scholarly journals The microsomal epoxide hydrolase has a single membrane signal anchor sequence which is dispensable for the catalytic activity of this protein

1994 ◽  
Vol 303 (3) ◽  
pp. 967-972 ◽  
Author(s):  
T Friedberg ◽  
B Löllmann ◽  
R Becker ◽  
R Holler ◽  
F Oesch
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Fusion Protein ◽  
Epoxide Hydrolase ◽  
Signal Sequence ◽  
Translation System ◽  
Amino Acid Residues ◽  
Membrane Anchor ◽  
Free Translation ◽  
A Cell

The microsomal epoxide hydrolase (mEH) catalyses the hydrolysis of reactive epoxides which are formed by the action of cytochromes P-450 from xenobiotics. In addition it has been suggested that mEH might mediate the transport of bile acids. For the mEH it has been shown that it is co-translationally inserted into the endoplasmic reticulum. Here we demonstrate that the N-terminal 20 amino acid residues of this protein serve as its single membrane anchor signal sequence and that the function of this sequence can also be supplied by a cytochrome P-450 (CYP2B1) anchor signal sequence. The evidence supporting this conclusion is as follows: (i) the rat mEH and a CYP2B1-mEH fusion protein, in which the CYP2B1 membrane anchor signal sequence replaced the N-terminal 20 amino acid residues of mEH, was co-translationally inserted into dog pancreas microsomes in a cell-free translation system, whereas a truncated epoxide hydrolase with a deletion of the 20 N-terminal amino acid residues was not co-translationally inserted. (ii) The mEH and the CYP2B1-mEH fusion protein, but not the truncated epoxide hydrolase, were anchored in microsomes in a cell-free translation system and in membrane fractions derived from fibroblasts which expressed these proteins heterologously. These fibroblasts were also used to evaluate the significance of the mEH membrane anchor for the catalytic activity of mEH. The mEH, the truncated mEH and the CYP-EH fusion protein were found to be enzymically active. This result shows that the membrane anchor signal sequence of mEH is dispensable for the catalytic activity of this protein. However, truncated mEH was only expressed at low levels, which might indicate that this protein is unstable.

scholarly journals Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors

1997 ◽  
Vol 323 (2) ◽  
pp. 415-419 ◽  
Author(s):  
Lakshmi KASTURI ◽  
Hegang CHEN ◽  
Susan H. SHAKIN-ESHLEMAN
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Free Translation ◽  
A Cell ◽  
And Function ◽  
A Site ◽  
The Impact

N-linked glycosylation can profoundly affect protein expression and function. N-linked glycosylation usually occurs at the sequon Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro. However, many Asn-Xaa-Ser/Thr sequons are glycosylated inefficiently or not at all for reasons that are poorly understood. We have used a site-directed mutagenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino acid residues in sequons influence core-glycosylation efficiency. We recently demonstrated that certain Xaa amino acids inhibit core glycosylation of the sequon, Asn37-Xaa-Ser, in rabies virus glycoprotein (RGP). Here we examine the impact of different Xaa residues on core-glycosylation efficiency when the Ser residue in this sequon is replaced with Thr. The core-glycosylation efficiencies of RGP variants with different Asn37-Xaa-Ser/Thr sequons were compared by using a cell-free translation/glycosylation system. Using this approach we confirm that four Asn-Xaa-Ser sequons are poor oligosaccharide acceptors: Asn-Trp-Ser, Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr sequons are efficiently glycosylated, even when Xaa = Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-Xaa-Thr sequons in other glycoproteins confirms that sequons with Xaa = Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these sequons are unlikely to serve as glycosylation sites when introduced into proteins by site-directed mutagenesis.

scholarly journals Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes

1983 ◽  
Vol 212 (1) ◽  
pp. 161-165 ◽  
Author(s):  
E M Danielsen ◽  
O Norén ◽  
H Sjöström
Keyword(s):  
Signal Sequence ◽  
Aminopeptidase N ◽  
Soluble Form ◽  
Translation System ◽  
Translation Product ◽  
Free Translation ◽  
Microsomal Membranes ◽  
A Cell ◽  
Membrane Bound ◽  
Small Intestinal

The biosynthesis of small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane-bound rather than a soluble form, indicating that synthesis of the enzyme takes place on ribosomes attached to the rough endoplasmic reticulum. The microsomal fractions process the Mr-115 000 polypeptide, which is the primary translation product of aminopeptidase N, to a polypeptide of Mr 140 000. This was found to be sensitive to the action of endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), showing that aminopeptidase N undergoes transmembrane glycosylation during synthesis. The position of the signal sequence in aminopeptidase N was determined by a synchronized translation experiment. It was found that microsomal fractions should be added before about 25% of the polypeptide was synthesized to ensure processing to the high-mannose glycosylated form. This suggests that the signal sequence is situated in the N-terminal part of the aminopeptidase N. The size of the cell-free translation product in the absence of microsomal fractions was found to be similar to that on one of the forms of the enzyme obtained from tunicamycin-treated organ-cultured intestinal explants.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

scholarly journals Identification of amino acid residues essential for catalytic activity of gentisate 1,2-dioxygenase fromPseudomonas alcaligenesNCIB 9867

2001 ◽  
Vol 204 (1) ◽  
pp. 141-146 ◽  
Author(s):  
Chi Hung Chua ◽  
Yongmei Feng ◽  
Chew Chieng Yeo ◽  
Hoon Eng Khoo ◽  
Chit Laa Poh
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Amino Acid Residues

Analysis of essential amino acid residues for catalytic activity of cis-epoxysuccinate hydrolase from Bordetella sp. BK-52

2013 ◽  
Vol 98 (4) ◽  
pp. 1641-1649 ◽  
Author(s):  
Wenna Bao ◽  
Haifeng Pan ◽  
Zhenhong Zhang ◽  
Yongqing Cheng ◽  
Zhipeng Xie ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Essential Amino Acid ◽  
Amino Acid Residues

scholarly journals The 3' untranslated region of satellite tobacco necrosis virus RNA stimulates translation in vitro.

1993 ◽  
Vol 13 (6) ◽  
pp. 3340-3349 ◽  
Author(s):  
X Danthinne ◽  
J Seurinck ◽  
F Meulewaeter ◽  
M Van Montagu ◽  
M Cornelissen
Keyword(s):  
Tobacco Necrosis Virus ◽  
Translation System ◽  
Coding Region ◽  
Necrosis Virus ◽  
Untranslated Sequence ◽  
Virus Rna ◽  
Free Translation ◽  
Order Of Magnitude ◽  
A Cell

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA. Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornavirus box A, which is involved in cap-independent translation but which is located upstream of the coding region.

scholarly journals A murine fer testis-specific transcript (ferT) encodes a truncated Fer protein.

1990 ◽  
Vol 10 (1) ◽  
pp. 146-153 ◽  
Author(s):  
K Fischman ◽  
J C Edman ◽  
G M Shackleford ◽  
J A Turner ◽  
W J Rutter ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Translation System ◽  
Appearance Time ◽  
Acid Protein ◽  
Mouse Tissues ◽  
Free Translation ◽  
Alternatively Spliced ◽  
A Cell ◽  
Testis Cdna ◽  
Carboxy Terminal

A cDNA for a potential tyrosine kinase-encoding mRNA was isolated from a mouse testis cDNA library. In a survey of eight mouse tissues, a transcript of 2.4 kilobases restricted to testis tissue was found. The mRNA encodes a 453-amino-acid protein of 51,383 daltons, the smallest tyrosine kinase protein ever described. RNA synthesized from the cDNA template directs the synthesis of a 51,000-Mr protein in a cell-free translation system. The carboxy-terminal 409 amino acids are 98 and 90% identical to the carboxy halves of the rat and human Fer proteins, respectively. This suggests that the cDNA represents an alternatively spliced testis-specific fer mRNA and is therefore termed by us ferT. On the basis of the appearance time of the fer mRNA in the testis of maturing neonatal mice, we speculate on the role played by this protein in the development of this organ.

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