A procedure for eliminating interferences in the lowry method of protein determination

Rodríguez‐Vico, Felipe; Martínez-Cayuela, Marina; Garcia-Peregrin, E.; Ramirez, H.

doi:10.1016/0003-2697(89)90479-x

Cited by 75 publications

(33 citation statements)

References 17 publications

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“…The crude membrane fraction was washed once with the suspension buffer, sedimented by centrifugation (100,000 ϫ g, 15 min, 4°C), and resuspended in the suspension buffer at a final protein concentration of 10 -20 mg/ml. Microsomal fractions from S. cerevisiae were prepared as described by Fernandez et al (13), and protein was determined by the method of Rodriquez-Vico et al (25) using BCA (Pierce).…”

Section: Construction and Expression Of Hdk And The Mutants In Yeast mentioning

confidence: 99%

Human Dolichol Kinase, a Polytopic Endoplasmic Reticulum Membrane Protein with a Cytoplasmically Oriented CTP-binding Site

Shridas¹,

Waechter²

2006

J. Biol. Chem.

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Dolichol kinase (DK) catalyzes the CTP-dependent phosphorylation of dolichol in the biosynthesis de novo and possibly the recycling of dolichyl monophosphate in yeast and mammals. A cDNA clone from human brain encoding the mammalian homologue, hDKp, of the yeast enzyme has recently been identified. In this study hDK has been overexpressed in Chinese hamster ovary cells and shown to be a polytopic membrane protein localized in the endoplasmic reticulum with an N terminus extended into the lumen and a cytoplasmically oriented C terminus. A conserved sequence, DXXAXXXGXXXGX 8 KKTXEG, found in several enzymes utilizing CTP as substrate including DKs, phytol kinases, and several CDP-diacylglycerol synthetases has been identified, and the possibility that it is part of the CTP-binding domain of hDKp has been investigated. Topological studies indicate that the loop between transmembrane domains (TMD) 11 and TMD12 of hDKp, containing the putative CTP binding domain, faces the cytoplasm. Deletion of the loop between TMD11-12, hDK(⌬459 -474), or mutation of selected conserved residues within the cytoplasmic loop results in either a partial or total loss of activity and significant reductions in the affinity for CTP. In addition, the SEC59 gene in the yeast DK mutant was sequenced, and a G420D substitution was found. Conversion of the corresponding residue Gly-443 in hDKp to aspartic acid resulted in inactivation of the mammalian enzyme. These results extend the information on the topological arrangement of hDKp and indicate that the cytoplasmic loop between TMDs 11-12, containing the critical conserved residues, lysine 470 and lysine 471 in the 470 KKTXEG 475 motif, is part of the CTP-binding site in hDK. Dolichyl monophosphate (Dol-P)2 serves an essential function as a glycosyl carrier lipid in the assembly of N-linked glycoproteins, glycosylphosphatidylinositol anchors, and the Cand O-mannosylation of proteins in the endoplasmic reticulum (ER) of yeast and animal cells (1, 2). The enzymatic transfer of phosphoryl groups from CTP to dolichol catalyzed by microsomal fractions from mammalian cells was first detected more than 20 years ago (3, 4). Developmental changes in dolichol kinase (DK) activity corresponding to an increased capacity for protein N-glycosylation have been reported for sea urchin embryos (5), estrogen-treated chick oviducts (6), Dictyostelium discoideum (7), and pig (8) and rat brain (9, 10). If the enzymatic reduction of the ␣-isoprene unit in dolichol occurs at the free polyprenol level as proposed by Sagami et al. (11), DK would catalyze the final step in the de novo pathway for Dol-P biosynthesis (1).In 1992 Heller et al. (12) identified the SEC59 gene product as a protein essential for the expression of yeast DK. Recently (13) we have identified a cDNA clone from a human brain library that encodes the mammalian homologue of DK (hDKp). The identification of the mammalian homologue of SEC59 was based on its ability to 1) complement the growth defect, 2) increase DK activity and, consequently, Dol-P l...

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Section: Construction and Expression Of Hdk And The Mutants In Yeast mentioning

confidence: 99%

Human Dolichol Kinase, a Polytopic Endoplasmic Reticulum Membrane Protein with a Cytoplasmically Oriented CTP-binding Site

Shridas¹,

Waechter²

2006

J. Biol. Chem.

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“…Analytical Methods-Protein was determined by the method of Rodriquez-Vico et al (25) or Bradford (26) using bovine serum albumin as standard.…”

Section: Preparation Of Sealed Microsomal Vesicles and Estimation Of mentioning

confidence: 99%

The CWH8 Gene Encodes a Dolichyl Pyrophosphate Phosphatase with a Luminally Oriented Active Site in the Endoplasmic Reticulum of Saccharomyces cerevisiae

Fernández¹,

Rush

Toke

et al. 2001

Journal of Biological Chemistry

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Mutations in the CWH8 gene, which encodes an ER transmembrane protein with a phosphate binding pocket in Saccharomyces cerevisiae, result in a deficiency in dolichyl pyrophosphate (Dol-P-P)-linked oligosaccharide intermediate synthesis and protein N-glycosylation (van Berkel, M. A., Rieger, M., te Heesen, S., Ram, A. F., van den Ende, H., Aebi, M., and Klis, F. M. (1999) Glycobiology 9, 243-253). Genetic, enzymological, and topological approaches were taken to investigate the potential role of Cwh8p in Dol-P-P/Dol-P metabolism. Overexpression of Cwh8p in the yeast double mutant strain, lacking LPP1/ DPP1, resulted in an impressive increase in Dol-P-P phosphatase activity, a relatively small increase in Dol-P phosphatase activity, but no change in phosphatidate (PA) phosphatase activity in microsomal fractions. The Dol-P-P phosphatase encoded by CWH8 is optimally active in the presence of 0.5% octyl glucoside and relatively unstable in Triton X-100, distinguishing this activity from the lipid phosphatases encoded by LPP1 and DPP1. Stoichiometric amounts of P i and Dol-P are formed during the enzymatic reaction indicating that Cwh8p cleaves the anhydride linkage in Dol-P-P. Membrane fractions from Sf-9 cells expressing Cwh8p contained a 30-fold higher level of Dol-P-P phosphatase activity, a slight increase in Dol-P phosphatase activity, but no increase in PA phosphatase relative to controls. This is the first report of a lipid phosphatase that hydrolyzes Dol-P-P/Dol-P but not PA. In accord with this enzymatic function, Dol-P-P accumulated in cells lacking the Dol-P-P phosphatase. Topological studies using different approaches indicate that Cwh8p is a transmembrane protein with a luminally oriented active site. The specificity, subcellular location, and topological orientation of this novel enzyme are consistent with a role in the re-utilization of the glycosyl carrier lipid for additional rounds of lipid intermediate biosynthesis after its release during protein N-glycosylation reactions.Although the biosynthesis of dolichyl-linked saccharide intermediates is initiated on the cytoplasmic side of the endoplasmic reticulum (ER), 1 dolichyl pyrophosphate (Dol-P-P) and several dolichyl monophosphate (Dol-P) molecules are released on the luminal surface during protein N-glycosylation reactions, glycosylphosphatidylinositol anchor synthesis, C-and O-mannosylation of proteins (see reviews in Refs. 1-3). For the dolichyl moiety of Dol-P-P to be re-utilized for additional rounds of lipid intermediate biosynthesis, it must be converted to Dol-P before or after returning to the cytoplasmic leaflet of the ER.There have been many reports of Dol-P-P phosphatase activities in microsomal fractions from mammalian tissues (see reports cited in Ref.3). However, the subcellular location and topological arrangement of the active sites of these enzymes have not been definitively established. A Dol-P-P phosphatase activity reported in brain (4) is curiously enriched in Golgi fractions and is an unlikely candidate to be involved in the ...

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“…The protein concentration of samples containing octylglucoside were determined by a modification of a recently published protocol described by Rodriguez-Vico et al (18). For samples containing OG, up to 0.5 mL of aqueous solutions were diluted with 10 mL of 3:2 (v:v) hexane:isopropanol.…”

Section: Protein Determinationmentioning

confidence: 99%

N-Cyclo-N′-(4-Dimethylamino-α-Naphthyl)Carbodiimide Inhibits Membrane-Bound and Partially Purified Tonoplast ATPase from Maize Roots

Bräuer

Tu²

1991

Plant Physiol.

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Certain carboxylic acid groups within the primary structure of proton translocating proteins are thought to be involved in the proton pathway. In this report, the effects of a lipophilic carboxylic acid reactive reagent, N-cyclo-N'(4-dimethylamino-a-naphthyl)carbodiimide (NCD-4), on the two types of proton pumps in maize (Zea mays L.) root microsomes were investigated. NCD-4 was found to inhibit the vacuolar-type H+-ATPase in microsomal preparations; however, the plasma membrane-type H+-ATPase was unaffected. The H+-ATPase in highly purified tonoplast vesicles was also inhibited by NCD-4. Inhibition was dependent on the concentration and length of exposure to the reagent. However, there was little, if any, increase in the fluorescence of treated vesicles, indicating few carboxylic acid residues were reacting. Inhibition of the tonoplast H+-ATPase by NCD-4 was examined further with a partially purified preparation. The partially purified H+-ATPase also showed sensitivity to the NCD-4, supporting the hypothesis that this carboxylic acid reagent is an inhibitor of the tonoplast ATPase from maize roots.

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A procedure for eliminating interferences in the lowry method of protein determination

Cited by 75 publications

References 17 publications

Human Dolichol Kinase, a Polytopic Endoplasmic Reticulum Membrane Protein with a Cytoplasmically Oriented CTP-binding Site

Human Dolichol Kinase, a Polytopic Endoplasmic Reticulum Membrane Protein with a Cytoplasmically Oriented CTP-binding Site

The CWH8 Gene Encodes a Dolichyl Pyrophosphate Phosphatase with a Luminally Oriented Active Site in the Endoplasmic Reticulum of Saccharomyces cerevisiae

N-Cyclo-N′-(4-Dimethylamino-α-Naphthyl)Carbodiimide Inhibits Membrane-Bound and Partially Purified Tonoplast ATPase from Maize Roots

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