Metabolism and subcellular localization of angiotensin converting enzyme in cultured human monocytes

The enhancement of monocyte ACE activity during culture by autologous T-lymphocytes was shown to be due to a stimulation of the rate of ACE synthesis. The rate of synthesis increased from 0.020 mU/10(6) monocytes/hr in monocytes cultured alone to 0.063 mU/10(6) monocytes/hr in monocytes co-cultured with T-lymphocytes. The presence of T-lymphocytes during culture did not alter the rate of ACE degradation observed in monocytes cultured alone. The ACE induced in monocytes by T-lymphocytes appears to be an ecto-enzyme. Brief exposure to diazosulfanilic acid (10(-3) M) and papain (250 micrograms/ml) reduced ACE activity 89% and 66%, respectively, without appreciably altering the activity of the cytosolic enzyme, lactate dehydrogenase.     

Comparison of the mineralocorticoid activity of 19-oxygenated and 19-nor derivatives of deoxycorticosterone.

19-Nordeoxycorticosterone (19-nor-DOC) is a mineralocorticoid with several unresolved physiologic questions. First, is 19-nor-DOC synthesized in the kidney from a circulating adrenocortical precursor (19-oicdeoxycorticosterone [19-oic-DOC] or 19-oxodeoxycorticosterone [19-oxo-DOC])? Second, does 19-nor-DOC, synthesized in the kidney, have mineralocorticoid activity or is it excreted in the urine without biologic activity? To answer this question, we administered two of the putative 19-nor-DOC precursors (19-oxo-DOC and 19-oic-DOC) to adrenalectomized rats and measured the formation of 19-nor-DOC and bioactivity as the urinary Na+ to K+ ratio. Each of the 10-microgram steroid treatments produced an elevation of urinary-free 19-nor-DOC (0 to 2 hours), whereas at the 1-micrograms dose only 19-oic-DOCA produced an increased UF 19-nor-DOC. None of the treatments led to an increase of conjugated 19-nor-DOC except 10 microgram 19-oic-DOCA. Increased mineralocorticoid activity (decreased urinary Na+ to K+ ratio) was produced by aldosterone, 1 and 10 micrograms 19-nor-DOC, and 10 micrograms 19-oic-DOCA over the same time period. An anti-mineralocorticoid effect (increased urinary Na+ to K+ ratio) was produced by 1 microgram 19-oxo-DOC. Urinary-free 19-nor-DOC, but not conjugated 19-nor-DOC, correlated with the urinary mineralocorticoid effect (decreased Na+ to K+ ratio). These data support the contention that 19-oic-DOC is the circulating 19-nor-DOC precursor and that, at least at the higher dose, it has a mineralocorticoid action on the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

Small RNA species of the HeLa cell: Metabolism and subcellular localization

The small molecular weight RNAs of the HeLa cell have been located in specific subcellular fractions. SnA is located in the nucleolus and is partially bonded to nucleolar 28S RNA. SnD, the most abundant of the small nuclear RNAs, is partially released from the nucleus when the nuclear preparation is briefly warmed. SnF is released from the nuclei when chromatin is digested with the micrococcal nuclease and not when pancreatic DNAase is used. The remainder of the small nuclear species remain in the nucleus following the digestion of chromatin and are concluded to be elements of the “nuclear skeleton.” SnK is found predominantly in the cytoplasm, but migrates quantitatively to the nuclear fraction in the presence of high levels of actinomycin D. ScL is totally cytoplasmic and is partially bound to cell membranes. It is the 7S RNA found in oncornavirus virions. All the small nuclear RNAs appear initially in the cytoplasmic fraction before fixation in the nucleus. Two short-lived cytoplasmic species behave kinetically as precursors to the stable nuclear RNAs.     

Proteolytic activity assayed by subcellular localization switching of a substrate

An approach to assay proteolytic activity in vivo by altering the subcellular localization of a labelled substrate was demonstrated. The assay included a protein shuttling between different cellular compartments and a site-specific recombinant protease. The shuttle protein used was the human immunodeficiency virus type 1 (HIV-1) Rev protein tandemly fused to the enhanced green fluorescent protein (EGFP) and the red fluorescent protein (RFP), while the protease was the site-specific protease VP24 from the herpes simplex virus type 1 (HSV-1). The fluorescent proteins in the Rev fusion protein were separated by a cleavage site specific for the VP24 protease. When co-expressed in COS-7 cells proteolysis was observed by fluorescence microscopy as a shift from a predominantly cytoplasmic localization of the fusion protein RevEGFP to a nuclear localization while the RFP part of the fusion protein remained in the cytoplasm. The cleavage of the fusion protein by VP24 was confirmed by Western blot analysis. The activity of VP24, when tagged N-terminally by the Myc-epitope, was found to be comparable to VP24. These results demonstrates that the activity and localization of a recombinantly expressed protease can be assessed by protease-mediated cleavage of fusion proteins containing a specific protease cleavage site.     

BaCelLo: a balanced subcellular localization predictor

MOTIVATION: The knowledge of the subcellular localization of a protein is fundamental for elucidating its function. It is difficult to determine the subcellular location for eukaryotic cells with experimental high-throughput procedures. Computational procedures are then needed for annotating the subcellular location of proteins in large scale genomic projects. RESULTS: BaCelLo is a predictor for five classes of subcellular localization (secretory pathway, cytoplasm, nucleus, mitochondrion and chloroplast) and it is based on different SVMs organized in a decision tree. The system exploits the information derived from the residue sequence and from the evolutionary information contained in alignment profiles. It analyzes the whole sequence composition and the compositions of both the N- and C-termini. The training set is curated in order to avoid redundancy. For the first time a balancing procedure is introduced in order to mitigate the effect of biased training sets. Three kingdom-specific predictors are implemented: for animals, plants and fungi, respectively. When distributing the proteins from animals and fungi into four classes, accuracy of BaCelLo reach 74% and 76%, respectively; a score of 67% is obtained when proteins from plants are distributed into five classes. BaCelLo outperforms the other presently available methods for the same task and gives more balanced accuracy and coverage values for each class. We also predict the subcellular localization of five whole proteomes, Homo sapiens, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae and Arabidopsis thaliana, comparing the protein content in each different compartment. AVAILABILITY: BaCelLo can be accessed at http://www.biocomp.unibo.it/bacello/.     

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.     

The metabolism of 19-nor contraceptive progestins modulates their biological activity at the neuroendocrine level

In this communication, a series of studies from our laboratory dealing with the mechanism of action of 17 alpha-ethinyl derivatives of 19-nor testosterone are reviewed. The administration of norethisterone (NET) to long-term castrated female rats induces the nuclear translocation of pituitary estradiol receptors and is followed by some estrogenic-like effects at the hypothalamic-pituitary unit. It is established that an A-ring reduced metabolite of NET, the 3 beta,5 alpha-tetrahydro NET derivative, is responsible for the observed in vivo estrogenic effects of the parent compound. 3 beta,5 alpha-NET binds to the estrogen receptor and is efficient in inducing the pituitary estrogen-dependent progesterone receptor and in increasing the uterine weight in long-term castrated rats. Furthermore, administration of 3 beta,5 alpha-NET and the 5 alpha-reduced metabolite of NET (5 alpha-NET) are able to inhibit the release of gonadotropins in the castrated animal to a greater extent than NET. Moreover, pretreatment with tamoxifen, an estrogen binding site competitor, results in a significant diminution of the antigonadotropic potency of 3 beta,5 alpha-NET but not of the 5 alpha-NET, which is only inhibited by the administration of cyproterone acetate. These findings underline the importance of the metabolic rate of NET for the expression of its biological effects at the hypothalamic-pituitary unit.     

Proclotting enzyme from horseshoe crab hemocytes. cDNA cloning, disulfide locations, and subcellular localization

Proclotting enzyme is an intracellular serine protease zymogen closely associated with an endotoxin-sensitive hemolymph coagulation system in limulus. Its active form, clotting enzyme, catalyzes conversion of coagulogen to insoluble coagulin gel. We present here the cDNA and amino acid sequences, disulfide locations, and subcellular localization of proclotting enzyme. The isolated cDNA for proclotting enzyme consists of 1,501 base pairs. The open reading frame of 1,125 base pairs encodes a sequence comprising 29 amino acid residues of prepro-sequence and 346 residues of the mature protein with a molecular mass of 38,194 Da. Three potential glycosylation sites for N-linked carbohydrate chains were confirmed to be glycosylated. Moreover, the zymogen contains six O-linked carbohydrate chains in the amino-terminal light chain generated after activation. The cleavage site that accompanies activation catalyzed by trypsin-like active factor B, proved to be an Arg-Ile bond. The resulting carboxyl-terminal heavy chain is composed of a typical serine protease domain, with a sequence similar to that of human coagulation factor XIa (34.5%) or factor Xa (34.1%). The light chain has a unique disulfide-knotted domain which shows no significant homology with any other known proteins. Thus, this proclotting enzyme has a mammalian serine protease domain and a structural domain not heretofore identified in coagulation and complement factors. Immunohistochemical studies showed that the proclotting enzyme is localized in large granules of hemocytes.     

Pyruvate dehydrogenase phosphate (PDHb) phosphatase in brain: activity, properties, and subcellular localization

The activity of pyruvate dehydrogenase phosphate (PDHb) phosphatase in rat brain mitochondria and homogenate was determined by measuring the rate of activation of purified, phosphorylated (i.e., inactive) pyruvate dehydrogenase complex (PDHC), which had been purified from bovine kidney and inactivated by phosphorylation with Mg . ATP. The PDHb phosphatase activity in purified mitochondria showed saturable kinetics with respect to its substrate, the phospho-PDHC. It had a pH optimum between 7.0 and 7.4, depended on Mg and Ca, and was inhibited by NaF and K-phosphate. These properties are consistent with those of the highly purified enzyme from beef heart. On subcellular fractionation, PDHb phosphatase copurified with mitochondrial marker enzymes (fumarase and PDHC) and separated from a cytosolic marker enzyme (lactate dehydrogenase) and a membrane marker enzyme (acetylcholinesterase), suggesting that it, like its substrate, is located in mitochondria. PDHb phosphatase had similar kinetic properties in purified mitochondria and in homogenate: dependence on Mg and Ca, independence of dichloroacetate, and inhibition by NaF and K-phosphate. These results are consistent with there being only one type of PDHb phosphatase in rat brain preparations. They support the validity of the measurements of the activity of this enzyme in brain homogenates.     

Low-molecular-weight RNA of eukaryotes: biogenesis, subcellular localization, functions

The experimental data on the organization and expression of the low-molecular-weight RNA genes on the subcellular localization, metabolism and evolution of these RNA are summarized. The processes in which they are involved are discussed. The biological role of the low-molecular-weight RNA is paid a special attention. A hypothesis concerning the involvement of these molecules in the mechanism of active transport of proteins from the cytoplasm into the nucleus of the cell is put forward.     

Protein transduction of an antioxidant enzyme: subcellular localization of superoxide dismutase fusion protein in cells

In protein therapy, it is important for exogenous protein to be delivered into the target subcellular localization. To transduce a therapeutic protein into its specific subcellular localization, we synthesized nuclear localization signal (NLS) and membrane translocation sequence signal (MTS) peptides and produced a genetic in-frame SOD fusion protein. The purified SOD fusion proteins were efficiently transduced into mammalian cells with enzymatic activities. Immunofluorescence and Western blot analysis revealed that the SOD fusion proteins successfully transduced into the nucleus and the cytosol in the cells. The viability of cells treated with paraquat was markedly increased by the transduced fusion proteins. Thus, our results suggest that these peptides should be useful for targeting the specific localization of therapeutic proteins in various human diseases.     

Role of N-terminal hydrophobic region in modulating the subcellular localization and enzyme activity of the bisphosphate nucleotidase from Debaryomyces hansenii

3', 5'-Bisphosphate nucleotidase is a ubiquitous enzyme that converts 3'-phosphoadenosine-5'-phosphate to adenosine-5'-phosphate and inorganic phosphate. These enzymes are highly sensitive to sodium and lithium and, thus, perform a crucial rate-limiting metabolic step during salt stress in yeast. Recently, we have identified a bisphosphate nucleotidase gene (DHAL2) from the halotolerant yeast Debaryomyces hansenii. One of the unique features of Dhal2p is that it contains an N-terminal 54-amino-acid-residue hydrophobic extension. In this study, we have shown that Dhal2p exists as a cytosolic as well as a membrane-bound form and that salt stress markedly influences the accumulation of the latter form in the cell. We have demonstrated that the N-terminal hydrophobic region was necessary for the synthesis of the membrane-bound isoform. It appeared that an alternative translation initiation was the major mechanism for the synthesis of these two forms. Moreover, the two forms exhibit significant differences in their substrate specificity. Unlike the cytosolic form, the membrane-bound form showed very high activity against inositol-1,4-bisphosphate. Thus, the present study for the first time reports the existence of multiple forms of a bisphosphate nucleotidase in any organism.     

Characterization and subcellular localization of debranching enzyme and endoamylase from leaves of sugar beet

Sugar beet leaves (Beta vulgaris L.) contained up to five endoamylases, two exoamylases, and a single debranching enzyme. Four of the endoamylases and the debranching enzyme were present in the chloroplast. The chloroplastic starch-debranching enzyme and an apoplastic endoamylase were copurified from mature leaves of sugar beet by 35 to 50% ammonium sulfate precipitation and chromatography on diethylaminoethyl-Sephacryl, β-cyclodextrin Sepharose 6B, and Sephadex G-150. The debranching enzyme, which was purified to homogeneity, had a molecular mass of 100 kilodaltons and a pH optimum of 5.5. It showed a high activity with pullulan as a substrate, low activity with soluble starch and amylopectin, and no activity with native starch grains isolated from sugar beet leaves. The endoamylase, which was partially purified, had a molecular mass of 43,000 kilodaltons, a pH optimum of 6.5, required calcium for activity and thermal stability, and showed an ability to hydrolyze native starch grains.     

Phospholipase C activity in palate mesenchyme cells: calcium and pH requirements, substrate specificity, and subcellular localization

Primary cultures of mouse embryo palate mesenchyme cells were incubated with [3H]arachidonic acid and [14C]stearic acid in order to radiolabel their lipids. The cells were then washed, collected by centrifugation, and homogenized. Incubation of the homogenates under various conditions revealed that deoxycholate inhibited phospholipase A activity and stimulated a phospholipase C activity in these cells which preferentially degraded phosphatidylinositol (PI) compared to phosphatidylcholine (PC), -ethanolamine (PE), and -serine (PS). Expression of this phospholipase C (E.C. 3.1.4.10) activity was dependent on Ca2+ and had a pH optimum of no more than 7.0-7.5. Centrifugation of the homogenates at 105,000g for 30 min produced a membranous fraction that contained phospholipase C activity with characteristics similar to those of the enzyme found in the supernatant. Such a dual distribution of this enzyme may reflect that mouse embryo palate mesenchyme cells are neural crest in origin.