Pyruvate kinase of Neurospora crassa: purification and some properties

1972 ◽  
Vol 18 (6) ◽  
pp. 805-815 ◽  
Author(s):  
M. Kapoor ◽  
T. M. Tronsgaard
Keyword(s):  
Neurospora Crassa ◽  
Conformational Change ◽  
Pyruvate Kinase ◽  
Phosphate Buffer ◽  
Ternary Complex ◽  
Sedimentation Coefficient ◽  
Tris Buffer ◽  
Sedimentation Analysis ◽  
Kinetics Studies ◽  
Ping Pong

Pyruvate kinase of Neurospora crassa has been purified and some of its properties are reported. The procedure used for purification consists of five steps yielding a 90 to 95% purified protein. Preliminary sedimentation analysis yielded a sedimentation coefficient of 9.5 for this enzyme. Maximal stabilization of the enzyme is achieved in phosphate buffer; Tris buffer induces a conformational change in the enzyme leading to inactivation. Inactivation can be reversed by incubation with substrates, PEP and ADP. Preliminary kinetics studies suggest the formation of a ternary complex rather than a Ping Pong type of a mechanism.

Purification and studies of some physicochemical properties of glutamine synthetase of Neurospora crassa

1978 ◽  
Vol 56 (10) ◽  
pp. 927-933 ◽  
Author(s):  
W. S. Lin ◽  
M. Kapoor
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Glutamine Synthetase ◽  
Neurospora Crassa ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Affinity Column ◽  
Subunit Structure ◽  
Α Helix

Glutamine synthetase (EC 6.3.1.2) of Neurospora crassa was purified to near homogeneity by chromatography on a glutamate–Sepharose affinity column. Its properties, including molecular weight, subunit structure, amino acid composition, and approximate α-helix content, have been examined. In the native state, this enzyme has been demonstrated by gel filtration to be an octamer of molecular weight 360 000 and as having a sedimentation coefficient of 13.2 S by sedimentation velocity measurements. Circular dichroism spectra in the far ultraviolet range suggest an approximate α-helix content of 23–24%. The subunit generated by treatment with urea was found to be 45 000 daltons by gel filtration methods and a molecular weight of 46 000 was calculated for the monomer obtained by sodium dodecyl sulphate (SDS) treatment and electrophoresis in SDS-polyacrylamide gels. Interprotomeric cross-linking experiments, using diimidoesters, suggest the presence of two noncovalently linked tetramers comprising the native octameric structure. Amino acid analyses revealed the presence of six tryptophans, four half cystines, and nine methionine residues per monomer of 45 000 daltons.

A study of the messenger RNA encoding pyruvate kinase of Neurospora crassa

1986 ◽  
Vol 6 (2) ◽  
pp. 201-208
Author(s):  
M. Devchand ◽  
M. Kapoor
Keyword(s):  
Neurospora Crassa ◽  
Pyruvate Kinase ◽  
Messenger Rna ◽  
Blot Hybridization ◽  
Dot Blot ◽  
Kinase Gene ◽  
S1 Nuclease ◽  
Coding Regions

In Neurospora crassa, there is a single pyruvate kinase (PK) consisting of four identical subunits of ∼60k daltons. Northern and dot blot hybridization studies, using most of the yeast pyruvate kinase gene as a probe, suggest the presence of two distinct mRNA species for pyruvate kinase, separable on the basis of the length of their polyadenylated tails, by oligo(dT)cellulose chromatography. These messages are present in polysomes, immuno-precipitated by anti-PK antibodies, indicating probable translation in vivo. Fractions containing both messages were translated in vitro in the heterologous systems as well as in a homologous N. crassa lysate, the newly-synthesized PK being detected by immunoadsorption. Protection studies using S1-nuclease suggest no major structural differences in the 5′-untranslated and most of the coding regions of the two messages.

Schnellmarkierte RNS in heterotroph kultivierten Geweben von Nicotiana tabacum / Rapidly Labelled RNA in Heterotrophically Cultured Tissues of Nicotiana tabacum

1972 ◽  
Vol 27 (10) ◽  
pp. 1205-1215 ◽  
Author(s):  
Wolfgang Beisenherz
Keyword(s):  
Nicotiana Tabacum ◽  
Thin Layer ◽  
Chromatographic Separation ◽  
Sedimentation Coefficient ◽  
Life Time ◽  
Rna Metabolism ◽  
Tissue Cultures ◽  
Differential Centrifugation ◽  
Sedimentation Analysis ◽  
Tissue Homogenates

The RNA metabolism in tissue cultures of Nicotiana tabacum has been studied by incorporation of radioactive phosphate. The RNA was extracted by phenol-kresol-hydroxychinon and fractionated by MAK columns. A rapidly labelled RNA fraction was eluted from the MAK column after the rRNA components. Its base ratio differed from the latter in that the AMP content (28,6%) was higher than the GMP content (26,0%). Sedimentation analysis on sucrose gradients showed a sedimentation coefficient of 37 -38s. The life time of the 37-38s RNA fraction in chase experiments was 3,5-4 hours. Differential centrifugation of tissue homogenates revealed that the labelled 37-38s RNA was associated with particles sedimented by low speed centrifugation. No 37-38s RNA could be detected in the ribosomal fraction and the supernatant. During chase experiments label appeared in the rRNA components. It could be excluded by thin layer chromatographic separation of 32P labelled compounds that this label originated from 32P-o-phosphate or labelled organic phosphates except of the rapidly labelled 37-38s RNA. From this it was concluded that the 37-38s RNA is a precursor of the rRNA.

PHYSICOCHEMICAL STUDIES OF ALKALI LIGNINS: II. ULTRACENTRIFUGAL SEDIMENTATION ANALYSIS

1960 ◽  
Vol 38 (2) ◽  
pp. 259-269 ◽  
Author(s):  
P. R. Gupta ◽  
R. F. Robertson ◽  
D. A. I. Goring
Keyword(s):  
Standard Deviation ◽  
Linear Relationship ◽  
Intrinsic Viscosity ◽  
Sedimentation Coefficient ◽  
Wide Distribution ◽  
Small Decrease ◽  
Sedimentation Analysis ◽  
Qualitative Interpretation ◽  
Physicochemical Studies ◽  
Lamm Equation

Ultracentrifugal sedimentation of alkali lignin fractions revealed marked boundary spreading at low and high centrifugal fields. An analysis of the gradient diagrams indicated a wide distribution of sedimentation coefficients ranging from 0.5 to over 400 × 10−13 second. There was only a small decrease in the polydispersity on subfractionation. The distributions were continuous with a single peak and positive skewness. A linear relationship was found between the intrinsic viscosity and the standard deviation of the weight-average sedimentation coefficient. The sedimentation coefficient based on the movement of the maximum ordinate at a field of 11.5 × 103 g was several times greater than the corresponding coefficient for the same fraction at 200 × 103 g. A qualitative interpretation of this phenomenon is given in terms of the Faxen and the Archibald solution to the Lamm equation.

scholarly journals Kinetics and mechanisms of catalyzed dual-E (antithetic) controllers

2021 ◽  
Author(s):  
Qaiser Waheed ◽  
Huimin Zhou ◽  
Peter Ruoff
Keyword(s):  
Ternary Complex ◽  
Reaction Kinetic ◽  
Zero Order ◽  
Control Engineering ◽  
Environmental Disturbances ◽  
Integral Control ◽  
Analysis And Design ◽  
Zero Order Kinetics ◽  
Ping Pong ◽  
Kinetic Mechanisms

Homeostasis plays a central role in our understanding how cells and organisms are able to oppose environmental disturbances and thereby maintain an internal stability. During the last two decades there has been an increased interest in using control engineering methods, especially integral control, in the analysis and design of homeostatic networks. Several reaction kinetic mechanisms have been discovered which lead to integral control. In two of them integral control is achieved, either by the removal of a single control species E by zero-order  kinetics ("single-E controllers"), or by the removal of two control species  by second-order kinetics ("antithetic or dual-E control"). In this paper we show results when the control species E 1  and E 2  in antithetic control are removed enzymatically by ping-pong or ternary-complex mechanisms. Our findings show that enzyme-catalyzed dual-E controllers can work in two control modes. In one mode, one of the two control species is active, but requires zero-order kinetics in its removal. In the other mode, both controller species are active and both are removed enzymatically. Conditions for the two control modes are put forward and biochemical examples with the structure of enzyme-catalyzed dual-E controllers are discussed.

Comparison of pH Changes and Elicitor Induced Production of Glyceollin Isomers in Soybean Cotyledons

1985 ◽  
Vol 40 (7-8) ◽  
pp. 477-481 ◽  
Author(s):  
Wolfgang F. Osswald ◽  
Sigrid Zieboll ◽  
E. F. Elstner
Keyword(s):  
Phosphate Buffer ◽  
Tris Buffer ◽  
Normal Pattern ◽  
Ph Changes ◽  
Ph Shift

Abstract During the incubation of soybean cotyledons with Pmg-elicitor for 22 hours the pH of the diffusion droplets increases from 7.2 to 8.3. This pH-shift is a precondition for the formation of the typical red colour of the diffusion droplet. After inhibiting the pH-shift by the use of 100 mм phosphate or Tris buffer instead of 10 mм buffer as solvent for the elicitor, the red colour is no longer formed with the exeption of 100 mM Ammediol buffer. However, the normal pattern of pterocarpan induction can be measured in the absence of the red colour in the diffusion droplet. Tris and Ammediol buffers exhibited a smaller pterocarpan induction as compared to phosphate buffer as solvent for the Pmg-elicitor.

The Synthesis of 6-Aminomethyl-5,6,7,8-Tetrahydropterin

1988 ◽  
Vol 41 (5) ◽  
pp. 667 ◽  
Author(s):  
P Waring
Keyword(s):  
Acid Hydrolysis ◽  
Phosphate Buffer ◽  
Selenium Dioxide ◽  
Tris Buffer ◽  
Side Chain ◽  
Neutral Ph ◽  
Side Chain Loss ◽  
Hydrolysis Of

6-Aminomethyl-5,6,7,8-tetrahydropterin has been prepared by reduction of 2-acetamido-6-cyanopteridin-4(3H)-one* to 2-acetamido-6-aminomethyl- 5,6,7,8-tetrahydropteridin-4(3H)-one followed by acid hydrolysis. The hitherto undescribed 6-cyanopterin was prepared by careful hydrolysis of the 2-acetamido compound prepared by dehydration of the oxime derived from 2-acetamido-6-formylpteridin-4(3H)-one. The latter was prepared by selenium dioxide oxidation of the methyl compound. Oxidation of 6-aminomethyl-5,6,7,8-tetrahydropterin at neutral pH appears to proceed with significant side-chain loss in Tris buffer but not in phosphate buffer.

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