scholarly journals Purification and properties of the uroporphyrinogen decarboxylase from Rhodobacter sphaeroides

1993 ◽  
Vol 293 (3) ◽  
pp. 703-712 ◽  
Author(s):  
R M Jones ◽  
P M Jordan
Keyword(s):  
Amino Acids ◽  
Acetic Acid ◽  
Rhodobacter Sphaeroides ◽  
Uroporphyrinogen Decarboxylase ◽  
Ph Optimum ◽  
Purification And Properties ◽  
N Terminus ◽  
Arginine Residues ◽  
High Degree ◽  
Degree Of Similarity

Uroporphyrinogen decarboxylase (EC 4.1.1.37) was purified 600-fold from Rhodobacter sphaeroides grown anaerobically in the light. The enzyme, under both denaturing and non-denaturing conditions, is a monomer of M(r) 41,000. The Km values are 1.8 microM and 6.0 microM for the conversion of uroporphyrinogen I and III to coproporphyrinogen I and III respectively. The enzyme is susceptible to inhibition by both uroporphyrinogen and uroporphyrin. The pH optimum is 6.8 and the isoelectric point is 4.4. The importance of cysteine and arginine residues is implicated from studies with inhibitors. The sequence of the first 29 amino acids of the N-terminus shows a high degree of similarity to the primary structures of other uroporphyrinogen decarboxylases. Studies on the order of decarboxylation of the four acetic acid side chains of uroporphyrinogen III suggest that at high substrate levels a random route is preferred.

Glutamate Dehydrogenase from Pea Roots: Purification and Properties of the Enzyme

1971 ◽  
Vol 49 (1) ◽  
pp. 127-138 ◽  
Author(s):  
E. Pahlich ◽  
K. W. Joy
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Glutamate Dehydrogenase ◽  
Constant Ratio ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Activity Ratios ◽  
Single Protein ◽  
Michaelis Constants ◽  
Pea Roots

Glutamate dehydrogenase (L-glutamate: NAD+ oxidoreductase (deaminating), EC 1.4.1.2) has been purified 1250-fold from pea roots. The preparation contains only a single protein, and the molecular weight was estimated to be 208 000 ± 10 000. The enzyme shows NADH (aminating) and NAD+ (deaminating) activities, but the ratio of these activities is not constant and can be changed experimentally. NADPH activity is also present and shows a relatively constant ratio to NAD+ activity. EDTA inhibits NADH activity in intermediate concentrations, but reactivates at higher concentrations. NAD+ (and NADPH) activity is only slightly changed by EDTA. The effects of dioxane and the coenzymes on the enzyme are also reported. Mechanisms which could explain the different activity ratios, in terms of two interconvertible enzyme forms, are discussed.The pH optimum for NADH and NAD+ activities is about pH 8.0. Michaelis constants were found to be: α-ketoglutarate, 3.3 × 10−3 M; ammonium (sulfate), 3.8 × 10−2 M; glutamate, 7.3 × 10−3 M; NADH, 8.6 × 10−4 M; NAD+, 6.5 × 10−4 M. The enzyme is highly specific for the substrates glutamate and α-ketoglutarate, showing no alanine or aspartate dehydrogenase activity, and no deamination with a range of amino acids.

scholarly journals Structural studies on wheat (Triticum aestivum) proteins lacking phenylalanine and histidine residues

1975 ◽  
Vol 149 (3) ◽  
pp. 725-732 ◽  
Author(s):  
D G Redman
Keyword(s):  
Amino Acids ◽  
Triticum Aestivum ◽  
Amino Acid ◽  
Structural Studies ◽  
Amino Acid Residues ◽  
Sequencing Studies ◽  
Arginine Residues ◽  
Terminal Amino ◽  
Methionine Residues ◽  
High Degree

1. Three very similar proteins, each of approx. 120 amino acid residues but lacking phenylalanine and histidine, were isolated from wheat (Triticum aestivum) flour in sufficient quantities for further structural studies. 2. Each protein, after reduction and carboxymethylation, was cleaved at the three methionine residues with CNBr to give four major peptides, which were isolated. These peptides are suitable for future sequencing studies, as the sums of their amino acid compositions are in good agreement with those of the whole proteins. 3. The N- and C-terminal peptides were identified. 4. Evidence from amino acid analyses, N-terminal amino acids and electrophoretic mobilities of the peptides suggests a high degree of homology between the proteins. Definite differences in C-terminal amino acids and the number of glycine, alanine and arginine residues were found in the C-terminal peptides.

scholarly journals A Second and Unusual pucBA Operon of Rhodobacter sphaeroides 2.4.1: Genetics and Function of the Encoded Polypeptides

2003 ◽  
Vol 185 (20) ◽  
pp. 6171-6184 ◽  
Author(s):  
Xiaohua Zeng ◽  
Madhu Choudhary ◽  
Samuel Kaplan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Complex Formation ◽  
Rhodobacter Sphaeroides ◽  
Sequence Data ◽  
Sole Source ◽  
Amino Acid Derivative ◽  
Dna Sequence Data ◽  
And Function ◽  
High Degree

ABSTRACT A new operon (designated the puc2BA operon) displaying a high degree of similarity to the original pucBA genes of Rhodobacter sphaeroides 2.4.1 (designated puc1) was identified and studied genetically and biochemically. The puc2B-encoded polypeptide is predicted to exhibit 94% identity with the original β-apoprotein. The puc2A-encoded polypeptide is predicted to be much larger (263 amino acids) than the 54-amino-acid puc1A-encoded polypeptide. In the first 48 amino acids of the puc2A-encoded polypeptide there is 58% amino acid sequence identity to the original puc1A-encoded polypeptide. We found that puc2BA is expressed, and DNA sequence data suggested that puc2BA is regulated by the PpsR/AppA repressor-antirepressor and FnrL. Employing genetic and biochemical approaches, we obtained evidence that the puc2B-encoded polypeptide is able to enter into LH2 complex formation, but neither the full-length puc2A-encoded polypeptide nor its N-terminal 48-amino-acid derivative is able to enter into LH2 complex formation. Thus, the sole source of α-polypeptides for the LH2 complex is puc1A. The role of the puc1C-encoded polypeptide was also determined. We found that the presence of this polypeptide is essential for normal levels of transcription and translation of the puc1 operon but not for transcription and translation of the puc2 operon. Thus, the puc1C gene product appears to have both transcriptional and posttranscriptional roles in LH2 formation. Finally, the absence of any LH2 complex when puc1B was deleted in frame was surprising since we know that in the presence of functional puc2BA, approximately 30% of the LH2 complexes normally observed contain a puc2B-encoded β-polypeptide.

scholarly journals Identification and characterization of Saccharomyces cerevisiae yapsin 3, a new member of the yapsin family of aspartic proteases encoded by the YPS3 gene

1999 ◽  
Vol 339 (2) ◽  
pp. 407-411 ◽  
Author(s):  
Vicki OLSEN ◽  
Niamh X. CAWLEY ◽  
Jakob BRANDT ◽  
Michi EGEL-MITANI ◽  
Y. Peng LOH
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Cleavage Sites ◽  
Basic Residue ◽  
Aspartic Proteases ◽  
N Terminus ◽  
High Degree ◽  
Identification And Characterization ◽  
Degree Of Similarity

A new aspartic protease from Saccharomyces cerevisiae, with a high degree of similarity with yapsin 1 and yapsin 2 and a specificity for basic residue cleavage sites of prohormones, has been cloned. This enzyme was named yapsin 3. Expression of a C-terminally truncated non-membrane anchored yapsin 3 in yeast yielded a heterogeneous protein between 135–200 kDa which, upon treatment with endoglycosidase H, migrated as a 60 kDa form. Amino-acid analysis of the N-terminus of expressed yapsin 3 revealed two different N-terminal residues, serine-48 and phenylalanine-54, which followed a dibasic and a monobasic residue respectively. Cleavage of several prohormones by non-anchored yapsin 3 revealed a specificity distinct from that of yapsin 1.

scholarly journals Structure of the human MLH1 N-terminus: implications for predisposition to Lynch syndrome

2015 ◽  
Vol 71 (8) ◽  
pp. 981-985 ◽  
Author(s):  
Hong Wu ◽  
Hong Zeng ◽  
Robert Lam ◽  
Wolfram Tempel ◽  
Iain D. Kerr ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Lynch Syndrome ◽  
Mismatch Repair ◽  
Base Pairs ◽  
X Ray ◽  
X Ray Crystallography ◽  
N Terminus ◽  
High Degree ◽  
Degree Of Similarity

Mismatch repair prevents the accumulation of erroneous insertions/deletions and non-Watson–Crick base pairs in the genome. Pathogenic mutations in theMLH1gene are associated with a predisposition to Lynch and Turcot's syndromes. Although genetic testing for these mutations is available, robust classification of variants requires strong clinical and functional support. Here, the first structure of the N-terminus of human MLH1, determined by X-ray crystallography, is described. The structure shares a high degree of similarity with previously determined prokaryoticMLH1homologs; however, this structure affords a more accurate platform for the classification ofMLH1variants.

scholarly journals Purification and properties of peroxisomal pyruvate (glyoxylate) aminotransferase from rat liver

1978 ◽  
Vol 175 (2) ◽  
pp. 765-768 ◽  
Author(s):  
T Noguchi ◽  
Y Takada
Keyword(s):  
Amino Acids ◽  
Kinetic Parameters ◽  
Rat Liver ◽  
Isoelectric Point ◽  
Enzyme Preparation ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Amino Donor ◽  
Glyoxylate Aminotransferase ◽  
Liver Peroxisomes

Pyruvate (glyoxylate) aminotransferase from rat liver peroxisomes was highly purified and characterized. The enzyme preparation has a mol.wt. of approx. 80,000 with two identical subunits, and isoelectric point of 8.0 and a pH optimum between 8.0 and 8.5. The enzyme catalysed transamination between a number of L-amino acids and pyruvate or glyoxylate. The effective amino acceptors were pyruvate, phenylpyruvate and glyoxylate with serine, and glyoxylate and phenylpyruvate with alanine as amino donor. These properties and kinetic parameters of the enzyme are remarkably similar to those previously described for mitochondrial alanine-glyoxylate aminotransferase isoenzyme 1 from glucagon-injected rat liver [Noguchi, Okuno, Takada, Minatogawa, Okai & Kido (1978, Biochem. J. 169, 113-122].

cDNA cloning of a renal Na(+)-Ca2+ exchanger

1992 ◽  
Vol 262 (6) ◽  
pp. F1105-F1109 ◽  
Author(s):  
R. F. Reilly ◽  
C. A. Shugrue
Keyword(s):  
Amino Acids ◽  
Northern Blot Analysis ◽  
Renal Cortex ◽  
Cytoplasmic Domain ◽  
Rabbit Kidney ◽  
Hydrophobic Membrane ◽  
Relative Molecular Weight ◽  
Polymerase Chain ◽  
High Degree ◽  
Degree Of Similarity

In the present study, the polymerase chain reaction (PCR) and library screening were used to clone a cDNA for a rabbit kidney Na(+)-Ca2+ exchanger on the basis of homology with the canine cardiac sarcolemmal sequence (D. A. Nicoll, S. Longoni, and K. D. Philipson. Science Wash. DC 250:562-565, 1990). There is a high degree of similarity between the two sequences, with nucleotide identities of 95, 89, and 90% in the hydrophobic membrane-associated domain, cytoplasmic domain, and 3'-untranslated region, respectively. The rabbit kidney cDNA encodes a predicted protein of 941 amino acids, 29 amino acids shorter than the canine sequence, with a relative molecular weight of 105,121. The deduced amino acid sequence is 96% identical in the membrane-associated domain and 94% identical in the cytoplasmic domain. Northern blot analysis reveals that the cDNA is expressed in the renal cortex. No expression is detected in the medulla. This result is in agreement with micropuncture studies that show Na(+)-Ca2+ exchanger activity in cortical but not medullary nephron segments.

NRD convertase: a putative processing endoprotease associated with the axoneme and the manchette in late spermatids

1996 ◽  
Vol 109 (11) ◽  
pp. 2737-2745 ◽  
Author(s):  
V. Chesneau ◽  
A. Prat ◽  
D. Segretain ◽  
V. Hospital ◽  
A. Dupaix ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Amino Acids ◽  
Germ Cells ◽  
Basic Amino Acids ◽  
N Terminus ◽  
Arginine Residues ◽  
Marked Accumulation ◽  
Low Levels ◽  
Morphological Transformations ◽  
Microtubular Structures

N-arginine dibasic convertase is a novel metalloendopeptidase which selectively cleaves at the N terminus of arginine residues in paired basic amino acids. Although present in brain and several other tissues, NRD convertase is particularly abundant in testis, where its expression appeared to be restricted to germ cells. Low levels of both mRNA and its corresponding protein were detected early in spermatogenesis. However, a marked accumulation of the protein was observed during late steps (14 to 19) of spermiogenesis. By electron microscopy, the NRD convertase immunoreactivity was localized in the cytoplasm of elongating and elongated spermatids, with a noticeable concentration at the level of two microtubular structures, i.e. the manchette and the axoneme. These observations strongly support the hypothesis that NRD convertase is involved in processing events potentially associated with the morphological transformations occurring during spermiogenesis.

scholarly journals Influenza A Virus Virulence Depends on Two Amino Acids in the N-Terminal Domain of Its NS1 Protein To Facilitate Inhibition of the RNA-Dependent Protein Kinase PKR

2017 ◽  
Vol 91 (10) ◽  
Author(s):  
Kristina L. Schierhorn ◽  
Fabian Jolmes ◽  
Julia Bespalowa ◽  
Sandra Saenger ◽  
Christin Peteranderl ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Antiviral Activity ◽  
Influenza A Virus ◽  
Influenza A ◽  
Ns1 Protein ◽  
Dependent Protein Kinase ◽  
N Terminus ◽  
Arginine Residues ◽  
Dependent Protein

ABSTRACT The RNA-dependent protein kinase (PKR) has broad antiviral activity inducing translational shutdown of viral and cellular genes and is therefore targeted by various viral proteins to facilitate pathogen propagation. The pleiotropic NS1 protein of influenza A virus acts as silencer of PKR activation and ensures high-level viral replication and virulence. However, the exact manner of this inhibition remains controversial. To elucidate the structural requirements within the NS1 protein for PKR inhibition, we generated a set of mutant viruses, identifying highly conserved arginine residues 35 and 46 within the NS1 N terminus as being most critical not only for binding to and blocking activation of PKR but also for efficient virus propagation. Biochemical and Förster resonance energy transfer (FRET)-based interaction studies showed that mutation of R35 or R46 allowed formation of NS1 dimers but eliminated any detectable binding to PKR as well as to double-stranded RNA (dsRNA). Using in vitro and in vivo approaches to phenotypic restoration, we demonstrated the essential role of the NS1 N terminus for blocking PKR. The strong attenuation conferred by NS1 mutation R35A or R46A was substantially alleviated by stable knockdown of PKR in human cells. Intriguingly, both NS1 mutant viruses did not trigger any signs of disease in PKR+/+ mice, but replicated to high titers in lungs of PKR−/− mice and caused lethal infections. These data not only establish the NS1 N terminus as highly critical for neutralization of PKR's antiviral activity but also identify this blockade as an indispensable contribution of NS1 to the viral life cycle. IMPORTANCE Influenza A virus inhibits activation of the RNA-dependent protein kinase (PKR) by means of its nonstructural NS1 protein, but the underlying mode of inhibition is debated. Using mutational analysis, we identified arginine residues 35 and 46 within the N-terminal NS1 domain as highly critical for binding to and functional silencing of PKR. In addition, our data show that this is a main activity of amino acids 35 and 46, as the strong attenuation of corresponding mutant viruses in human cells was rescued to a large extent by lowering of PKR expression levels. Significantly, this corresponded with restoration of viral virulence for NS1 R35A and R46A mutant viruses in PKR−/− mice. Therefore, our data establish a model in which the NS1 N-terminal domain engages in a binding interaction to inhibit activation of PKR and ensure efficient viral propagation and virulence.

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