Involvement of stearoyl-CoA desaturase in the reduction of amidoxime prodrugs.

1. This study investigates the enzymatic reduction of N-hydroxylated amidines by porcine adipose tissue and the possible involvement of stearoyl-CoA desaturase (SCD). 2.

Appropriate function of 11beta-hydroxysteroid dehydrogenase type 1 in the endoplasmic reticulum lumen is dependent on its N-terminal region sharing similar topological determinants with 50-kDa esterase.

By interconverting glucocorticoids, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) exerts an important pre-receptor function and is currently considered a promising therapeutic target. In addition, 11beta-HSD1 plays a potential role in 7-ketocholesterol metabolism. Here we investigated the role of the N-terminal region on enzymatic activity and addressed the relevance of 11beta-HSD1 orientation into the endoplasmic reticulum (ER) lumen.

Regulation of stearoyl-CoA desaturase-1 after central and peripheral nerve lesions.

Background: Interruption of mature axons activates a cascade of events in neuronal cell bodies which leads to various outcomes from functional regeneration in the PNS to the failure of any significant regeneration in the CNS. One factor which seems to play an important role in the molecular programs after axotomy is the stearoyl Coenzyme A-desaturase-1 (SCD-1). This enzyme is needed for the conversion of stearate into oleate.

A plasminogen-like protein selectively degrades stearoyl-CoA desaturase in liver microsomes.

Stearoyl-CoA desaturase (SCD) is an integral membrane protein of the endoplasmic reticulum that is rapidly and selectively degraded when isolated liver microsomes are incubated at 37 degrees C. We previously reported the purification of a 90-kDa microsomal protein with SCD protease activity and characterized the inhibitor sensitivity of the protease. Here we show that the 90-kDa protein is a microsomal form of plasminogen (Pg) and that the purified SCD protease contains a spectrum of plasmin-like derivatives.

A microsomal endopeptidase from liver that preferentially degrades stearoyl-CoA desaturase.

A protease was purified some 700-fold from rat liver microsomes by a combination of differential detergent solubilization, hydroxyapatite column chromatography, and gel filtration. The protease exhibits substrate selectivity for stearoyl-CoA desaturase (SCD). The purified protease rapidly degraded SCD while other microsomal proteins including cytochrome b(5) and 11beta-hydroxysteroid dehydrogenase were degraded slowly or not at all.

Stearoyl-CoA desaturase, a short-lived protein of endoplasmic reticulum with multiple control mechanisms.

Stearoyl-CoA desaturase (SCD) is a short-lived, polytopic membrane-bound non-heme iron enzyme localized primarily in the endoplasmic reticulum. SCD is required for the biosynthesis of monounsaturated fatty acids, and plays a key role in hepatic synthesis of triglycerides and very-low-density lipoproteins. The intracellular concentration of SCD fluctuates in a wide range in response to complex and often competing hormonal and dietary factors.

Selective mutagenesis of lysyl residues leads to a stable and active form of delta 9 stearoyl-CoA desaturase.

Stearoyl-CoA desaturase (SCD) is a short-lived integral membrane protein of the endoplasmic reticulum (ER) that catalyzes the insertion of a double bond in the delta 9 position of saturated fatty acids. Its expression has been difficult in heterologous systems. In this study, recombinant adenovirus vector was used to express both wild-type (wt) and engineered forms of rat SCD in human transformed kidney cells.

Induction of stearoyl CoA desaturase is associated with high-level induction of emerin RNA.

The genes encoding enzymes involved in fatty acid metabolism are regulated by sterols. Stearoyl CoA desaturase, a key enzyme in the synthesis of unsaturated fatty acyl-CoAs is transcriptionally regulated by fat-free diet and sterols. To identify other genes that are induced in rat liver by fat-free diet we performed an in vivo gene expression profile analysis using DNA microarrays.

The N terminus of microsomal delta 9 stearoyl-CoA desaturase contains the sequence determinant for its rapid degradation.

Stearoyl-CoA desaturase (SCD) is a key regulator of membrane fluidity, turns over rapidly, and represents a model for selective degradation of short-lived proteins of the endoplasmic reticulum (ER). The mechanism whereby specific ER proteins are targeted for degradation in the midst of stable proteins coexisting in the same membrane is unknown. To investigate the intracellular fate of SCD and to identify the determinants involved in the rapid turnover of SCD, we created chimeras of SCD tagged at the C terminus with the green fluorescent protein (GFP).

Targeting proteins to the lumen of endoplasmic reticulum using N-terminal domains of 11beta-hydroxysteroid dehydrogenase and the 50-kDa esterase.

Previous studies identified two intrinsic endoplasmic reticulum (ER) proteins, 11beta-hydroxysteroid dehydrogenase, isozyme 1 (11beta-HSD) and the 50-kDa esterase (E3), sharing some amino acid sequence motifs in their N-terminal transmembrane (TM) domains. Both are type II membrane proteins with the C terminus projecting into the lumen of the ER. This finding implied that the N-terminal TM domains of 11beta-HSD and E3 may constitute a lumenal targeting signal (LTS).

Isolation and complete covalent structure of liver microsomal paraoxonase.

Paraoxonase (PON1) is a serum esterase exclusively associated with high-density lipoproteins; it might confer protection against coronary artery disease by destroying pro-inflammatory oxidized lipids in oxidized low-density lipoproteins. Here I show that rabbit liver microsomes contain a PON analogue (MsPON) and report the isolation and complete covalent structure of MsPON. In detergent-solubilized microsomes, MsPON co-purifies with the microsomal triacylglycerol transfer protein (MTP) complex.

Degradation of stearoyl-coenzyme A desaturase: endoproteolytic cleavage by an integral membrane protease.

Stearoyl-coenzyme A desaturase (SCD) is a key regulator of membrane fluidity, turns over rapidly, and represents a prototype for selective degradation of resident proteins of the endoplasmic reticulum. Using detergent-solubilized, desaturase-induced rat liver microsomes we have characterized a protease that degrades SCD. Degradation of SCD in vitro is highly selective, has a half-life of 3-4 h, and generates a 20-kDa C-terminal fragment of SCD.

Determination of lumenal orientation of microsomal 50-kDa esterase/N-deacetylase.

The amino acid arrangements responsible for the insertion and specific lumenal orientation of proteins having an uncleaved signal-peptide-like anchor are poorly understood. A 50-kDa protein having a hydrophobic N-terminus similar to the lumenal glycoprotein 11beta-hydroxysteroid dehydrogenase [Ozols, J. (1995) J.

Interleukin-2 induces N-glycosylation in T-cells: characterization of human lymphocyte oligosaccharyltransferase.

We have investigated the enzyme mediating N-glycosylation in "resting" and activated lymphocytes. Normal peripheral blood lymphocytes (PBLs) were found to have low activity for glycosylation of a synthetic glycan acceptor peptide. N-glycosylation activity increased 10-fold after mitogen activation of PBLs.

Isolation and structural analysis of microsomal membrane proteins.

Investigation of the endoplasmic reticulum requires a methodological background in the solubilization, purification, and structural analysis of membrane proteins. The experience of one laboratory with microsomal proteins over 30 years is summarized in this review. We focus on the isolation and structure of the major proteins of rabbit liver microsomes.

Degradation of hepatic stearyl CoA delta 9-desaturase.

delta 9-Desaturase is a key enzyme in the synthesis of desaturated fatty acyl-CoAs. Desaturase is an integral membrane protein induced in the endoplasmic reticulum by dietary manipulations and then rapidly degraded. The proteolytic machinery that specifically degrades desaturase and other short-lived proteins in the endoplasmic reticulum has not been identified.

Posttranslational modification of tubulin by palmitoylation: II. Identification of sites of palmitoylation.

As shown in the companion article, tubulin is posttranslationally modified in vivo by palmitoylation. Our goal in this study was to identify the palmitoylation sites by protein structure analysis. To obtain quantities of palmitoylated tubulin required for this analysis, a cell-free system for enzymatic [3H]palmitoylation was developed and characterized in our companion article.

Xenopus lipovitellin 1 is a Zn(2+)- and Cd(2+)-binding protein.

This report discusses the identification of a Zn(2+)- and Cd(2+)-binding protein of Xenopus laevis that is abundant in vitellogenic oocytes and in embryos from fertilization to stage 46. Oocyte or embryo homogenates were fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with 65Zn2+ or 109Cd2+. The resulting autoradiograms showed binding of both radionuclides to a protein, designated pCdZn.

Induction of antigen-specific cytolytic T cells in situ in human melanoma by immunization with synthetic peptide-pulsed autologous antigen presenting cells.

Human melanoma cells can process the MAGE-1 gene product and present the processed nonapeptide EADPTGHSY on their major histocompatibility complex class I molecules, HLA-A1, as a determinant for cytolytic T lymphocytes (CTLs). Considering that autologous antigen presenting cells (APCs) pulsed with the synthetic nonapeptide might, therefore, be immunogenic, melanoma patients whose tumor cells express the MAGE-1 gene and who are HLA-A1+ were immunized with a vaccine made of cultured autologous APCs pulsed with the synthetic nonapeptide. Analyses of the nature of the in vivo host immune response to the vaccine revealed that the peptide-pulsed APCs are capable of inducing autologous melanoma-reactive and the nonapeptide-specific CTLs in situ at the immunization site and at distant metastatic disease sites.

Human oligosaccharyltransferase: isolation, characterization, and the complete amino acid sequence of 50-kDa subunit.

Oligosaccharyltransferase (OT) catalyzes the glycosylation of asparagine residues in nascent polypeptides in the endoplasmic reticulum. In a previous communication we reported the purification and characterization of this enzyme from chicken oviduct. Here we describe the purification and sequence analysis of OT from human liver microsomes.

Purification and characterization of hepatic oligosaccharyltransferase.

Oligosaccharyltransferase transfers a preformed oligosaccharide from a dolichol carrier molecule to specific asparaginyl residues of proteins synthesized in the endoplasmic reticulum. We have isolated a protein complex with this activity from chicken liver microsomes with 850 fold purification. The purification procedure involved removal of peripheral and lumenal proteins, solubilization of the membranes by non-ionic detergent and glycerol gradient centrifugation.

The 40 kDa 63Ni(2+)-binding protein (pNiXc) on western blots of Xenopus laevis oocytes and embryos is the monomer of fructose-1,6-bisphosphate aldolase A.

A Ni(2+)-binding protein (pNiXc, 40 kDa), present in Xenopus laevis oocytes and embryos, was isolated from mature oocytes by chromatography on DEAE-cellulose and cellulose phosphate, followed by FPLC on Ni-iminodiacetate-Agarose, or reverse-phase HPLC on a C-4 column. Size-exclusion HPLC showed that intact pNiXc is approximately 155 kDa, consistent with tetrameric structure. After cleavage with Lys-C proteinase or cyanogen bromide, six peptides were separated by HPLC and sequenced by Edman degradation, providing sequence data for 83 residues.

Lumenal orientation and post-translational modifications of the liver microsomal 11 beta-hydroxysteroid dehydrogenase.

The topology and post-translational modifications of microsomal 11 beta-hydroxysteroid dehydrogenase (11 beta-DH) was investigated using the approaches of protein structure analysis. Sequence analysis of peptides generated by chemical and enzymatic cleavages revealed that carbohydrate is attached at Asn-122, -161, and -206. Enzymatic deglycosylation reactions of the protein identified the attached glycans as high mannose carbohydrates, implying that the bulk of the protein molecule is oriented on the lumenal side of the endoplasmic membrane.

Lipovitellin 2 beta is the 31 kD Ni(2+)-binding protein (pNiXb) in Xenopus oocytes and embryos.

An Ni(2+)-binding protein (pNiXb, 31 kD) present in mature Xenopus laevis oocytes and in embryos from fertilization in N/F stage 42, was isolated and characterized. After oocytes or embryos were fractionated by PAGE, electroblotted onto nitrocellulose, and probed with 63Ni2+, pNiXb was detected by autoradiography. pNiXb, a yolk protein located in the embryonic gut, was purified from yolk platelets by ammonium sulfate precipitation, delipidation, gel filtration chromatography, and HPLC analysis.