scholarly journals Rabbit DNase I: purification from urine, immunological and proteochemical characterization, nucleotide sequence, expression in tissues, relationships with other mammalian DNases I and phylogenetic analysis

1997 ◽  
Vol 325 (2) ◽  
pp. 465-473 ◽  
Author(s):  
Toshihiro YASUDA ◽  
Haruo TAKESHITA ◽  
Tamiko NAKAJIMA ◽  
Osamu HOSOMI ◽  
Yoshimitsu NAKASHIMA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Parotid Gland ◽  
Signal Sequence ◽  
Dnase I ◽  
Glycosylation Site ◽  
Enzymic Activity ◽  
Actin Binding ◽  
Immunological Properties ◽  
Apparent Homogeneity ◽  
I Gene

DNase I from rabbit urine was purified approx. 3600-fold to apparent homogeneity with a 41% yield by affinity chromatography utilizing DNA–cellulose; the purity of the final preparation was assessed by SDS/PAGE, lack of contamination by other nucleases and production of a monospecific antibody against the enzyme. Although the proteochemical and enzymological properties of the purified enzyme resembled those of other mammalian DNases I, the enzymic activity of rabbit DNase I was less efficiently inhibited by monomeric actin than was that of human DNase I, probably due to substitution of an amino acid residue involved in actin binding (Tyr-65 to Phe). The effects of specific antibodies to human, rabbit and rat DNases I on the activities of the corresponding purified enzymes revealed that human DNase I lies between the rat and rabbit enzymes with regard to its immunological properties. An 1158 bp full-length cDNA encoding rabbit DNase I was constructed from the total RNA of rabbit pancreas using a combination of reverse transcriptase-PCR and rapid amplification of cDNA ends, followed by sequencing. This identified a 17- or 21-amino-acid signal sequence, with the mature enzyme containing 260 amino acids and a single N-glycosylation site at Asn-18. The amino acid sequence deduced from the cDNA sequence exactly matched that determined proteochemically from the purified enzyme up to residue 20. A systematic survey of DNase I distribution as measured by both enzymic activity and DNase I gene transcripts in 12 rabbit tissues showed the pancreas and parotid gland to produce equivalent levels, higher than those in other tissues. Enzymic activity and DNase I gene expression levels in each tissue correlated well. The results of phylogenetic and sequence identity analysis, immunological properties and tissue-distribution patterns of DNase I indicated a closer relationship between the rabbit and human enzymes than for other mammalian DNases I. Furthermore, differences between the enzymic activities expressed in mammalian parotid gland and pancreas suggest that the distribution of DNase I in mammalian tissue is species-specific.

scholarly journals Rat mammary-gland transferrin: nucleotide sequence, phylogenetic analysis and glycan structure

1995 ◽  
Vol 307 (1) ◽  
pp. 47-55 ◽  
Author(s):  
H Escrivá ◽  
A Pierce ◽  
B Coddeville ◽  
F González ◽  
M Benaissa ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Mammary Gland ◽  
Sialic Acid ◽  
Amino Acid ◽  
Signal Sequence ◽  
Glycosylation Site ◽  
Glycan Structure ◽  
Sialic Acid Content ◽  
Native Page ◽  
Rat Mammary Gland

The complete cDNA for rat mammary-gland transferrin (Tf) has been sequenced and also the native protein isolated from milk in order to analyse the structure of the main glycan variants present. A lactating-rat mammary-gland cDNA library in lambda gt10 was screened with a partial cDNA copy of rat liver Tf and subsequently rescreened with 5′ fragments of the longest clones. This produced a 2275 bp insert coding for an open reading frame of 695 amino acid residues. This includes a 19-amino acid signal sequence and the mature protein containing 676 amino acids and one N-glycosylation site in the C-terminal domain at residue 490. Phylogenetic analysis was carried out using 14 translated Tf nucleotide sequences, and the derived evolutionary tree shows that at least three gene duplication events have occurred during Tf evolution, one of which generated the N- and C-terminal domains and occurred before separation of arthropods and chordates. The two halves of human melanotransferrin are more similar to each other than to any other sequence, which contrasts with the pattern shown by the remaining sequences. Native rat milk Tf is separated into four bands on native PAGE that differ only in their sialic acid content: one biantennary glycan is present containing either no sialic acid residues or up to three. The complete structures of the two major variants were determined by methylation, m.s. and 400 MHz 1H-n.m.r. spectroscopy. They contain either one or two neuraminic acid residues (alpha 2-->6)-linked to galactose in conventional biantennary N-acetyl-lactosamine-type glycans. Most contain fucose (alpha 1-->6)-linked to the terminal non-reducing N-acetylglucosamine.

scholarly journals Molecular basis of N-acetylglucosaminyltransferase I deficiency in Arabidopsis thaliana plants lacking complex N-glycans

2005 ◽  
Vol 387 (2) ◽  
pp. 385-391 ◽  
Author(s):  
Richard STRASSER ◽  
Johannes STADLMANN ◽  
Barbara SVOBODA ◽  
Friedrich ALTMANN ◽  
Josef GLÖSSL ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Amino Acid ◽  
Heterologous Expression ◽  
Molecular Cloning ◽  
Molecular Basis ◽  
Insect Cells ◽  
Glycosylation Site ◽  
Enzymic Activity ◽  
Critical Amino Acid ◽  
High Mannose

GnTI (N-acetylglucosaminyltransferase I) is a Golgi-resident enzyme essential for the processing of high-mannose to hybrid and complex N-glycans. The Arabidopsis thaliana cgl mutant lacks GnTI activity and as a consequence accumulates oligomannosidic structures. Molecular cloning of cgl GnTI cDNA revealed a point mutation, which causes a critical amino acid substitution (Asp144→Asn), thereby creating an additional N-glycosylation site. Heterologous expression of cgl GnTI in insect cells confirmed its lack of activity and the use of the N-glycosylation site. Remarkably, introduction of the Asp144→Asn mutation into rabbit GnTI, which does not result in the formation of a new N-glycosylation site, led to a protein with strongly reduced, but still detectable enzymic activity. Expression of Asn144 rabbit GnTI in cgl plants could partially restore complex N-glycan formation. These results indicate that the complete deficiency of GnTI activity in cgl plants is mainly due to the additional N-glycan, which appears to interfere with the proper folding of the enzyme.

The Influence of Carbohydrate Structure on the Clearance of Recombinant Tissue-Type Plasminogen Activator

1988 ◽  
Vol 60 (02) ◽  
pp. 255-261 ◽  
Author(s):  
A Hotchkiss ◽  
C J Refino ◽  
C K Leonard ◽  
J V O'Connor ◽  
C Crowley ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Sodium Periodate ◽  
Glycosylation Site ◽  
Tissue Type ◽  
Carbohydrate Structure ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
High Mannose ◽  
Oligosaccharide Structures

SummaryModification of the carbohydrate structures of recombinant tissue-type plasminogen activator (rt-PA) can increase or decrease its rate of clearance in rabbits. When rt-PA was treated with sodium periodate to oxidize carbohydrate residues, the rate of clearance was decreased from 9.6 ± 1.9 ml min−1 kg−1 to 3.5 ± 0.6 ml min−1 kg−1 (mean ± SD, n = 5). A similar change in the clearance of rt-PA was introduced by the use of endo-β-N-acetyl- glucosaminidase H (Endo-H), which selectively removes high mannose asparagine-linked oligosaccharides; the clearance of Endo-H-treated rt-PA was 5.0 ± 0.5 ml min−1 kg−1. A mutant of rt-PA was produced with an amino acid substitution at position 117 (Asn replaced with Gin) to remove a potential glycosylation site that normally contains a high mannose structure. The clearance of this material was also decreased, similar to the periodate and Endo-H-treated rt-PA. Conversely, when rt-PA was produced in the CHO 15B cell line, which can produce only high mannose oligosaccharide structures on glycoproteins, the clearance was increased by a factor of 1.8. These results demonstrate that the removal of rt-PA from the blood depends significantly upon the nature of its oligosaccharide structures.

scholarly journals Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa

Genome ◽  
1991 ◽  
Vol 34 (4) ◽  
pp. 644-651 ◽  
Author(s):  
Kenneth Koo ◽  
W. Dorsey Stuart
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Neurospora Crassa ◽  
Rna Binding ◽  
Signal Sequence ◽  
Average Length ◽  
Open Reading Frame ◽  
Mrna Transcript ◽  
S1 Nuclease ◽  
Reading Frame

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential α-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.Key words: sequence, membranes, ribonucleoprotein.

scholarly journals SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (5) ◽  
pp. 2154-2164 ◽  
Author(s):  
D J DeMarini ◽  
M Winey ◽  
D Ursic ◽  
F Webb ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Gene Product ◽  
Signal Sequence ◽  
Null Alleles ◽  
Nucleoside Triphosphate ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

scholarly journals Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

2001 ◽  
Vol 183 (6) ◽  
pp. 1954-1960 ◽  
Author(s):  
Grit Zarnt ◽  
Thomas Schräder ◽  
Jan R. Andreesen
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Tertiary Structure ◽  
Ralstonia Eutropha ◽  
Signal Sequence ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Gene Encoding

ABSTRACT The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparentk cat/Km andK i values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k cat/Km value of 788 × 104 M−1 s−1. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.

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