scholarly journals Properties and structural requirements for substrate specificity of cytochrome P-450-dependent obtusifoliol 14 α-demethylase from maize (Zea mays) seedlings

1991 ◽  
Vol 277 (2) ◽  
pp. 483-492 ◽  
Author(s):  
M Taton ◽  
A Rahier
Keyword(s):  
Zea Mays ◽  
Substrate Specificity ◽  
Binding Site ◽  
Critical Role ◽  
Biochemical Properties ◽  
Product Specificity ◽  
Enzyme Substrate ◽  
Absolute Requirement ◽  
High Degree ◽  
Cytochrome P 450

The biochemical properties of cytochrome P-450-dependent obtusifoliol 14 alpha-demthylase (P-450OBT.14DM) from maize (Zea mays) seedlings were defined. In particular, the enzyme was shown by differential centrifugation to be localized in the endoplasmic reticulum. P-450OBT.14DM had an apparent Km of 160 +/- 5 microM and an apparent Vmax of 65 +/- 5 pmol/min per mg of protein for its best substrate, obtusifoliol. The substrate specificity of P-450OBT.14DM was thoroughly investigated by comparing the demethylation of obtusifoliol with that of a series of 15 natural or novel synthetic analogues of obtusifoliol. The results obtained clearly indicate that three distinct domains of the sterol substrate are governing obtusifoliol demethylation by P-450OBT.14DM. They revealed that (i) P-450OBT.14DM has probably a specific apolar binding site for the side chain, (ii) the delta 8-double bond is an absolute requirement for substrate demethylation and (iii) the 3-hydroxy group plays a critical role in the enzyme-substrate interaction. Interestingly the binding site, beyond the C-3 position, contains a cleft which cannot accommodate a 4 beta-methyl substituent present in lanosterol or eburicol, the precursors of 14-desmethylsterols respectively in mammals and yeast. This result indicates that P-450OBT.14DM is a novel constitutive cytochrome P-450 with a high degree of substrate and product specificity.

scholarly journals A reinvestigation of the substrate specificity of pig kidney diamine oxidase

1970 ◽  
Vol 117 (1) ◽  
pp. 169-176 ◽  
Author(s):  
W. G. Bardsley ◽  
C. M. Hill ◽  
R. W. Lobley
Keyword(s):  
Substrate Specificity ◽  
Binding Site ◽  
Enzyme Preparation ◽  
Diamine Oxidase ◽  
Substrate Binding ◽  
Pig Kidney ◽  
Substrate Interaction ◽  
Enzyme Substrate ◽  
Rf Values

1. The substrate specificity of pig kidney diamine oxidase was reinvestigated with a purer enzyme preparation than has previously been used for this purpose. 2. All substrates were extensively purified before use, and methods of preparation or sources are given, together with RF values. 3. The substrate specificity determined differed somewhat from that reported by previous workers and, in addition, the behaviour of several compounds not previously used as substrates is described. 4. A model for enzyme–substrate interaction embodying these observations is formulated. It is suggested that a negatively charged substrate-binding group is situated at 6.0–9.0 Å from the oxidizing site. The binding and oxidizing sites are separated by a hydrophobic or methylene-binding site.

Hilar cholangiocarcinoma

2018 ◽  
Vol 1 (3) ◽  
pp. 28-30
Author(s):  
Tanita Suttichaimongkol
Keyword(s):  
Magnetic Resonance ◽  
Hilar Cholangiocarcinoma ◽  
Critical Role ◽  
Magnetic Resonance Cholangiopancreatography ◽  
Resonance Imaging ◽  
Extrahepatic Cholangiocarcinoma ◽  
Case Presentation ◽  
Duct Epithelium ◽  
Bile Duct Epithelium ◽  
High Degree

Cholangiocarcinoma is a primary biliary tract tumor arising from the bile duct epithelium. Classically, these tumors have been categorized according to their anatomic location as intrahepatic and extrahepatic. Hilar cholangiocarcinoma is the most common type of extrahepatic cholangiocarcinoma. It is the most difficult cancer to diagnose and therefore carries a poor prognosis with a 5-year survivalrate of less than 10%. Diagnostic imaging, coupled with a high degree of clinical suspicion, play a critical role in timely diagnosis, staging, and evaluation for surgical resectability. The most common imagingmodalities used for diagnosis and staging of hilar cholangiocarcinoma include ultrasound (US), computed tomography (CT), magnetic resonance imaging/magnetic resonance cholangiopancreatography(MRI/MRCP). This article showed a case presentation and reviewed the imaging appearance of hilar cholangiocarcinoma.   Figure 1  Greyscale sonography at the level of hepatic hilum revealed an ill-defined hilar mass (asterisk)resulting in upstream dilatation of right (arrow) and left (arrow head) main intrahepatic duct.  

scholarly journals VARIATION IN BIOCHEMICAL PROPERTIES OF ALLOZYMES OF XANTHINE DEHYDROGENASE IN DROSOPHILA PSEUDOOBSCURA

Genetics ◽  
1980 ◽  
Vol 96 (4) ◽  
pp. 927-938
Author(s):  
David R Wilcox ◽  
Satya Prakash
Keyword(s):  
Substrate Specificity ◽  
Binding Affinity ◽  
Electrophoretic Mobility ◽  
Significant Variation ◽  
Xanthine Dehydrogenase ◽  
Biochemical Properties ◽  
Variant Allele ◽  
Drosophila Pseudoobscura ◽  
Mesa Verde

ABSTRACT Twenty-six D. pseudoobscura strains isogenic for xanthine dehydrogenase alleles from Mesa Verde, Colorado, were tested for differences in the biochemical properties of different allelic forms of xanthine dehydrogenase. No significant differences in binding affinity (Km) or substrate specificity of the enzyme were found. Significant variation among strains, in activity (V  max) and among electromorphs, as well as among strains, in thermolability was found. For the few strains tested, the activity and thermolability differences were shown to co-segregate with the electrophoretic mobility of the variant allele.

scholarly journals Cell Cycle-Dependent Expression of Mammalian E2-C Regulated by the Anaphase-Promoting Complex/Cyclosome

2000 ◽  
Vol 11 (8) ◽  
pp. 2821-2831 ◽  
Author(s):  
Atsushi Yamanaka ◽  
Shigetsugu Hatakeyama ◽  
Kin-ichiro Kominami ◽  
Masatoshi Kitagawa ◽  
Masaki Matsumoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Substrate Specificity ◽  
Critical Role ◽  
Anaphase Promoting Complex ◽  
Polyubiquitin Chain ◽  
M Phase ◽  
Ubiquitin Conjugating Enzyme ◽  
Recognition Motifs ◽  
Cycle Dependent ◽  
Conjugating Enzyme

Progression through mitosis requires the precisely timed ubiquitin-dependent degradation of specific substrates. E2-C is a ubiquitin-conjugating enzyme that plays a critical role with anaphase-promoting complex/cyclosome (APC/C) in progression of and exit from M phase. Here we report that mammalian E2-C is expressed in late G2/M phase and is degraded as cells exit from M phase. The mammalian E2-C shows an autoubiquitinating activity leading to covalent conjugation to itself with several ubiquitins. The ubiquitination of E2-C is strongly enhanced by APC/C, resulting in the formation of a polyubiquitin chain. The polyubiquitination of mammalian E2-C occurs only when cells exit from M phase. Furthermore, mammalian E2-C contains two putative destruction boxes that are believed to act as recognition motifs for APC/C. The mutation of this motif reduced the polyubiquitination of mammalian E2-C, resulting in its stabilization. These results suggest that mammalian E2-C is itself a substrate of the APC/C-dependent proteolysis machinery, and that the periodic expression of mammalian E2-C may be a novel autoregulatory system for the control of the APC/C activity and its substrate specificity.

KIF3B gene silent variant leading to sperm morphology and motility defects and male infertility

2021 ◽  
Author(s):  
Raheleh Heydari ◽  
Mehrshad Seresht-Ahmadi ◽  
Shahab Mirshahvaladi ◽  
Marjan Sabbaghian ◽  
Anahita Mohseni-Meybodi
Keyword(s):  
Male Infertility ◽  
Protein Expression ◽  
Binding Site ◽  
Sperm Count ◽  
Critical Role ◽  
Coiled Coil ◽  
Control Group ◽  
Coding Region ◽  
Exon 2 ◽  
Coiled Coil Domain

Abstract Sperm structural and functional defects are leading causes of male infertility. Patients with immotile sperm disorders suffer from axoneme failure and show a significant reduction in sperm count. The kinesin family member 3B (KIF3B) is one of the genes involved in the proper formation of sperm with a critical role in intraflagellar and intramanchette transport. A part of exon 2 and exons 3–5 of the KIF3B encodes a protein coiled-coil domain that interacts with IFT20 from the IFT protein complex. In the present study, the coding region of KIF3B coiled-coil domain was assessed in 88 oligoasthenoteratozoospermic patients, and the protein expression was evaluated in the mature spermatozoa of the case and control groups using immunocytochemistry and western blotting. According to the results, there was no genetic variation in the exons 3–5 of the KIF3B, but a new A > T variant was identified within the exon 2 in 30 patients, where nothing was detected in the control group. In contrast to healthy individuals, significantly reduced protein expression was observable in oligoasthenoteratozoospermic (OAT) patients carrying variation where protein organization was disarranged, especially in the principal piece and midpiece of the sperm tail. Besides, the protein expression level was lower in the patients’ samples compared to that of the control group. According to the results of the present study the NM_004798.3:c.1032A > T, p.Pro344 = variant; which has been recently submitted to the Clinvar database; although synonymous, causes alterations in the transcription factor binding site, exon skipping, and also exonic splicing enhancer-binding site. Therefore, KIF3B can play an important role in spermatogenesis and the related protein reduction can cause male infertility.

scholarly journals Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase α subunit

2010 ◽  
Vol 135 (2) ◽  
pp. 115-134 ◽  
Author(s):  
Susan Meier ◽  
Neslihan N. Tavraz ◽  
Katharina L. Dürr ◽  
Thomas Friedrich
Keyword(s):  
Binding Site ◽  
Critical Role ◽  
Aromatic Amino Acids ◽  
Cation Binding ◽  
Carboxy Terminus ◽  
Leakage Currents ◽  
Α Subunit ◽  
Inward Currents ◽  
The Common ◽  
Carboxy Terminal

The Na+/K+-ATPase mediates electrogenic transport by exporting three Na+ ions in exchange for two K+ ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb+ ions in the crystal structures of the Na+/K+-ATPase has defined two “common” cation binding sites, I and II, which accommodate Na+ or K+ ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this “unique” Na+ binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na+/K+-ATPase α2 subunit decreases the affinity for extracellular and intracellular Na+, in agreement with previous biochemical studies. Apparently, the ΔYY deletion changes Na+ affinity at site III but leaves the common sites unaffected, whereas the more extensive ΔKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K+, the ΔYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na+ dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na+/Na+ exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na+ release/binding mechanism. In analogy to the mechanism proposed for the H+ leak currents of the wild-type Na+/K+-ATPase, we suggest that the ΔYY deletion lowers the energy barrier for the intracellular Na+ occlusion reaction, thus destabilizing the Na+-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na+/K+-ATPase α subunit represents a structural and functional relay between Na+ binding site III and the intracellular cation occlusion gate.

scholarly journals Polymorphisms and gene expression in the almond IGT family are not correlated to variability in growth habit in major commercial almond cultivars

2021 ◽  
Author(s):  
Álvaro Montesinos ◽  
Chris Dardick ◽  
María José Rubio-Cabetas ◽  
Jérôme Grimplet
Keyword(s):  
Gene Expression ◽  
Critical Role ◽  
Gene Families ◽  
Fruit Trees ◽  
Hormonal Responses ◽  
Breeding Programs ◽  
Branch Angle ◽  
Conserved Domain ◽  
Family Gene ◽  
High Degree

Almond breeding programs aimed at selecting cultivars adapted to intensive orchards have recently focused on the optimization of tree architecture. This multifactorial trait is defined by numerous components controlled by processes such as hormonal responses, gravitropism and light perception. Gravitropism sensing is crucial to control the branch angle and therefore, the tree habit. A gene family, denominated IGT family after a share conserved domain, has been described as involved in the regulation of branch angle in several species, including rice and Arabidopsis, and even in fruit trees like peach. Here we identified six members of this family in almond: LAZY1 , LAZY2 , TAC1 , DRO1 , DRO2 , IGT-like . After analyzing their protein sequences in forty-one almond cultivars and wild species, little variability was found, pointing a high degree of conservation in this family. Gene expression was analyzed in fourteen cultivars of agronomical interest comprising diverse tree habit phenotypes. Only LAZY1 , LAZY2 and TAC1 were expressed in almond shoot tips during the growing season. No relation was established between the expression profile of these genes and the tree habit. However, some insight has been gained in how LAZY1 and LAZY2 are regulated, identifying the IPA1 almond homologues and other transcription factors involved in hormonal responses as regulators of their expression. Besides, we have found various polymorphisms that could not be discarded as involved in a potential polygenic origin of regulation of architectural phenotypes. Therefore, we have established that unlike many species, IGT family genes do not play a critical role in the control of tree habit in currently commercialized almond cultivars, with other gene families contributing to the variability of these traits.

scholarly journals Fragment-derived modulators of an industrial β-glucosidase

2020 ◽  
Vol 477 (22) ◽  
pp. 4383-4395
Author(s):  
Eleni Makraki ◽  
John F. Darby ◽  
Marta G. Carneiro ◽  
James D. Firth ◽  
Alex Heyam ◽  
...  
Keyword(s):  
Binding Site ◽  
Glycoside Hydrolase ◽  
Mixed Model ◽  
Specific Binding ◽  
Fold Increase ◽  
Kinetic Assay ◽  
Nmr Spectrum ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Maximum Activation

A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate-binding site, generating a productive conformation for the enzyme–substrate complex.

In silico Discovery of Enzyme–Substrate Specificity-determining Residue Clusters

2005 ◽  
Vol 352 (5) ◽  
pp. 1105-1117 ◽  
Author(s):  
Gong-Xin Yu ◽  
Byung-Hoon Park ◽  
Praveen Chandramohan ◽  
Rajesh Munavalli ◽  
Al Geist ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
In Silico ◽  
Enzyme Substrate
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