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PubMed2000

Subcellular localization of Bic-D::GFP is linked to an asymmetric oocyte nucleus.

Bicaudal-D (Bic-D) is essential for the establishment of oocyte fate and subsequently for polarity formation within the developing Drosophila oocyte. To find out where in the germ cells Bic-D performs its various functions we made transgenic flies expressing a chimeric Bic-D::GFP fusion protein. Once Bic-D::GFP preferentially accumulates in the oocyte, it shows an initial anterior localization in germarial region 2. In the subsequent egg chamber stages 1-6 Bic-D::GFP preferentially accumulates between the oocyte nucleus and the posterior cortex in a focus that is consistently aligned with a crater-like indentation in the oocyte nucleus. After stage 6 Bic-D::GFP fluorescent signal is predominantly found between the oocyte nucleus and the dorso-anterior cortex. During the different phases several genes have been found to be required for the establishment of the new Bic-D::GFP distribution patterns. Dynein heavy chain (Dhc), spindle (spn) genes and maelstrom (mael) are required for the re-localization of the Bic-D::GFP focus from its anterior to its posterior oocyte position. Genes predicted to encode proteins that interact with RNA (egalitarian and orb) are required for the normal subcellular distribution of Bic-D::GFP in the germarium, and another potential RNA binding protein, spn-E, is required for proper transport of Bic-D::GFP from the nurse cells to the oocyte in later oogenesis stages. The results indicate that Bic-D requires the activity of mRNA binding proteins and a negative-end directed microtubule motor to localize to the appropriate cellular domains. Asymmetric subcellular accumulation of Bic-D and the polarization of the oocyte nucleus may reflect the function of this localization machinery in vectorial mRNA localization and in tethering of the oocyte nucleus. The subcellular polarity defined by the Bic-D focus and the nuclear polarity marks some of the first steps in antero-posterior and subsequently in dorso-ventral polarity formation.

Animals↗

PubMed1987

Subcellular localization of enzymes in Streptomyces aureofaciens and its alteration by benzyl thiocyanate. I. Phosphatases and ATP-glucokinase.

Mycelia of a low- and a high-production strain of Streptomyces aureofaciens were converted into protoplasts and divided into five subcellular fractions in order to localize exopolyphosphatases (EC 3.6.1.11), triphosphatase (EC 3.6.1.25), inorganic diphosphatase (EC 3.6.1.1), apyrase (EC 3.6.1.5) and glucokinase (EC 2.7.1.2). The highest specific activity of enzymes hydrolyzing polyphosphates was found in cytoplasmic vesicles and membranes. Triphosphatase was detected in the periplasmic fraction. Periplasmic vesicles and cytoplasm exhibited a high activity of diphosphatase. Apyrase was found mainly in the fractions of membranes and cytoplasmic vesicles. Glucokinase was a cytoplasmic enzyme. The enzymes were released from membrane structures into cytoplasm or periplasmic space if benzyl thiocyanate (10 microM) was present in the growth medium.

Cations↗

PubMed1995

Differential expression and subcellular localization of protein kinase C isoforms in neuronal and cardiac tissues of Limulus polyphemus.

The presence and subcellular distribution of specific protein kinase C (PKC) isoforms in Limulus neuronal and cardiac tissue extracts were determined using polyclonal antibodies to individual PKC isoforms. Western blot analysis revealed that the brain, abdominal ganglia, cardiac ganglia and heart all contained PKC's alpha, beta, gamma, epsilon and zeta. PKC delta was detected in all neuronal tissues. PKC theta was found only in the cardiac ganglia and heart. PKC's gamma and delta were predominantly localized in membrane fractions while the alpha, beta, epsilon, zeta and theta isoforms were found in both fractions. Antibodies to PKC's epsilon and zeta detected relatively high molecular weight bands in membrane fractions and lower molecular weight bands in the cytosolic fractions. The results demonstrate that Limulus neuronal and cardiac tissues differentially express the conventional, novel and atypical isoforms of protein kinase C.

Animals↗

PubMed1991

Subcellular localization of calcium release in isolated rat myocardial cells.

Independent determinations of Ca2+ by two indicators showed that subcellular Ca2+ activity (intracellular free calcium concentration) was heterogeneous in rat myocardial cells. Arsenazo III (Az III), a membrane-impermeant absorbance indicator for Ca2+, was loaded into cardiac muscle via liposomes and calcium quantitated at two wavelengths through a focal point diaphragm in 5-100 microns 2 regions by a photometer. Fura-2, a high-affinity fluorescent calcium indicator, was loaded into cells as the ester form, and the light intensity was measured by digital-imaging microscopy using a photon-counting camera. Calcium activity at each picture element was quantitated by division of fluorescence excited by two ultraviolet wavelengths, and corrected for concentration differences in fura-2 by a third, Ca(2+)-insensitive wavelength. Both methods revealed hot spots of Ca2+ release and uptake that could be enlarged, added to, or altered. Addition of 1-10 nM norepinephrine caused up to a 500% increase in Ca2+ in localized regions, although whole cell average Ca2+ increased by only 0-80%, suggesting the importance of localized intracellular Ca2+ release for contraction.

Animals↗

PubMed1990

Differential subcellular localization of particular mRNAs in hippocampal neurons in culture.

In situ hybridization was used to assess the subcellular distribution of mRNAs encoding several important neuronal proteins in hippocampal neurons in culture. mRNA encoding GAP-43, a protein that is largely excluded from dendrites, was restricted to nerve cell bodies, as were mRNAs encoding neurofilament-68 and beta-tubulin, which are prominent constituents of dendrites and of axons. In contrast, mRNA encoding MAP-2, a protein that is selectively distributed in dendrites and cell bodies, was present in both dendrites and cell bodies. These results demonstrate that different mRNAs are differentially distributed within individual hippocampal neurons. Taken together with previous findings from other laboratories, our results suggest that only a limited set of mRNAs are available for local translation within dendrites.

Animals↗

PubMed2004

Rat sperm AS-A: subcellular localization in testis and epididymis and surface distribution in epididymal sperm.

In this study, we investigated the subcellular compartmentalization of arylsulfatase-A (AS-A) in the testis and epididymis as well as the surface distribution in rat epididymal sperm. Testicular AS-A was compartmentalized specifically to the area underneath the outer acrosomal membrane of the acrosomal granule and to the dorsal aspect of the sperm acrosome. Epididymal AS-A was synthesized in the endoplasmic reticular (ER) network of principal cells and secreted into epididymal lumen as evident by its reactivity in the apical cytoplasm and vesicles therein underneath stereocilia. In clear cells, AS-A reactivity was found throughout the cytoplasmic machineries involved in endocytosis. Surface distribution of AS-A was initially detectable at the concave ridge as early as in sperm of the initial segment (IS). AS-A was additionally localized to the post-acrosomal region in caput (CP), corpus (CO) and cauda (CD) epididymal sperm. The expression levels of surface AS-A gradually increased during sperm transit from IS to CD epididymidis. These results favored the adsorption of AS-A from epididymal fluid onto the sperm surface, rather than shunting from the acrosome as a consequence of capacitation-associated membrane priming.

Animals↗

PubMed1984

Subcellular localization of vitamin D3 25-hydroxylase in human liver.

Vitamin D3 25-hydroxylase activity was measured in subcellular and submitochondrial fractions of human liver. Quantitation of 25-hydroxyvitamin D3 was based on high performance liquid chromatography. Vitamin D3 25-hydroxylase activity was detected in the mitochondrial fraction only. The mitochondrial 25-hydroxylase activity was linear with time up to 60 min and with mitochondrial protein up to 1 mg/ml. An apparent Km value of about 10(-5) M was found. Substrate satuation level was not reached. In the presence of 2.4 X 10(-4) M vitamin D3, the rate of 25-hydroxyvitamin D3 formation was 0.19 nmol X mg of protein-1 X h-1 After fractionation of the mitochondria, 86% of the 25-hydroxylase activity was recovered in the mitoplast fraction. The outer membrane fraction was devoid of activity. It is concluded that human liver contains only one detectable vitamin D3 25-hydroxylase enzyme localized to the mitochondrial inner membrane.

Cell Fractionation↗

PubMed1999

Subcellular localization of chromogranins, calcium channels, amine carriers, and proteins of the exocytotic machinery in bovine splenic nerve.

Subcellular fractionation of bovine splenic nerves, which consist mainly of sympathetic nerve fibers, has been useful for characterizing cellular organelles en route to the terminal. In the present study we have characterized the subcellular distribution of both secretory and membrane proteins. A newly discovered chromogranin-like protein, NESP55, was found in large dense-core vesicles. The endogenous processing of NESP55 was comparable to that of chromogranins but more limited than that of secretogranin II and chromogranin B. For membrane proteins three major types of distribution were found. The amine carrier VMAT2 was confined to large dense-core vesicles. VAMP or synaptobrevin was present both in large dense-core vesicles and in lighter vesicles, whereas SNAP-25, syntaxin, and two types (N and L) of Ca2+ channels were found in a special population of lighter vesicles but were not present in large dense-core vesicles or at the most in very low concentrations. The plasma membrane norepinephrine transporter was apparently present in a separate type of vesicle, but this requires further study. These results further characterize vesicles en route to the terminal and establish for the first time that peptides involved in exocytosis (syntaxin, SNAP-25, and N- and L-type Ca2+ channels) are apparently transported to the terminal in a special type of vesicle. The exclusive presence of the amine carrier in large dense-core vesicles indicates that the formation of small dense-core vesicles in the terminals requires a reuse of membrane components of large dense-core vesicles.

Amines↗

PubMed1995

Subcellular localization and molecular pharmacology of distinct populations of [3H]-AMPA binding sites in rat hippocampus.

1. The subcellular distributions of [3H]-alpha-amino-3-hydroxy-5- methylisoxazolepropionate ([3H]-AMPA) and [3H]-kainate binding sites in rat hippocampus were investigated by cell fractionation techniques. 2. Two major populations of [3H]-AMPA sites were detected with the majority of binding located intracellularly in the microsomal (P3) fraction. Most of the remaining sites were in the synaptosomal membrane fraction but some were also present in the nuclear fraction. In contrast, essentially all of the [3H]-kainate binding sites were in the synaptosomal membrane fraction. 3. Saturation binding analysis yielded KD and Bmax values for [3H]-AMPA of 147 nM and 2.6 pmol mg-1 protein respectively for the synaptosomal membrane-associated sites and 129 nM and 5.3 pmol mg-1 protein respectively for the microsomal sites. 4. Both main populations of [3H]-AMPA sites displayed the same rank order of inhibition by competitive ligands, the apparent Mr values of GluR1 subunits were equivalent, suggesting the same degree of post-translational modification and the hydrodynamic properties of the receptor complexes were identical. 5. These data are consistent with the hypothesis that the movement of AMPA receptors between cellular compartments in the postsynaptic neurone could constitute one mechanism underlying long-term potentiation in the hippocampus.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1966

The subcellular localization of kallikrein, amylase and acetylcholine in the submaxillary gland of the guinea-pig.

1. The subcellular distribution pattern of kallikrein was compared with that of amylase and acetylcholine in submaxillary gland homogenates of the guinea-pig.2. Kallikrein was located in particles which sedimented in the nuclear (46%) and mitochondrial (17%) fractions. These particles were probably similar if not identical to zymogen granules.3. The above observations would support a digestive role for salivary kallikrein.

Acetylcholine↗

PubMed1981

Subcellular localization and biochemical properties of rat testicular alcohol- and aldehyde-dehydrogenase.

The biochemical properties and the subcellular distribution of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the rat testis is reported. Both enzymes are NAD-dependent. Testicular ADH activity was found in the nuclear and in the cytosolic fractions. Measurable ALDH was determined in the nuclear, mitochondrial, cytosolic and in the microsomal preparations. The major activity of ADH and ALDH were assayed in cytosol fraction. Cytosolic ALDH possesses lower Km than that determined in the mitochondrial and in the microsomal fractions. The results suggest the presence of multiple active form of testicular ALDH, possibly isoenzymes.

Aging↗

PubMed1963

THE SUBCELLULAR LOCALIZATION OF DOPAMINE AND ACETYLCHOLINE IN THE DOG CAUDATE NUCLEUS.

The distribution of dopamine (3,4-dihydroxyphenylethylamine) between the subcellular fractions from homogenates of dog caudate nucleus was compared with the distributions of acetylcholine, 5-hydroxytryptamine and lactate dehydrogenase activity. The distributions of noradrenaline and 5-hydroxytryptamine between the subcellular fractions from homogenates of dog hypothalamus were also determined. Most of the dopamine, in contrast to acetylcholine, occurred in the soluble supernatant fraction; the remainder was associated with the fractions rich in pinched-off nerve ending particles, but localization in any one fraction was not as sharp as that of acetylcholine. Evidence is presented that suggests that the dopamine occurs in a free or easily released form throughout cell cytoplasm.

Acetylcholine↗

PubMed1985

Endo-N-acetyl-beta-D-glucosaminidase activity in rat liver. Studies on substrate specificity, enzyme inhibition, subcellular localization and partial purification.

Endo-N-acetyl-beta-D-glucosaminidase (EC 3.2.1.96, endoglucosaminidase) has been partially purified (520-fold with respect to the cytoplasmic activity) by using concanavalin A-Sepharose, CM-Sephadex and Bio-Gel P-150 chromatography. From the influence of exogenous glycopeptides on the endoglucosaminidase activity it can be concluded that this activity consists of one enzyme hydrolysing both N-acetyl-lactosaminic-type and oligomannosidic-type substrates. Glycoproteins present in the homogenate inhibit the endoglucosaminidase activity. On re-examination of the subcellular distribution of endoglucosaminidase (after removal of inhibiting glycoproteins from the respective subcellular fractions), its cytoplasmic localization was confirmed.

Acetylglucosaminidase↗

PubMed1994

Subcellular localization of the F5 protein to the neuronal membrane-associated cytoskeleton.

F5 was identified originally as an interleukin-2-regulated gene in the murine helper T-lymphocyte clone L2. Subsequent studies demonstrated high levels of F5 mRNA and protein in mature neurons in adult mouse central and peripheral nervous systems. The F5 protein was present in dendrites and perikarya but not in axons. In the present studies, the intracellular localization of the F5 protein in adult mouse brain was determined by subcellular fractionation and Western blotting. Although the deduced F5 sequence predicts a soluble protein, virtually no F5 immunoreactivity was found in the cytosol. The F5 protein was restricted to the P2 crude mitochondrial and P3 crude microsomal particulate fractions. Within the P2 fraction, F5 protein was enriched in the P2B synaptosomal subfraction. The results of temperature-dependent phase separation with Triton X-114 and alkaline extraction with sodium carbonate of the P2 and P3 fractions were consistent with the F5 protein being an extrinsic membrane-associated protein. Although essentially all of the F5 protein in the P3 fraction was membrane-associated, a substantial proportion of P2-associated F5 protein and nearly all of the synaptosomal F5 protein was detergent-insoluble. Direct isolation and subfractionation of brain cytoskeleton confirmed colocalization of F5 immunoreactivity with the membrane-associated cytoskeleton and postsynaptic densities. These studies suggest that the F5 protein, which has a large number of potential phosphorylation sites, plays a role in membrane-cytoskeletal interactions and in dynamic aspects of synaptic structure or function.

Animals↗

PubMed1970

The subcellular localization of di- and tri-peptide hydrolase activity in guinea-pig small intestine.

1. Two different subcellular fractionation techniques were applied to guinea-pig intestinal mucosa and the composition of the brush borders prepared by the two methods were compared. 2. By using a kinetic assay system the subcellular distribution of activity against ten dipeptides and five tripeptides was studied. 3. Only small amounts (5-10%) of activity against dipeptides were found in the brush-border region, the enzymes being concentrated in the cytosol. 4. Significant amounts (10-60%) of activity against tripeptides were found in the brush border with the remainder largely present in the soluble fraction. 5. The relevance of these studies to the localization in vivo and the possible role of peptidases in protein digestion is discussed.

Animals↗

PubMed1970

Subcellular localization of renin and kininogenase in the rat kidney.

1. The distribution of enzymatic activities was determined in subcellular fractions of rat kidney cortex homogenates after various homogenization procedures. The specific activities of kininogenase (KGA), BAEE esterase (pH 8.5), alkaline phosphatase and glucose-6-phosphatase were, on average, 3.4 times higher in the microsomal fraction than in the whole homogenate. The total amount of these activities in the microsomal fraction after gentle, ordinary and forced homogenization were about 15, 40 and 65% of total recovered activities, respectively. These results confirmed the localization of KGA in the microsomal fraction.2. Renin activity was primarily recovered in the heavy mitochondrial fraction. When the force of the homogenization was increased some renin activity was shifted to the soluble fraction.3. When a mixture of renin and purified urinary KGA was given intravenously to an anaesthetized rat, a hypotensive response due to the KGA was followed by a hypertensive renin response. Over a certain range of concentrations KGA and renin could be measured simultaneously. In fractions of kidney homogenates, however, KGA activity was too low to be measured by this method.

Acid Phosphatase↗

PubMed1976

Subcellular localization of the heparin-neutralizing factor in blood platelets.

1. The distribution of the heparin-neutralizing factor (platelet factor 4, PF4) in subcellular organelles of blood platelets of rabbits and man was investigated. 2. In both species the organelles storing 5-hydroxytryptamine (5-HT storage organelles) contained only trivial amounts of PF4. 3. In contrast, the content of PF4 was highest in the subcellular fractions rich in alpha-granules. 4. In conclusion, PF4 is probably localized in the alpha-granules and therefore the platelets contain at least two types of organelles (5-HT organelles and alpha-granules) capable of releasing their contents in response to the same stimuli, such as exposure to collagen, thrombin, etc.

Animals↗

PubMed1992

Identification and subcellular localization of human rab5b, a new member of the ras-related superfamily of GTPases.

Members of the mammalian rab family of GTPases are associated with specific subcellular compartments, where these proteins are postulated to function in vesicular transport. By screening a human umbilical vein endothelial cell library with degenerate oligonucleotide probes, we have isolated a 1.6-kb cDNA clone encoding a 215-amino-acid protein belonging to the rab family of GTPases. This newly identified rab protein is 81% identical to human rab5, the canine counterpart of which has been localized to the plasma membrane and early endosomes. In light of this homology, we have named this new member of the GTPase superfamily "rab5b." Northern analysis using the rab5b cDNA as a probe revealed a 3.6-kb mRNA in a variety of cell types, including human umbilical vein endothelial cells, K562 erythroleukemia cells, U937 monoblastic cells, and HeLa cells. A fusion protein between glutathione-S-transferase (GST) and rab5b was expressed in bacteria and purified to homogeneity. The recombinant protein was shown to bind GTP and GDP. As is typical of other recombinant rab proteins, the rab5b-GST fusion protein displayed a low intrinsic rate of GTP hydrolysis (0.005/min). An antiserum to rab5b was prepared and used to determine the apparent molecular size and subcellular distribution of the protein. Western blotting with this antibody revealed a 25-kD protein in COS cells transfected with rab5b and in nontransfected HeLa cells. Indirect immunofluorescence and subcellular fractionation showed that rab5b localizes to the plasma membrane. We speculate that rab5b plays a role in vesicular trafficking at the plasma membrane in various cell types.

Amino Acid Sequence↗