scholarly journals PROPERDIN FACTOR D: CHARACTERIZATION OF ITS ACTIVE SITE AND ISOLATION OF THE PRECURSOR FORM

1974 ◽  
Vol 139 (2) ◽  
pp. 355-366 ◽  
Author(s):  
Douglas T. Fearon ◽  
K. Frank Austen ◽  
Shaun Ruddy
Keyword(s):  
Active Site ◽  
Gel Filtration ◽  
Serine Esterase ◽  
Factor B ◽  
Alternate Pathway ◽  
Serine Esterases ◽  
Definition Of ◽  
Chromatographic Isolation

The activity of properdin factor D was measured by the generation of the hemolytically active cellular intermediate, EAC43B(D), bearing the C3b-dependent alternate pathway C3 convertase. Treatment of factor D with DFP prevented formation of EAC43B(D); thus, a serine esterase is essential for the generation of the alternate pathway C3 convertase, a situation analogous to the role of C1 in the formation of the classical C3 convertase, C42. The definition of factor D as a serine esterase prompted a search for its proenzyme form, and resulted in the chromatographic isolation from plasma of a single peak of trypsin-inducible factor D activity, distinct from activated factor D. Analytical gel filtration indicated an apparent mol wt of 25,000. This protein from which trypsin elaborated factor D activity, as assessed by the formation of EAC43B(D), the generation of the CoVF-dependent C3 convertase, and the cleavage of factor B in the presence of C3b, was designated "precursor factor D." The DFP resistance of precursor factor D, and the susceptibility of its trypsin-activated form to inactivation by DFP is analogous to the behavior of other plasma serine esterases, including C1.

Characterization of anEscherichia coliSulfite Oxidase Homologue Reveals the Role of a Conserved Active Site Cysteine in Assembly and Function†

Biochemistry ◽  
2005 ◽  
Vol 44 (30) ◽  
pp. 10339-10348 ◽  
Author(s):  
Stephen J. Brokx ◽  
Richard A. Rothery ◽  
Guijin Zhang ◽  
Derek P. Ng ◽  
Joel H. Weiner
Keyword(s):  
Active Site ◽  
Active Site Cysteine ◽  
And Function

Paradigm Function Morphology: Assumptions and Innovations

2020 ◽  
Author(s):  
Gregory Stump
Keyword(s):  
Inflectional Morphology ◽  
Formal Representation ◽  
The Core ◽  
Recent Innovation ◽  
Morphological Theory ◽  
Per Se ◽  
Fundamental Insight ◽  
Definition Of

Paradigm Function Morphology (PFM) is an evolving approach to modeling morphological systems in a precise and enlightening way. The fundamental insight of PFM is that words have both content and form and that in the context of an appropriately organized lexicon, a language’s morphology deduces a complex word’s form from its content. PFM is therefore a realizational theory: a language’s grammar and lexicon are assumed to provide a precise characterization of a word’s content, from which the language’s morphology then projects the corresponding form. Morphemes per se have no role in this theory; by contrast, paradigms have the essential role of defining the content that is realized by a language’s morphology. At the core of PFM is the notion of a paradigm function, a formal representation of the relation between a word’s content and its form; the definition of a language’s paradigm function is therefore the definition of its inflectional morphology. Recent elaborations of this idea assume a distinction between content paradigms and form paradigms, which makes it possible to account for a fact that is otherwise irreconcilable with current morphological theory—the fact that the set of morphosyntactic properties that determines a word’s syntax and semantics often differs from the set of properties (some of them morphomic) that determines a word’s inflectional form. Another recent innovation is the assumption that affixes and rules of morphology may be complex in the sense that they may be factored into smaller affixes and rules; the evidence favoring this assumption is manifold.

scholarly journals Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase).

1994 ◽  
Vol 127 (2) ◽  
pp. 467-478 ◽  
Author(s):  
J P Tassan ◽  
S J Schultz ◽  
J Bartek ◽  
E A Nigg
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Cell Cycle Control ◽  
Gel Filtration ◽  
Multiprotein Complex ◽  
Cyclin Dependent Kinases ◽  
Critical Residue ◽  
Cdk Activating Kinase

The activity of cyclin-dependent kinases (cdks) depends on the phosphorylation of a residue corresponding to threonine 161 in human p34cdc2. One enzyme responsible for phosphorylating this critical residue has recently been purified from Xenopus and starfish. It was termed CAK (for cdk-activating kinase), and it was shown to contain p40MO15 as its catalytic subunit. In view of the cardinal role of cdks in cell cycle control, it is important to learn if and how CAK activity is regulated during the somatic cell cycle. Here, we report a molecular characterization of a human p40MO15 homologue and its associated CAK activity. We have cloned and sequenced a cDNA coding for human p40MO15, and raised specific polyclonal and monoclonal antibodies against the corresponding protein expressed in Escherichia coli. These tools were then used to demonstrate that p40MO15 protein expression and CAK activity are constant throughout the somatic cell cycle. Gel filtration suggests that active CAK is a multiprotein complex, and immunoprecipitation experiments identify two polypeptides of 34 and 32 kD as likely complex partners of p40MO15. The association of the three proteins is near stoichiometric and invariant throughout the cell cycle. Immunocytochemistry and biochemical enucleation experiments both demonstrate that p40MO15 is nuclear at all stages of the cell cycle (except for mitosis, when the protein redistributes throughout the cell), although the p34cdc2/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis. The absence of obvious changes in CAK activity in exponentially growing cells constitutes a surprise. It suggests that the phosphorylation state of threonine 161 in p34cdc2 (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin substrates, and by phosphatase(s).

scholarly journals Beyond the Binary: Rethinking Gender Neutrality in Indian Rape Law

2016 ◽  
Vol 11 (2) ◽  
pp. 367-397
Author(s):  
Harshad PATHAK
Keyword(s):  
Human Rights ◽  
Rape Law ◽  
The Common ◽  
Definition Of ◽  
Human Rights Based Approach ◽  
Gender Neutrality ◽  
Gender Specific ◽  
Indian Penal Code

AbstractDespite expanding the definition of rape under the Indian Penal Code to include non-penile-vaginal acts of penetration, the said definition continues to conform to a gender-specific notion of rape, based on a predetermined characterization of the victim-perpetrator framework on the basis of their genders. Herein, I will critique this idea of gender specificity in Indian rape law on the grounds that it reinforces a binary notion of gender, and results in gross underinclusion. Instead, it is more appropriate to adopt a human-rights-based approach in defining the offence of rape, and negate the role of gender in identifying the victims and perpetrators of an act of rape. The argument is pillared on a state’s obligation to not discriminate on the basis of sex, the recognition of transgender rights, and an assessment of the common grounds for opposing gender neutrality in Indian rape law.

scholarly journals FMN-dependent oligomerization of putative lactate oxidase from Pediococcus acidilactici

2019 ◽  
Author(s):  
Yashwanth Ashok ◽  
Mirko M. Maksimainen ◽  
Tuija Kallio ◽  
Pekka Kilpeläinen ◽  
Lari Lehtiö
Keyword(s):  
Hydrogen Peroxide ◽  
Active Site ◽  
Pediococcus Acidilactici ◽  
Active Site Residues ◽  
Lactate Oxidase ◽  
Monooxygenase Activity ◽  
Aerococcus Viridans ◽  
Lactate Levels

AbstractLactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate oxidases relies largely to studies conducted with that particular enzyme. Pediococcus acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but this enzyme has not been characterized before in detail. Here we report structural characterization of the recombinant enzyme and its co-factor dependent oligomerization. The crystal structures revealed two distinct conformations in the loop closing the active site, consistent with previous biochemical studies implicating the role of loop in catalysis. Despite the structural conservation of active site residues when compared to Aerococcus viridans lactate oxidase we were not able to detect either oxidase or monooxygenase activity when L-lactate or other potential alpha hydroxyl acids were used as a substrate. Pediococcus acidilactici lactate oxidase is therefore an example of a misannotation of an FMN-dependent enzyme, which catalyzes likely a so far unknown oxidation reaction.

scholarly journals Structural Characterization of an S-enantioselective Imine Reductase from Mycobacterium Smegmatis

Biomolecules ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1130
Author(s):  
Timo Meyer ◽  
Nadine Zumbrägel ◽  
Christina Geerds ◽  
Harald Gröger ◽  
Hartmut H. Niemann
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Mycobacterium Smegmatis ◽  
Catalytic Mechanism ◽  
Building Blocks ◽  
Secondary Amines ◽  
Hydrophobic Amino Acids ◽  
High Degree

NADPH-dependent imine reductases (IREDs) are enzymes capable of enantioselectively reducing imines to chiral secondary amines, which represent important building blocks in the chemical and pharmaceutical industry. Since their discovery in 2011, many previously unknown IREDs have been identified, biochemically and structurally characterized and categorized into families. However, the catalytic mechanism and guiding principles for substrate specificity and stereoselectivity remain disputed. Herein, we describe the crystal structure of S-IRED-Ms from Mycobacterium smegmatis together with its cofactor NADPH. S-IRED-Ms belongs to the S-enantioselective superfamily 3 (SFam3) and is the first IRED from SFam3 to be structurally described. The data presented provide further evidence for the overall high degree of structural conservation between different IREDs of various superfamilies. We discuss the role of Asp170 in catalysis and the importance of hydrophobic amino acids in the active site for stereospecificity. Moreover, a separate entrance to the active site, potentially functioning according to a gatekeeping mechanism regulating access and, therefore, substrate specificity is described.

scholarly journals Carbon dioxide binding at a Ni/Fe center: synthesis and characterization of Ni(η1-CO2-κC) and Ni-μ-CO2-κC:κ2O,O′-Fe

2017 ◽  
Vol 8 (1) ◽  
pp. 600-605 ◽  
Author(s):  
Changho Yoo ◽  
Yunho Lee
Keyword(s):  
Carbon Dioxide ◽  
Active Site ◽  
Synthesis And Characterization ◽  
Fe Species

A heterobimetallic Ni-μ-CO2-κC:κ2O,O′-Fe species reminiscent of the CODH active site was synthesized, helping to elucidate the role of the unique iron.

scholarly journals Synthesis and characterization of a biotinylated organophosphorus ester for detection and affinity purification of a brain serine esterase: neuropathy target esterase

1994 ◽  
Vol 301 (2) ◽  
pp. 551-556 ◽  
Author(s):  
P Glynn ◽  
D J Read ◽  
R Guo ◽  
S Wylie ◽  
M K Johnson
Keyword(s):  
Affinity Purification ◽  
Order Rate Constant ◽  
Single Step ◽  
Neuropathy Target Esterase ◽  
Serine Esterase ◽  
Western Blots ◽  
Sds Page ◽  
Covalent Inhibitors ◽  
Serine Esterases

We have synthesized a novel stable precursor, saligenin phosphorotrichloridate, which, on reaction with N-monobiotinyldiamines, generates a series of biotinylated covalent inhibitors of serine esterases. A homologue designated S9B [1-(saligenin cyclic phospho)-9-biotinyldiaminononane] was selected to allow detection and rapid isolation of neuropathy target esterase (NTE). This enzyme is the primary target site for those organophosphorus esters (OPs) which cause delayed neuropathy. NTE comprises about 0.03% of the total protein in brain microsomal fractions and has resisted purification attempts over many years. S9B is a potent progressive inhibitor of NTE esteratic activity (second-order rate constant 1.4 x 10(7) M-1.min-1). Incubation of S9B with brain microsomes led to specific covalent labelling of NTE as determined by detection of a biotinylated 155 kDa polypeptide on Western blots. Specificity of S9B labelling was further demonstrated by inhibition with the neuropathic OP mipafox. Biotinyl-NTE in SDS-solubilized S9B-labelled microsomes was adsorbed on to avidin-Sepharose and subsequently eluted, yielding a fraction enriched approx. 1000-fold in NTE by a single step with recoveries of 30%. Essentially pure NTE was obtained after separation from two endogenous biotinylated polypeptides (120 and 70 kDa) in avidin-Sepharose eluates by preparative SDS/PAGE. Other biotinylated saligenin phosphoramidates derived from the same precursor may be useful for detection and isolation of other serine esterases and proteinases.

scholarly journals Partial purification and characterization of a peroxidase activity from human placenta

1990 ◽  
Vol 268 (3) ◽  
pp. 739-743 ◽  
Author(s):  
J L Nelson ◽  
A P Kulkarni
Keyword(s):  
Peroxidase Activity ◽  
Potential Role ◽  
Gel Filtration ◽  
Partial Purification ◽  
Human Haemoglobin ◽  
Prostaglandin Synthase ◽  
Membrane Bound ◽  
Cytochrome P 450

Peroxidases can metabolize a variety of xenobiotics to reactive intermediates capable of binding to protein or DNA. The potential role of these enzymes in fetotoxicity has not been explored. In this study, the presence of peroxidase activity was observed in human term and pre-term placenta. Human term placental peroxidase activity (HTPP) was partially purified by concanavalin A affinity chromatography from CaCl2 extracts of the particulate fraction. HTPP appears to be a membrane-bound glycoprotein. Arachidonic acid-dependent oxidation of guaiacol was not observed, suggesting that the peroxidase activity was not due to prostaglandin synthase. Moreover, HTPP preparations were devoid of catalase and spectrally dissimilar from human haemoglobin, cytochrome P-450, eosinophil peroxidase and myloperoxidase, suggesting an endogenous origin. An Mr of approx. 119,000 was determined for HTPP by gel filtration. Cathodic slab-PAGE of cetyltrialkylammonium bromide-solubilized HTPP yielded two peroxidase-staining bands.

Cytochrome cbb3 oxidase and bacterial microaerobic metabolism

2002 ◽  
Vol 30 (4) ◽  
pp. 653-658 ◽  
Author(s):  
R. S. Pitcher ◽  
T. Brittain ◽  
N. J. Watmugh
Keyword(s):  
Active Site ◽  
Reduction Potential ◽  
Oxygen Affinity ◽  
Pseudomonas Stutzeri ◽  
Spectroscopic Characterization ◽  
High Oxygen Affinity ◽  
Copper Oxidase ◽  
Reduced State

Cytochrome cbb3 oxidase is a member of the haem-copper oxidase superfamily. It is characterized by its high oxygen affinity, while retaining the ability to pump protons. These attributes are central to its proposed role in bacterial microaerobic metabolism. Recent spectroscopic characterization of both the cytochrome cbb3 oxidase complex from Pseudomonas stutzeri and the dihaem ccoP subunit expressed separately in Escherichia coli has revealed the presence of a low-spin His/His co-ordinated c-type cytochrome. The low midpoint reduction potential of this haem (Em < + 100 mV), together with its unexpected ability to bind CO in the reduced state at the expense of the distal histidine ligand, raises questions about the role of the ccoP subunit in the delivery of electrons to the active site.

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