles for this process.

Amino Acid Motifs↗

PubMed1986

Relationships between free cadmium ion activity in seawater, cadmium accumulation and subcellular distribution, and growth in polychaetes.

We have examined the relationships between Cd ion activity [Cd2+], in seawater, Cd accumulation and subcellular distribution and growth in the polychaete Neanthes arenaceodentata. Organisms were exposed for 3 weeks to a range of [Cd2+] in a Cd-chelate buffer system. Cadmium accumulation and growth were monitored weekly for each exposure group and subcellular Cd distributions were determined at the end of the 3-week period. We found Cd associated with all of the subcellular fractions except the very low molecular weight ligands. Total Cd accumulation was greatest at day 7 and decreased over time in all but the highest [Cd2+] where it remained constant. For each point in time, however, there was a linear relationship between total Cd and [Cd2+] in seawater. Linear relationships were also observed between [Cd2+] and Cd loading in each subcellular ligand pool. Specific growth rates varied with both [Cd2+] and time in a nonlinear manner.

Animals↗

PubMed2005

Cell-specific subcellular localization of soluble epoxide hydrolase in human tissues.

Soluble epoxide hydrolase (sEH) is a phase-I xenobiotic metabolizing enzyme having both an N-terminal phosphatase activity and a C-terminal epoxide hydrolase activity. Endogenous hydrolase substrates include arachidonic acid epoxides, which have been involved in regulating blood pressure and inflammation. The subcellular localization of sEH has been controversial. Earlier studies using mouse and rat liver suggested that sEH may be cytosolic and/or peroxisomal. In this study we applied immunofluorescence and confocal microscopy using markers for different subcellular compartments to evaluate sEH colocalization in an array of human tissues. Results showed that sEH is both cytosolic and peroxisomal in human hepatocytes and renal proximal tubules and exclusively cytosolic in other sEH-containing tissues such as pancreatic islet cells, intestinal epithelium, anterior pituitary cells, adrenal gland, endometrium, lymphoid follicles, prostate ductal epithelium, alveolar wall, and blood vessels. sEH was not exclusively peroxisomal in any of the tissues evaluated. Our data suggest that human sEH subcellular localization is tissue dependent, and that sEH may have tissue- or cell-type-specific functionality. To our knowledge, this is the first report showing the subcellular localization of sEH in a wide array of human tissues.

Amino Acid Sequence↗

PubMedDate not supplied

Subcellular fractionation of bone marrow-derived macrophages: localization of phospholipase A1 and A2 and acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase.

Analysis of the subcellular distribution of lipid-metabolizing enzymes was carried out in bone marrow-derived macrophages with special respect to a comparison of the subcellular localisation of phospholipase A1 and A2 and to acyl-CoA:1-1-acylglycero-3-phosphorylcholine-0-acetyltransferase. After cell disruption differential centrifugation was followed by additional sucrose gradient purification of three main fractions. Satisfactory enrichment factors were obtained by this method for the following marker enzymes. The plasma-membrane enzyme alkaline phosphodiesterase I was enriched up to 25-fold and the acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase was enriched up to 30-fold. The marker enzyme for the endoplasmic reticulum, NADPH-cytochrome c reductase showed a similar enrichment and distribution as the acyltransferase. Therefore it was concluded that the acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase of bone marrow-derived macrophages is mainly located in the endoplasmic reticulum. Phospholipase A1 and A2 occurred in a high proportion together with the lysosomal marker enzyme N-acetyl-beta-glucosaminidase in the soluble supernatant and in the gradient fractions. In the endoplasmic reticulum phospholipase A2 occurred only in trace activities whereas phospholipase A1 was maximally enriched in this subcellular fraction. No subcellular fraction could be obtained where phospholipase A2 was enriched exclusively. However, it can be concluded that the two enzymes which are responsible for the balance of fatty acid liberation and re-acylation are located in two different cellular compartments. Furthermore it can be claimed that in the cell there has to exist an exchange of substrates and products between these compartments to achieve a complete metabolic cycle of the de- and re-acylation reaction of phospholipids in bone marrow-derived macrophages.

Acyltransferases↗

PubMedDate not supplied

Aggregatory behaviour of platelets incubated with subcellular fractions of normal and chagasic human syncytiotrophoblast.

The surface of human syncytiotrophoblast does not induce maternal blood platelet aggregation even though it is not an endothelium. It can be surmised that as occurs in endothelial injury the subcellular components of the syncytiotrophoblast may have pro- or antiaggregatory activity. During congenital Chagas' disease which is associated to trophoblast lesions, platelets may play a role in the development of T. cruzi-induced placentitis. In the present work the aggregatory behaviour of normal human blood platelets was recorded after their challenging with subcellular fractions of syncytiotrophoblast isolated from normal and chagasic women. Nuclear, Mitochondrial, Microsomal and Supernatant fractions isolated from normal and chagasic syncytiotrophoblast failed to induce per se any aggregatory reaction on platelets. When samples of platelet-rich plasma (PRP) were preincubated with normal and chagasic nuclear fractions and then stimulated with collagen at threshold level (CT-PRP) an inhibition of the aggregatory response was observed. Treatment of CT-PRP with normal and chagasic mitochondrial fractions induced inhibition of platelet aggregation whereas only chagasic fraction reduced latency time. Microsomal fraction from normal placentas showed no significant effects on platelet aggregation. It is concluded that subcellular fractions of normal human syncytiotrophoblast do not exhibit any effect on platelet aggregation, whereas those subcellular fractions enriched in intracellular membrane components isolated from chagasic placentas inhibit platelet aggregation.

Adult↗

PubMed2006

Biological significance of metals partitioned to subcellular fractions within earthworms (Aporrectodea caliginosa).

Metal ions in excess of metabolic requirements are potentially toxic and must be removed from the vicinity of important biological molecules to protect organisms from adverse effects. Correspondingly, metals are sequestrated in various forms, defining the accumulation pattern and the magnitude of steady-state levels reached. To investigate the subcellular fractions over which Ca, Mg, Fe, Cu, Zn, Cd, Pb, Ni, and As are distributed, earthworms (Aporrectodea caliginosa) collected from the field were analyzed by isolating metal-rich granules and tissue fragments from intracellular microsomal and cytosolic fractions (i.e., heat-stable proteins and heat-denatured proteins). The fractions showed metal-specific binding capacity. Cadmium was mainly retrieved from the protein fractions. Copper was equally distributed over the protein fraction and the fraction comprising tissue fragments, cell membranes, and intact cells. Zinc, Ca, Mg, and As were mainly found in this fraction as well. Lead, Fe, and Ni were mainly isolated from the granular fraction. To study accumulation kinetics in the different fractions, three experiments were conducted in which earthworms were exposed to metal-spiked soil and a soil contaminated by anthropogenic inputs and, indigenous earthworms were exposed to field soils. Although kinetics showed variation, linear uptake and steady-state types of accumulation patterns could be understood according to subcellular compartmentalization. For risk assessment purposes, subcellular distribution of metals might allow for a more precise estimate of effects than total body burden. Identification of subcellular partitioning appears useful in determining the biological significance of steady-state levels reached in animals.

Animals↗

PubMed1979

Effects of soluble fractions from untreated and SiO2-treated subcellular particles of macrophages on nucleic acid metabolism in isolated nuclei of experimental granulation tissue.

Nuclei isolated from proliferating granulation tissue were incubated with 20 000 g supernatants from untreated and SiO2-treated subcellular particles of rat peritoneal macrophages in the presence of radioactive nucleic acid precursors. The supernatant from SiO2-treated subcellular particles increased the incorporation of [3H]CTP into nuclear RNA maximally by 26% at 5 min, and that of [methyl-3H]dTTP into DNA by 16% at 20 min. The release of radioactivity from labeled DNA was suppressed simultaneously. An RNase preparation from rat peritoneal macrophages enhanced the release of radioactivity from labeled DNA similarly as the soluble fraction from untreated subcellular particles of macrophages. The results suggest that the effects of the soluble fractions upon DNA metabolism of granuloma cells are at least partly independent of the effects on RNA metabolism and that the soluble fraction from SiO2-treated subcellular particles of macrophages stabilizes DNA through inhibition of nuclease activity.

Animals↗

PubMed1976

Inactivation of Oxytocin and its analogues by subcellular fractions of hen tissues.

The enzymic inactivation of oxytocin by liver, kidney, uterus and pancreas homogenate subcellular fractions of hens was studied. Oxytocin was most rapidly degraded by the soluble fraction of tissues examined. All the subcellular fractions of liver and kidney inactivated oxytocin, but only the microsomal and soluble fractions of uterus and pancreas showed the oxytocin-inactivating activity. The location of enzymes inactivating oxytocin in subcellular fractions of hen tissues was investigated with the aid of synthetic analogues of oxytocin (deamino-oxytocin and deamino-carba1-oxytocin). The carboxamidopeptidase activity, hydrolyzing the amide bonds in the linear portion of oxytocin was located in the soluble fraction of hen liver, kidney and uterus. No carboxamidopeptidase activity in the pancreatic soluble fraction was found. These results showed that aminopeptidase activity is bound to heavy subcellular particles in the hen tissue. An action of unknown endopeptidases was observed in the microsomal fraction of uterus and pancreas.

Animals↗

PubMed2001

[Subcellular distribution of daunorubicin in the P-glycoprotein-mediated multidrug-resistant cell line K562/ADR].

OBJECTIVE: To examine subcellular distribution of daunorubicin (DNR) in P-glycoprotein-mediated multidrug-resistant cell line K562/ADR and its relation to multidrug resistance. METHODS: The subcellular distribution of DNR in K562/ADR was studied by confocal scanning laser microscopy (CSLM), fluorometry, RT-PCR. Rhodamine 123, NBD-ceramide and neutral red as fluorescent probes to stain the mitochondria, Golgi apparatus and lysosomes respectively were used to identify the subcellular compartments wherein DNR was sequestered. Effect of verapamil, chloroquine and brefeldin A on DNR distribution and accumulation was examined. RESULTS: Compared with the drug-sensitive cell K562/S in which DNR fluorescence diffusely appeared in the nucleus and cytoplasm, DNR in K562/ADR cells was distributed to the perinuclear region and peripheral cytoplasm, It was scenty in the nucleus and other cytoplasmic regions, as suggested by the distribution of Rhodamine123. Only verapamil, but not chloroquine and brefeldin A, could markedly restore diffuse cytoplasmic and nuclear fluorescence distribution in the resistant cell line. CONCLUSION: Altered subcellular distribution of DNR in drug resistant cell line may participate in the generation of multidrug resistance in which P-glycoprotein plays an important role.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1976

3H-delta9-tetrahydrocannabinol tissue and subcellular distribution in the central nervous system and tissue distribution in peripheral organs of tolerant and nontolerant dogs.

Tolerant and nontolerant dogs received one i.v. administration of 0.5 mg/kg of 3H-delta9-tetrahydrocannabinol 30 minutes before they were sacrificed. Plasma, peripheral and brain tissues, as well as subcellular fractions of brain tissues from both treatment groups, were analyzed for radioactivity. Throughout the time period before sacrifice, the plasma concentrations of radioactivity in the tolerant and nontolerant dogs were not significantly different. The percentage of radioactivity in brain and plasma that was due to either unchanged delta9-tetrahydrocannabinol or a major metabolite was the same in each group. Of the radioactivity in brain, 46% was identified as delta9-tetrahydrocannabinol. Regardless of treatment, there was a specific accumulation of radioactivity in adrenals, liver, kidney, heart and pancreas. The only significant differences in radioactivity between tolerant and nontolerant peripheral tissues were found in liver, kidney cortex, heart and lymph nodes. Although all brain areas from tolerant dogs contained less radioactivity than the comparable brain areas from nontolerant animals, only pituitary and putamen were significantly less. There was a specific accumulation of radioactivity in some brain areas that could be associated with behavioral effects. The concentration in cerebellar and cerebral gray was significantly greater than that in white, and there was a marked reduction in the concentration in gray after tolerance developed. The mean percentage of radioactivity in each subcellular fraction was as follows: 23% crude nuclei, 44% mitochondria, 8% cholinergic nerve endings, 7% noncholinergic nerve endings, 2% free mitochondria and 6% synaptic vesicles. The quantity of radioactivity in homogenates of brains from tolerant dogs was 17% less than brains of nontolerant animals, which was merely a reflection of the respective plasma concentrations. The distribution of radioactivity was similar in both groups, although most of the subcellular fractions from tolerant dogs contained a lesser amount of radioactivity. The most notable difference was observed in the synaptic vesicle fraction. The synaptic vesicle fraction of tolerant dogs contained 40% less radioactivity than did the same fraction from nontolerant dogs, which implied a possible mechanism of action. A comparison of the remaining subcellular fractions did not appear to explain the development of tolerance.

Animals↗

PubMed1992

Correlation between the conformational phenotype of p53 and its subcellular location.

In order to obtain insight into the parameters determining the subcellular localization of mutant and wild-type forms of p53, we analysed the subcellular distribution of p53 in four Balb/c mouse-derived cell lines ranging in their cellular phenotypes from normal (3T3), via minimal transformant (T3T3), to maximally transformed (3T3tx, Meth A). Epitope mapping showed the p53 proteins in 3T3 and in T3T3 cells to be in a wild-type conformation, as they reacted with PAb246, whereas p53 in 3T3tx and in Meth A cells were PAb246 negative and thus displayed a mutant conformation. Despite its reactivity with PAb246, p53 in T3T3 cells had an extended half-life and accumulated to abnormally high levels. We show that the conformationally wild-type p53 in 3T3 and T3T3 cells predominantly localized to the cell nucleus, with about half of it being tightly associated with nuclear structures. In contrast, approximately 60% of mutant p53 in 3T3tx and Meth A cells localized to the cytoplasm, the rest residing in the cell nucleus; all the nuclear p53 in these cells appeared to be structurally bound. The cytoplasmic location of mutant p53 in 3T3tx and Meth A cells was not seen by immunofluorescence microscopic analysis, and required cell fractionation for its detection. Both cytoplasmic and nuclear p53 of the mutant phenotype bound to hsc proteins with a similar stoichiometry, suggesting that hsc binding is not directly related to the subcellular distribution of these proteins. We suggest that the conformational phenotype of p53 is a major determinant of its subcellular location.

Animals↗

PubMed1985

Evidence for two subcellular pools and different kinetic behaviour of retinyl palmitate in rat liver.

Rats were fed vitamin A deficient diets (-A) or supplemented with vitamin A (+A) (4.4 mg retinol equivalents/kg diet), either without (-RA) or with retinoic acid (+RA) (12 mg/kg diet) supplementation for up to six weeks. Plasma and liver levels as well as the subcellular localization of vitamin A were determined. In rats reared on the vitamin A rich diet the localization of retinyl palmitate (principal reserve form) is shown to be dependent on age. Two pools exist, i.e. one consisting of the nuclear and mitochondrial-lysosomal fractions and the other containing the microsomal and cytosol fractions. A rapid replenishment of mitochondrial-lysosomal fractions occurs in the first weeks after the weaning. During six weeks of deficient diet an identical mobilization was seen from the different subcellular fractions. Supplementation with RA caused an immediate and sustained reduction of serum vitamin A levels but did not disturb the subcellular localization of retinyl palmitate. A relationship between these phenomena and the subcellular distribution of the retinyl palmitate hydrolase (RPH) and the cellular vitamin A binding proteins (CRBP) is likely to exist.

Animals↗

PubMedDate not supplied

The subcellular distribution of 14C-lidamidine and its metabolites.

Examination of the subcellular distribution of 14C-lidamidine and its metabolites in rat liver showed that the majority of radioactivity appeared in the postmicrosomal supernatant fraction, with lysosomes and microsomes having the highest relative specific activity (RSA) of the particulate fractions. When the subcellular distribution pattern was corrected for cross-contamination, based on the distribution of subcellular fraction marker enzymes, there was a significant decrease in the lysosomal RSA with an attendant increase in the microsomal RSA. Thin-layer chromatography of subcellular fraction extracts revealed different distribution patterns for lidamidine and metabolites in each fraction. The whole homogenate and cytosol fraction contained mostly polar metabolites (76-91%), whereas the particulate fractions contained 37-50% of their radioactivity as polar metabolites. The highest percentages of unchanged lidamidine and its more pharmacologically active, demethylated metabolite were associated with the mitochondrial and microsomal fractions.

Animals↗

PubMedDate not supplied

Subcellular alcohol and aldehyde-dehydrogenases in the genital system of the female rat.

Selected rat endocrine tissue, i.e., ovary, uterus and vagina were fractionated into nuclear (NC), mitochondrial (MT), and into their cytoplasmic components for the measurement of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in infantile and in adult female rats. The genital organs studied possessed ALDH in all subcellular fractions isolated with exception of MT-preparation of the vaginal tissue of adult rats. Measurable ADH was determined in ovarian NC fraction. Increased specific activity of ALDH as a function of age was noted in MT and CT preparations of the uterus. Kinetic studies performed on ALDH indicate a similar Vmax for all ovarian fractions of infantile and adult female rats. The NC preparation of the uterus showed higher Vmax than the remainder of the subcellular fractions studied. The apparent Km was also determined for ALDH in various genital tissues fractionated and compared to the corresponding subcellular component of the liver. The Vmax of MT and CT-ALDH of major reproductive tissues of infantile rats studied accounted for at least 30% to 50% and for 10% to 25% of that of hepatic CT-ALDH, respectively. This difference was further reduced in adult rats. The results indicate the presence of NAD-dependent ALDH in various subcellular fractions of the female genital system which may be important in the gonadal metabolic detoxification of ethanol derived acetaldehyde.

Age Factors↗

PubMed1984

[Phospholipids of the subcellular fractions of brain and liver tissues in rats in total oxygen starvation of the body].

The rate of phospholipid turnover in brain subcellular fractions differ from that in the liver tissue. Hypoxia of medium severity (pressure chamber 240 mm Hg) influences neither the content nor the turnover rate of phospholipids in liver subcellular fractions. Meanwhile in brain subcellular fractions, it results in a decreased phospholipid turnover rate. Severe hypoxia (less than 200 mm Hg) leads only to the suppression of the phospholipid turnover rate in the liver, whereas in the brain, it results in the decline of the phospholipid turnover rate as well as in the reduction of phospholipid content. Thus, the well-known concept of the higher brain sensitivity to oxygen deficiency as compared to the liver is exemplified by phospholipid turnover in subcellular fractions.

Animals↗

PubMed1983

Analytical subcellular distribution of calmodulin and calmodulin-binding proteins in normal and virus-transformed fibroblasts.

We report a quantitative subcellular localization study of calmodulin and the subcellular distribution of calmodulin-binding proteins in normal and virus-transformed chicken embryo fibroblasts. We developed homogenization conditions for fibroblasts which give maximal cell disruption with minimal organelle destruction, prepared and characterized subcellular fractions, and measured the levels and distribution of calmodulin in the fractions. We found that the majority of calmodulin is present in the soluble fraction, but that a small, reproducible amount of calmodulin is present in each of the particulate fractions. The amount of calmodulin in all subcellular fractions of transformed fibroblasts is greater than or equal to the amount of calmodulin in the corresponding fractions of normal fibroblasts. In contrast to the mostly soluble distribution of calmodulin, many of the calmodulin-binding proteins in fibroblasts are associated with the particulate fractions. Although there are no apparent qualitative differences between normal and transformed fibroblasts with respect to the number, distribution, or apparent molecular weight of the calmodulin-binding proteins, there may be quantitative changes in the levels of some calmodulin-binding proteins after transformation. These data suggest that many of the calmodulin-binding proteins may be particulate proteins rather than soluble proteins, and that several calmodulin-binding proteins in fibroblasts may bind calmodulin in a calcium-independent manner.

Animals↗