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PubMed1986

Uptake and subcellular processing of 59Fe-125I-labelled transferrin by rat liver.

The uptake of transferrin and iron by the rat liver was studied after intravenous injection or perfusion in vitro with diferric rat transferrin labelled with 125I and 59Fe. It was shown by subcellular fractionation on sucrose density gradients that 125I-transferrin was predominantly associated with a low-density membrane fraction, of similar density to the Golgi-membrane marker galactosyltransferase. Electron-microscope autoradiography demonstrated that most of the 125I-transferrin was located in hepatocytes. The 59Fe had a bimodal distribution, with a larger peak at a similar low density to that of labelled transferrin and a smaller peak at higher density coincident with the mitochondrial enzyme succinate dehydrogenase. Approx. 50% of the 59Fe in the low-density peak was precipitated with anti-(rat ferritin) serum. Uptake of transferrin into the low-density fraction was rapid, reaching a maximal level after 5-10 min. When livers were perfused with various concentrations of transferrin the total uptakes of both iron and transferrin and incorporation into their subcellular fractions were curvilinear, increasing with transferrin concentrations up to at least 10 microM. Analysis of the transferrin-uptake data indicated the presence of specific transferrin receptors with an association constant of approx. 5 X 10(6) M-1, with some non-specific binding. Neither rat nor bovine serum albumin was taken up into the low-density fractions of the liver. Chase experiments with the perfused liver showed that most of the 125I-transferrin was rapidly released from the liver, predominantly in an undegraded form, as indicated by precipitation with trichloroacetic acid. Approx. 40% of the 59Fe was also released. It is concluded that the uptake of transferrin-bound iron by the liver of the rat results from endocytosis by hepatocytes of the iron-transferrin complex into low-density vesicles followed by release of iron from the transferrin and recycling of the transferrin to the extracellular medium. The iron is rapidly incorporated into mitochondria and cytosolic ferritin.

Animals↗

PubMed1988

Investigation of endosomal compartments involved in endocytosis and transcytosis of polymeric immunoglobulin A by subcellular fractionation of perfused isolated rat liver.

1. A gamma camera was used to monitor continuously the uptake of radiolabelled polymeric immunoglobulin A (pIgA) into the rat body after intravenous injection. Uptake into liver was fast but, since the peak of liver labelling occurred only after 9-15 min, it was not sufficiently rapid to constitute a pulse dose. A perfused, isolated rat liver system was therefore established which could be given a single pass dose of pIgA; a variety of tests showed such livers remained viable for at least 3 h and could be subsequently fractionated on Ficoll and Nycodenz gradients with normal distributions of marker enzymes. 2. Subcellular fractionation at different times after a single pass dose of pIgA showed that whilst pIgA appeared sequentially in sinusoidal plasma membrane, light endosomes, dense endosomes, very dense endosomes and lysosomes as in vivo, the predominance of pIgA in the light endosome compartment disappeared much earlier than after injection in vivo of pIgA, presumably because this compartment was not being continuously loaded over the first 10-15 min. The time course of appearance of label in bile was unchanged. A large excess of unlabelled asialofetuin did not change these patterns, indicating that the asialoglycoprotein receptor was not involved. 3. Low doses of the microtubule agent colchicine reduced the proportion of pIgA reaching the bile, but subcellular fractionation of treated liver showed that distribution of label amongst liver fractions was little changed, although the overall liver pIgA content had increased. This would suggest that pIgA did not remain in the common compartment which could have supplied bile or lysosomes but rather flowed out of it as rapidly as in untreated liver but towards those compartments supplying the lysosomes. 4. Experiments with nocodazole, which reversibly disrupts microtubules, showed that very little of the pIgA taken into an inhibited liver appeared in the bile after nocodazole was removed 30 min later, even though a second dose of pIgA, given after nocodazole removal, appeared in bile with a normal time course. The first dose of pIgA must therefore have passed beyond the compartments competent to supply the bile before nocodazole was removed. Such compartments were undamaged since the second dose of pIgA appeared in bile normally. We therefore conclude that the bulk of pIgA must be supplied to the bile from light or dense endosomes rather than from very dense endosomes and lysosomes.

Animals↗

PubMed1989

The effects of fasting or hypoxia on rates of protein synthesis in vivo in subcellular fractions of rat heart and gastrocnemius muscle.

We measured rates of protein synthesis in vivo in subcellular fractions (soluble, myofibrillar and stromal fractions) of the heart and the gastrocnemius from rats after fasting or under hypoxic conditions (i.e. atmospheres containing 5% or 10% O2). Such interventions are known to inhibit protein synthesis under some circumstances. The recovery of tissue protein after fractionation was 80-100%. The proportions of protein present in the soluble and stromal fractions were different in the two muscles. The rates of protein synthesis in the myofibrillar and stromal fractions were less than those for total mixed tissue protein, whereas the rate for soluble protein was greater. Both fasting and moderate hypoxia (10% O2 for 24 h) inhibited protein synthesis in the gastrocnemius. In this tissue, the synthesis of the myofibrillar fraction was apparently the most sensitive to inhibition, and this resulted in some significant increases in the soluble-fraction/myofibrillar-fraction protein-synthesis rate ratios. In the heart, fasting inhibited protein synthesis, but moderate hypoxia (10% O2 for 24 h) did not. The rate of protein synthesis in the cardiac myofibrillar fraction was again more sensitive to fasting than were the rates in the other fractions, but it was not as sensitive as that in the gastrocnemius. Under severely hypoxic conditions (5% O2 for 1 or 2 h), protein synthesis was decreased in all fractions in both tissues. These results suggest that the rates of protein synthesis in these relatively crude subcellular fractions vary.

Animals↗

PubMed1990

Polarized subcellular distribution of the 1-, 4- and 5-phosphatase activities that metabolize inositol 1,4,5-trisphosphate in intestinal epithelial cells.

In intestinal epithelial cells, Ins(1,4,5)P3 is metabolized both by an intracellular 5-phosphatase and by less specific extracellular phosphatases [Rubiera, Velasco, Michell, Lazo & Shears (1988) Biochem. J. 255, 131-137]. A total of 91% of intracellular Ins(1,4,5)P3 5-phosphatase was particulate, and was preferentially associated with plasma membranes rather than with other subcellular organelles. A soluble Ins(1,4,5)P3 3-kinase activity was also characterized, further supporting the idea that inositol phosphates are important in enterocyte function. We have studied the distribution of Ins(1,4,5)P3 phosphatase activities in basolateral and brush-border domains of the plasma membrane. Compared with homogenates, the extracellular phosphatases were 13-17-fold enriched in brush-border membranes, but only 2-fold enriched in basolateral membranes. The 1- and 4-phosphates of Ins(1,4,5)P3 were hydrolysed at equal rates by the extracellular phosphatases; these enzymes are proposed to have digestive functions. The intracellular particulate 5-phosphatase was 2-fold enriched in brush-border membranes and 13-fold enriched in basolateral membranes, at the same pole of the cell where Ins(1,4,5)P3 is believed to be generated. This is opposite to the polarized distribution of particulate 5-phosphatase in hepatocytes [Shears, Evans, Kirk & Michell (1988) Biochem. J. 256, 363-369]; these differences in subcellular distribution may be important in determining cell-specific metabolism of Ins(1,4,5)P3.

Animals↗

PubMed1992

Subcellular distribution and characteristics of ciprofibroyl-CoA synthetase in rat liver. Its possible identity with long-chain acyl-CoA synthetase.

The subcellular distribution and characteristics of ciprofibroyl-CoA synthetase were studied in rat liver and compared with those of long-chain acyl-CoA synthetase (palmitate as substrate) which, as already known, is distributed among mitochondria, microsomes and peroxisomes. Upon differential centrifugation, the subcellular distribution of ciprofibroyl-CoA synthetase followed closely that of palmitoyl-CoA synthetase and was specifically inactivated in the mitochondrial fraction by freezing and thawing, a behaviour already described for palmitoyl-CoA synthetase. Both enzyme activities were found to co-purify through several steps from rat liver microsomes. By using a partially purified enzyme, the activation of ciprofibrate to its acyl-CoA ester followed Michaelis-Menten kinetics with an apparent Km of 0.63 +/- 0.1 mM. Ciprofibroyl-CoA synthetase was competitively inhibited by 25 and 50 microM-palmitic acid. Higher concentrations of the fatty acid resulted in a mixed type of inhibition. Conversely, ciprofibrate up to 0.5 mM was found to inhibit competitively palmitoyl-CoA synthetase, whereas higher concentrations also resulted in a mixed inhibition. The highest activity of ciprofibroyl-CoA synthetase was found in fat and liver homogenates. The distribution of the enzyme in different rat tissues was similar to that of palmitoyl-CoA synthetase. The present results suggest that long-chain acyl-CoA synthetase and ciprofibroyl-CoA synthetase activities reside in identical or closely related proteins.

Acyl Coenzyme A↗

PubMed1992

Subcellular localization and release of human neutrophil gelatinase, confirming the existence of separate gelatinase-containing granules.

An e.l.i.s.a. was developed using specific polyclonal rabbit antibodies against human neutrophil gelatinase. This assay, in contrast to the functional assay, is independent of activation of gelatinase, and is specific for the detection of gelatinase in both its reduced and unreduced forms. Using this assay, we were able to demonstrate a difference between the subcellular localization of gelatinase on the one hand, and the subcellular localization of vitamin B-12-binding protein, lactoferrin and cytochrome b558 on the other hand. The latter three co-localized in fractions of slightly higher density than gelatinase on a two-layer Percoll density gradient. Furthermore, the release of gelatinase exceeded the release of vitamin B-12-binding protein as well as lactoferrin by a factor of 3-6 following stimulation with formylmethionyl-leucyl-phenylalanine, leukotriene B4 and other soluble stimuli. Thus, although gelatinase has previously been found to co-localize with lactoferrin on immuno-electron microscopy, we confirm the existence of gelatinase-rich and lactoferrin- and vitamin B-12-binding-protein-poor granules, that are lighter and mobilized more easily than specific granules. These gelatinase-containing granules are not the store of cytochrome b558.

Cytoplasmic Granules↗

PubMed1993

Subcellular-membrane characterization of [3H]ryanodine-binding sites in smooth muscle.

The plant alkaloid ryanodine, known to interact selectively with the intracellular Ca(2+)-release channel in skeletal and cardiac muscles, has been repeatedly reported to affect smooth-muscle contractile functions that are consistent with its intracellular action at the Ca(2+)-release channel sites. Direct evidence for the binding of [3H]ryanodine to smooth-muscle membranes is sparse. Following our recent detailed characterization of functional effects of ryanodine and a preliminary report on the presence of [3H]ryanodine binding sites in rat vas deferens smooth muscle, we now report in this study a detailed characterization of binding of [3H]ryanodine to smooth muscle at the subcellular-membrane level. The ryanodine receptor in rat vas deferens muscle layer is primarily of smooth-muscle origin and is localized at the subcellular membrane site that is consistent with its role as a Ca(2+)-release channel in the sarcoplasmic reticulum (SR). Ryanodine binding to its receptor is Ca(2+)-dependent, with half-maximal binding occurring within the physiologically relevant cytosolic Ca2+ concentration. It is also sensitive to many factors, including change in Mg2+ concentration, ionic strength and osmolarity across the membrane vesicles. Agents known to inhibit (Ruthenium Red, Mg2+) or enhance (caffeine, Na+, K+) the Ca(2+)-induced Ca2+ release also inhibit or enhance the binding of ryanodine. Quantitative differences in ryanodine receptors exist among smooth muscles and do not seem to parallel their SR contents. Results from the present study indicate both the need and the basis for future investigations of the functional role of the ryanodine receptor in different smooth muscles.

Animals↗

PubMed1995

Accumulation of phosphatidylalcohol in cultured cells: use of subcellular fractionation to investigate phospholipase D activity during signal transduction.

Phosphatidylalcohol accumulates as a product of a phospholipase D (PLD)-catalysed transphosphatidylation reaction in cells incubated in the presence of a primary alcohol. In the presence of ethanol the phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated the accumulation of [3H]phosphatidylethanol (PEth) in HeLa cells prelabelled with [3H]palmitic acid. Radioactivity associated with PEth increased linearly during a 30 min incubation, indicating that a sustained activation of PLD is caused by PMA in these cells. This was accompanied by the membrane association of protein kinase C-alpha (PKC-alpha), the PKC isoform that recent studies indicate is involved in the activation of PLD. In similar experiments, the neuropeptide bradykinin stimulated an accumulation of PEth in 3T3 Li cells. The radioactivity associated with PEth increased to a maximal level at 30 s and plateaued after this time, suggesting that bradykinin induces only a transient activation of PLD in these cells. This is consistent with the effects of bradykinin on PKC-alpha, which underwent a rapid and transient association with cell membranes. The subcellular localization of PEth was examined using the technique of subcellular fractionation on Percoll density gradients to isolate organelle-enriched fractions from HeLa and 3T3 Li cells. An accumulation of [3H]PEth was measured in the plasma-membrane (PM)-enriched fractions of both HeLa and 3T3 Li cells after incubation with PMA and bradykinin respectively. This was accompanied by a time-dependent accumulation of [3H]PEth in the combined mitochondrial and endoplasmic reticulum (MER)-enriched fractions of both cell lines. PMA was also found to cause translocation of PKC-alpha to both the PM- and MER-enriched fractions in HeLa cells. However, bradykinin stimulated the translocation of PKC-alpha to the PM-enriched fractions only of 3T3 Li cells. The results show that PLD activation leads to the accumulation of PEth in both the PM and MER fractions. We therefore propose that either bradykinin activates a PM-associated PLD and the PLD reaction product is rapidly translocated to other membrane systems or it activates an MER-associated PLD by a mechanism that does not involve PKC-alpha.

3T3 Cells↗

PubMed1995

Epoxidation of plasmalogens: source for long-chain alpha-hydroxyaldehydes in subcellular fractions of bovine liver.

1. Masked long-chain alpha-hydroxyaldehydes were trapped in all subcellular fractions of bovine liver by application of pentafluorbenzyloxime derivatization [van Kuijk, Thomas, Stephens and Dratz (1986) Biochem. Biophys. Res. Commun. 139, 144-149] and quantified via GLC/MS using characteristic ion traces. 2. The chain-length profile of long-chain 2-hydroxyalkanales clearly indicates their relationship to plasmalogens as precursor molecules. 3. The previously postulated existence of alpha-acyloxyplasmalogens as precursor molecules of masked long-chain alpha-hydroxyaldehydes in bovine tissue lipids [Lutz and Spiteller (1991) Liebigs Ann. Chem. 1991, 563-567] was excluded. 4. The constant oxidation rate of plasmalogens in all subcellular fractions provides conclusive evidence for a non-enzymic plasmalogen epoxidation process (probably via hydroperoxy radicals). 5. The high reactivity of alpha-hydroxyaldehydes sheds some doubt on the postulation that plasmalogens protect mammalian cells against oxidative stress as postulated previously [Morand, Zoeller and Raetz (1988) J. Biol. Chem. 263, 11590-11596; Morand, Zoeller and Raetz (1988) J. Biol. Chem. 263, 11597-11606].

Aldehydes↗

PubMed1976

Analytical subcellular fractionation of jejunal biopsy specimens: methodology and characterization of the organelles in normal tissue.

1. Portions of closed jejunal biopsies were gently homogenized in isotonic sucrose or sorbitol and centrifuged at 800 g for 10 min to prepare a cell extract. 2. The extract was fractionated in a single-step procedure by isopycnic centrifugation on a continuous sucrose or sorbitol density gradient with the Beaufay automatic zonal rotor. 3. The subcellular organelles were located in the density gradient by assay of marker enzymes and previously unassigned enzymes were localized to particular organelles. 4. The following organelles were characterized by their modal equilibrium densities in sucrose density gradients: brush borders (1.21), peroxisomes (1.18), mitochondria (1.16), endoplasmic reticulum (1.16), basal-lateral membranes (1.12). At least three distinct populations of lysosomes with different modal densities and enzyme content were demonstrated. 5. This analytical fraction technique can be used to study the subcellular pathology of human jejunum.

Adult↗

PubMed1977

Analytical subcellular fractionation of guinea-pig myocardium.

1. Homogenates of guinea-pig left ventricle were fractionated by differential pelleting and by centrifugation on continuous sucrose density gradients. 2. The principal subcellular organelles of myocardium, characterized by their marker enzyme content, were resolved by density gradient centrifugation in a small-volume zonal rotor. The equilibrium densities (p) of the principal organelles are (with marker enzymes in parentheses): sarcolemma, 1-12 (5'-nucleotidase); lysosomes, 1-16 (N-acetyl-beta-glucosaminidase); mitochondria, 1-17 (cytochrome oxidase); peroxisomes, 1-18 (catalase); cytosol (lactate dehydrogenase). 3. The subcellular distribution of various adenosine triphosphatase activities and previously unassigned enzymes was determined. Leucyl-beta-naphthylamidase and gamma-glutamyl transpeptidase showed both cytosol and sarcolemma components. Ca2+-dependent adenosine triphosphatase showed dual localization to the mitochondria and to the sarcolemma.

Adenosine Triphosphatases↗

PubMed1978

Subcellular distribution of hydrolase activities for glycine and leucine homopeptides in human jejunum.

1. The subcellular distribution of peptidase activities in the normal human jejunum against glycine and leucine homopeptides has been investigated with an analytical fractionation technique. 2. An 8000 g-min supernatant was prepared from homogenates of Crosby capsule biopsy specimens and subjected to isopycnic centrifugation in a Beaufay automatic zonal rotor. 3. The distribution of subcellular organelles in the gradient was established by measurement of organelle-specific marker enzymes. 4. A sensitive fluorimetric assay for glycine peptidase was developed and used for the localization of peptidase activity with peptides composed of from two to five glycine residues as substrates. 5. Glycine peptidase activity was located in the cytosol and in the brush-border membrane but the distribution of activity varied markedly with the chain-length of substrate; the longer the peptide the greater the proportion of activity associated with the brush border. Leucine peptidase showed a similar variation in cytosol--brush border distributions. 6. The results are consistent with concepts that suggest absorption and intracellular hydrolysis of small peptides and brush-border digestion of larger peptides.

Cytosol↗

PubMed1979

Subcellular fractionation studies of the intestinal mucosa in congenital sucrase--isomaltase deficiency.

1. Jejunal biopsy specimens from three children with congenital sucrase-isomaltase deficiency were assayed for disaccharidase activity and were subjected to analytical subcellular fractionation with enzymic microanalysis. 2. By use of the highly sensitive fluorigenic modification of the disaccharidase assay, brush-border sucrase and isomaltase activities were depressed but nevertheless detectable in each child. 3. Apart from the expected decrease in brush-border alpha-glucosidase activity, the other enterocyte marker-enzyme activities were normal. 4. There were no abnormalities in the enterocytes of any child on analytical subcellular fractionation or on electron microsocopy.

Acetylglucosaminidase↗

PubMed1979

The adenylate cyclase system in human liver: characterization, subcellular distribution and hormonal sensitivity in normal or cirrhotic adult, and in foetal liver.

1. Adenylate cyclase (EC 4.6.1.1) activity was characterized in human liver, and its subcellular distribution compared with that of three other potential enzyme markers of the pericellular membrane: leucine aminopeptidase (EC 3.4.11.1), gamma-glutamyltransferase (EC 2.3.2.2) and 5'-nucleotidase (EC 3.1.3.5). Although these three enzyme activities were detected in each of the subcellular fractions studied, 85% of the total adenylate cyclase activity was found in the 1000 g pellet ('nuclear' fraction) with a threefold increase in specific activity as compared with the homogenate. No adenylate cyclase activity existed in the 150 000 g supernatant fraction. 2. In the 'nuclear' fraction, adenylate cyclase activity was increased in a dose-dependent fashion by glucagon with a half-maximal stimulation at 10 nmol/l and a maximal four- to seven-fold increase at 1 mumol/l. Catecholamines activated adenylate cyclase 2.5- to three-fold, with an order of potency (protokylol greater than isoprenaline greater than adrenaline greater than noradrenaline) typical of a beta 2-adrenoreceptor. Prostaglandin E1 and NaF also stimulated cyclase two- and four-fold respectively. Insulin, serotonin, dopamine, thyroid-stimulating hormone and ACTH had no effect. Adenosine provoked a weak inhibition at 0.1 mmol/l. Finally guanosine triphosphate and 5'-guanylyl imidodiphosphate induced a marked increase in basal activity, four- and eight-fold respectively, but both reduced the relative increase in enzyme activity due to glucagon or adrenaline. 3. Cyclase from foetal liver (12--16 weeks old) and cirrhotic adult liver appeared to behave similarly to that from normal liver; however, foetal cyclase was more active, and cirrhotic enzyme less active than normal adult liver. Both systems responded to catecholamines via a beta 2-adrenoreceptor. 4. These results validate the use of rat liver adenylate cyclase as a tool for pharmacological and physiological studies.

Adenosine↗

PubMed1980

Subcellular distribution of low- and high-molecular-weight renin and its relation to a renin inhibitor in pig renal cortex.

1. The subcellular distribution of low-molecular-weight and high-molecular weight forms of pig renin has been investigated. 2. Renin, in aqueous extracts of a 'renin granular fraction' prepared by differential centrifugation, after gel filtration on Sephadex G-100 displayed an apparent molecular weight of 40 000 and was not activated by acidification to pH 2.8. 3. Renin in the soluble fraction separated on Sephadex G-100 at neutral pH displayed a main peak of activity with an apparent molecular weight of 40 000. When eluates were acidified to pH 2.8 (2 degrees C, 60 min) a marked increase in renin activity was observed in the region corresponding to an apparent molecular weight of 50 000. 4. A renin inhibitory material was isolated from the soluble fraction by DEAE chromatography. This material displayed an apparent molecular weight of 50 000 and it was destroyed by acidification to pH 2.8. 5. The presence of the proteolytic inhibitor N-ethylmaleimide yielded an apparently high-molecular-weight form of renin (60 000--70 000) from the soluble fraction, but this was not found in the granular fraction. 6. We conclude that pig renal renin is stored within membrane-bounded subcellular organelles as the low-molecular-weight form. High-molecular-weight renin and renin inhibitory activity are localized to the cortical soluble fraction. In addition, the soluble fraction contains a material which in the presence of N-ethylmaleimide results in the formation of an apparently high-molecular-weight renin.

Animals↗

PubMed2000

Subcellular localization of the Agrobacterium tumefaciens T-DNA transport pore proteins: VirB8 is essential for the assembly of the transport pore.

Agrobacterium tumefaciens transforms plants by transferring DNA to the plant cell nucleus. The VirB membrane proteins are postulated to form a pore for the transport of the DNA across the bacterial membranes. Immunofluorescence and immunoelectron microscopy were used to study the transport pore complex. Three likely components of the transport pore, VirB8, VirB9 and VirB10, localized primarily to the inner membrane, outer membrane and periplasm respectively. A significant amount of VirB10 was also found associated with the outer membrane. When expressed alone VirB9 and VirB10 were randomly distributed along the cell membrane. Subcellular location of both proteins changed dramatically in the presence of the other VirB proteins. Both proteins localized to fewer sites and most of the gold particles representing protein molecules were found in clusters suggesting that the two proteins are in a protein complex. VirB8, on the other hand, localized to clusters even in the absence of the other VirB proteins. To investigate the role of VirB8 in the formation of VirB9 and VirB10 protein complexes, we studied the effect of deletion of virB8 on the subcellular location of VirB9 and VirB10. In a virB8 deletion mutant both proteins were distributed randomly on the cell membrane indicating that VirB8 is essential for complex assembly.

Agrobacterium tumefaciens↗

PubMed1995

The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments.

Here, a complete study is described of all the genes and isoenzymes for aspartate aminotransferase (AspAT) present in Arabidopsis thaliana. Four classes of cDNAs representing four distinct AspAT genes (ASP1-ASP4) have been cloned from Arabidopsis. Sequence analysis of the cDNAs suggests that the encoded proteins are targeted to different subcellular compartments. ASP1 encodes a mitochondrial form of AspAT, ASP3 encodes a chloroplastic/plastidic form of AspAT, whereas ASP2 and ASP4 each encode cytosolic forms of AspAT. Three distinct AspAT holoenzymes (AAT1-AAT3) were resolved by activity gel analysis. Organelle isolation reveals that AAT1 is mitochondrial-localized, AAT3 is plastid-localized, and AAT2 is cytosolic. Gene-specific Northern analysis reveals that each Asp mRNA accumulates differentially with respect to organ-type. However, the individual Asp mRNAs show no dramatic fluctuations in response to environmental stimuli such as light. Southern analysis reveals that four distinct nuclear genes probably represent the entire AspAT gene family in Arabidopsis. These molecular studies shed light on the subcellular synthesis of aspartate in Arabidopsis and suggest that some of the AspAT isoenzymes may play overlapping roles in plant nitrogen metabolism.

Amino Acid Sequence↗

PubMed2000

Spatial, temporal and subcellular localization of islet-brain 1 (IB1), a homologue of JIP-1, in mouse brain.

Islet-brain 1 (IB1) was recently identified as a DNA-binding protein of the GLUT2 gene promoter. The mouse IB1 is the rat and human homologue of the Jun-interacting protein 1 (JIP-1) which has been recognized as a key player in the regulation of c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. JIP-1 is involved in the control of apoptosis and may play a role in brain development and aging. Here, IB1 was studied in adult and developing mouse brain tissue by in situ hybridization, Northern and Western blot analysis at cellular and subcellular levels, as well as by immunocytochemistry in brain sections and cell cultures. IB1 expression was localized in the synaptic regions of the olfactory bulb, retina, cerebral and cerebellar cortex and hippocampus in the adult mouse brain. IB1 was also detected in a restricted number of axons, as in the mossy fibres from dentate gyrus in the hippocampus, and was found in soma, dendrites and axons of cerebellar Purkinje cells. After birth, IB1 expression peaks at postnatal day 15. IB1 was located in axonal and dendritic growth cones in primary telencephalon cells. By biochemical and subcellular fractionation of neuronal cells, IB1 was detected both in the cytosolic and membrane fractions. Taken together with previous data, the restricted neuronal expression of IB1 in developing and adult brain and its prominent localization in synapses suggest that the protein may be critical for cell signalling in developing and mature nerve terminals.

Adaptor Proteins, Signal Transducing↗