scholarly journals Synthesis of pyruvate carboxylase from its apoenzyme and (+)-biotin in Bacillus stearothermophilus. Mechanism and control of the reaction

1971 ◽  
Vol 122 (5) ◽  
pp. 663-669 ◽  
Author(s):  
T. K. Sundaram ◽  
J. J. Cazzulo ◽  
H. L. Kornberg
Keyword(s):  
Pyruvate Carboxylase ◽  
Thermal Denaturation ◽  
Bacillus Stearothermophilus ◽  
The Other ◽  
Acetyl Coa ◽  
Allosteric Effector ◽  
Allosteric Effectors ◽  
Hill Coefficients ◽  
And Control ◽  
The Hill

1. Acetyl-CoA acts as a positive allosteric effector in the formation of active pyruvate carboxylase from its apoenzyme, ATP and (+)-biotin which is catalysed by holoenzyme synthetase; this effect is counteracted by l-aspartate. 2. The Hill coefficients (apparent n values) were approximately 2 for acetyl-CoA and 4 for l-aspartate; the n value for each effector remained constant when the concentration of the other effector was varied. 3. Active pyruvate carboxylase was formed also when the apoenzyme was incubated with holoenzyme synthetase and synthetic biotinyl-5′-AMP; acetyl-CoA and l-aspartate affected this process as they did the overall reaction from (+)-biotin and ATP. 4. When hydroxylamine replaced the apoenzyme, holoenzyme synthetase catalysed the formation of biotinylhydroxamate from (+)-biotin and ATP. This reaction was not affected by the allosteric effectors. 5. The apoenzyme was protected against thermal denaturation by acetyl-CoA and, to a lesser degree, by l-aspartate. The holoenzyme synthetase was not markedly protected by these effectors. 6. It is concluded that the allosteric effectors act on the apoenzyme and not the synthetase.

scholarly journals The atypical velocity response by pyruvate carboxylase to increasing concentrations of acetyl-coenzyme A

1979 ◽  
Vol 179 (3) ◽  
pp. 497-502 ◽  
Author(s):  
Simon B. Easterbrook-Smith ◽  
Anne J. Campbell ◽  
D. Bruce Keech ◽  
John C. Wallace
Keyword(s):  
Velocity Profile ◽  
Pyruvate Carboxylase ◽  
Degree Of Saturation ◽  
Acetyl Coa ◽  
Reaction Velocity ◽  
Low Protein ◽  
Allosteric Effector ◽  
Velocity Response ◽  
Hill Coefficients ◽  
Hydrolysis Of

An investigation was made of the interaction of pyruvate carboxylase with its allosteric effector, acetyl-CoA, and the velocity profile of the deacylation of acetyl-CoA as a function of acetyl-CoA concentration indicated that this ligand does not bind to this enzyme in a positive homotropic co-operative manner. An examination was therefore made of the factors that contribute to the sigmoidicity of the rate curves obtained for pyruvate carboxylation with various concentrations of acetyl-CoA. Hill coefficients for acetyl-CoA obtained with both sheep and chicken liver pyruvate carboxylases were found to be dependent on the fixed pyruvate concentration used in the assay solution. Thus, by varying the acetyl-CoA concentration, the degree of saturation of the enzyme by pyruvate was also changed. A further consequence of non-saturating concentrations of pyruvate was that the non-productive hydrolysis of the enzyme– carboxybiotin complex increased, resulting in an under-estimate of the reaction velocity measured by oxaloacetate formation. Another factor contributing to the sigmoidicity is that, at non-saturating concentrations of acetyl-CoA, the enzyme undergoes inactivation upon dilution to low protein concentrations, again resulting in an under-estimate of the reaction velocity. Under conditions where none of the above factors was operating and the only effect of varying acetyl-CoA concentrations was to alter the proportion of the enzyme catalysing the carboxylation reaction at acetyl-CoA-dependent and -independent rates, the sigmoidicity of the acetyl-CoA velocity profile was completely eliminated.

Properties and regulation of pyruvate carboxylase from Bacillus stearothermophilus

1970 ◽  
Vol 176 (1042) ◽  
pp. 1-19 ◽  
Keyword(s):  
Enzyme Activity ◽  
Pyruvate Carboxylase ◽  
Bacillus Stearothermophilus ◽  
Kinetic Studies ◽  
Allosteric Effectors ◽  
Regulatory Properties

Pyruvate carboxylase has been purified 400-fold from the thermophile, Bacillus stearothermophilus; it resembles pyruvate carboxylases purified from mesophilic organisms in its general kinetic and regulatory properties. The enzyme is virtually inactive in the absence of acetylcoenzyme A ; this activating effect is antagonized by L-aspartate. Kinetic studies show that these two compounds act as allosteric effectors. ADP inhibits the enzyme activity competitively with ATP. Although the thermophile enzyme is appreciably more thermostable than similar mesophile enzymes, it is quite labile at the temperature at which the organism grows optimally, but can be stabilized by the two allosteric effectors and by some of the reactants.

scholarly journals Synthesis of pyruvate carboxylase from its apoenzyme and (+)-biotin in Bacillus stearothermophilus. Purification and properties of the apoenzyme and the holoenzyme synthetase

1971 ◽  
Vol 122 (5) ◽  
pp. 653-661 ◽  
Author(s):  
J. J. Cazzulo ◽  
T. K. Sundaram ◽  
Susan N. Dilks ◽  
H. L. Kornberg
Keyword(s):  
Molecular Weight ◽  
Electrophoretic Mobility ◽  
Pyruvate Carboxylase ◽  
Bacillus Stearothermophilus ◽  
Gel Filtration ◽  
Homogeneous State ◽  
Holocarboxylase Synthetase ◽  
Acetyl Coa ◽  
Purification And Properties

1. Methods are described for the assay and purification of pyruvate apocarboxylase and pyruvate holocarboxylase synthetase from biotin-deficient Bacillus stearothermophilus. 2. Pyruvate apocarboxylase was obtained 200-fold purified and in a nearly homogeneous state; it closely resembled the holoenzyme of the thermophile in fractionation properties, electrophoretic mobility and molecular weight (estimated to be 350000 by gel filtration). 3. Pyruvate holocarboxylase synthetase, purified more than 50-fold, was estimated to have a molecular weight of approx. 40000. 4. The conversion of the purified apoenzyme into the holoenzyme required the presence of the synthetase, ATP (Km3.3×10−7m), (+)-biotin (Km7.5×10−8m) and Mg2+; it differed from the conversions effected by systems forming other carboxylases in mesophilic organisms in also requiring the presence of acetyl-CoA.

scholarly journals Structure, mechanism and regulation of pyruvate carboxylase

2008 ◽  
Vol 413 (3) ◽  
pp. 369-387 ◽  
Author(s):  
Sarawut Jitrapakdee ◽  
Martin St Maurice ◽  
Ivan Rayment ◽  
W. Wallace Cleland ◽  
John C. Wallace ◽  
...  
Keyword(s):  
Active Site ◽  
Pyruvate Carboxylase ◽  
Polypeptide Chain ◽  
Tricarboxylic Acid Cycle ◽  
Biotin Carboxylase ◽  
Acetyl Coa ◽  
Specific Expression ◽  
Allosteric Effector ◽  
Bona Fide ◽  
And Function

PC (pyruvate carboxylase) is a biotin-containing enzyme that catalyses the HCO3−- and MgATP-dependent carboxylation of pyruvate to form oxaloacetate. This is a very important anaplerotic reaction, replenishing oxaloacetate withdrawn from the tricarboxylic acid cycle for various pivotal biochemical pathways. PC is therefore considered as an enzyme that is crucial for intermediary metabolism, controlling fuel partitioning toward gluconeogenesis or lipogenesis and in insulin secretion. The enzyme was discovered in 1959 and over the last decade there has been much progress in understanding its structure and function. PC from most organisms is a tetrameric protein that is allosterically regulated by acetyl-CoA and aspartate. High-resolution crystal structures of the holoenzyme with various ligands bound have recently been determined, and have revealed details of the binding sites and the relative positions of the biotin carboxylase, carboxyltransferase and biotin carboxyl carrier domains, and also a unique allosteric effector domain. In the presence of the allosteric effector, acetyl-CoA, the biotin moiety transfers the carboxy group between the biotin carboxylase domain active site on one polypeptide chain and the carboxyltransferase active site on the adjacent antiparallel polypeptide chain. In addition, the bona fide role of PC in the non-gluconeogenic tissues has been studied using a combination of classical biochemistry and genetic approaches. The first cloning of the promoter of the PC gene in mammals and subsequent transcriptional studies reveal some key cognate transcription factors regulating tissue-specific expression. The present review summarizes these advances and also offers some prospects in terms of future directions for the study of this important enzyme.

scholarly journals Ligand-induced conformational transitions and secondary-structure composition of chicken liver pyruvate carboxylase

1979 ◽  
Vol 177 (2) ◽  
pp. 697-705 ◽  
Author(s):  
Karen S. McGurk ◽  
H. Olin Spivey
Keyword(s):  
Secondary Structure ◽  
Pyruvate Carboxylase ◽  
Conformational Transitions ◽  
Sulphonic Acid ◽  
Chicken Liver ◽  
Stopped Flow ◽  
Acetyl Coa ◽  
Allosteric Effector ◽  
Fluorescence Measurements ◽  
Secondary Structure Composition

Apparent conformational transitions induced in chicken liver pyruvate carboxylase by substrates, KHCO3 and MgATP, and the allosteric effector, acetyl-CoA, were studied by using the fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid and c.d. Fluorescence measurements were made with both conventional and stopped-flow spectrophotometers. Additions of acetyl-CoA and/or ATP to the enzyme-probe solutions quenched fluorescence of the probe by the following cumulative amounts regardless of the sequence of additions: acetyl-CoA, 10–13%; ATP, 21–24%; acetyl-CoA plus ATP, about 35%. Additions of KHCO3 had no effect on the fluorescence. The rates of quenching by acetyl-CoA and MgATP (in the presence of acetyl-CoA) were too rapid to measure by stopped-flow kinetic methods, but kinetics of the MgATP effect (in the absence of acetyl-CoA) indicate three unimolecular transitions after the association step. The negligible effect of the probe on enzyme catalytic activity, a preservation of the near-u.v. c.d. effect of MgATP and acetyl-CoA in the presence of the probe and no observable unimolecular transitions after binding of the probe to the enzyme indicate that the probe had no deleterious effect on the enzyme. In contrast with results with 8-anilinonaphthalene-1-sulphonic acid, fluorescence of the ε-derivative of acetyl-CoA or ATP [fluorescent analogues; Secrist, Barrio, Leonard & Weber (1972) Biochemistry11, 3499–3506] was not changed when either one was added to the enzyme. Secondary-structure composition of chicken liver pyruvate carboxylase estimated from the far-u.v. c.d. spectrum of the enzyme is 27% helix, 7% β-pleated sheet and 66% other structural types.

Pyruvate carboxylase from Thiobacillus novellus: properties and possible function

1984 ◽  
Vol 30 (5) ◽  
pp. 532-539 ◽  
Author(s):  
A. M. Charles ◽  
D. W. Willer
Keyword(s):  
Initial Velocity ◽  
Pyruvate Carboxylase ◽  
Maximum Activity ◽  
Temperature Optimum ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Citrate Cycle ◽  
End Products ◽  
Sigmoidal Curve ◽  
Hill Coefficients

Pyruvate carboxylase (EC 6.4.1.1) from Thiobacillus novellus (ATCC 8093) was highly purified and found to have a pH optimum of 7.6, a temperature optimum of 25–35 °C, and a requirement for a monovalent and a divalent cation, as well as a CoA derivative, for maximum activity. These were best served by K+, Mg2+, and acetyl-CoA. Km values for pyruvate, ATP, HCO3− and Mg2+ were 0.25, 0.04, 0.27, and 0.44 mM, respectively. Initial velocity plots of increasing acetyl-CoA concentrations gave a sigmoidal curve with Ka of 4.2 μM, and Hill coefficients of 2.2. Plots of fixed acetyl-CoA concentrations against varying concentrations of pyruvate, ATP, or CO2 all gave rectangular hyperbolae. Apart from end products, only hydroxypyruvic acid was found to be inhibitory. The enzyme was very sensitive to mercurials. This enzyme is not believed to serve an anaplerotic function, because of the simultaneous presence of the highly regulated phosphoenolpyruvate carboxylase in the organism. Instead, it may function either to supply oxaloacetate to the citrate cycle or as part of the system that provides reduced NADP+.

scholarly journals Experiences and expectations regarding COVID-19 prevention and control measures among the hill tribe population of northern Thailand: a qualitative study

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Siwarak Kitchanapaibul ◽  
Anusorn Udplong ◽  
Tawatchai Apidechkul ◽  
Ratipark Tamornpark ◽  
Thanatchaporn Mulikaburt ◽  
...  
Keyword(s):  
Disease Prevention ◽  
Prevention And Control ◽  
Control Measures ◽  
The Other ◽  
Northern Thailand ◽  
Hill Tribe ◽  
Prevention And Control Measures ◽  
Hill Tribes ◽  
And Control ◽  
The Hill

Abstract Background COVID-19 has been a major human threat for a year. A large number of people have been infected and killed globally, including hill tribe people living in remote and border areas between Thailand and Myanmar. Different expectations of and experiences with the implemented disease prevention and control measures by local, national and international organizations have been widely reported. This study aimed to understand the experiences and expectations regarding the disease prevention and control measures that were implemented among hill tribe people in Thailand. Methods Qualitative data were collected from participants aged 20 and older who belonged to the hill tribes living on the border of northern Thailand and Myanmar. A semistructured questionnaire was used to guide interviews. Information was extracted for thematic analysis by the NVivo program. Results Fifty-seven participants (36 female, 21 male) were interviewed; 27 participants were Thai Yai, 14 participants were Yunnan Chinese, 8 participants were Akha, and 8 participants were from other tribes. The average age was 45.8 years (min = 20 years, max = 90 years). Thirty participants had never attended school, and the other 27 participants had received education at different levels, from primary school to higher education. Forty participants were unemployed, 13 worked as agriculturists, and the other 4 were attending school. Both positive experiences, such as improving personal hygiene practices, maintaining close contact and increasing relationships among family members and demonstrating the leadership of the villager leaders, and negative experiences, including interruption of social interactions, family financial problems, poor access to medical care services, and invisible people to the government, were found. Different expectations were observed regarding organizations at the local, national, and international levels. Expectations at the local level included villagers and community leaders taking action to strongly contribute to prevention and control measures and to prevent unscreened people from entering the village. Obtaining accurate information about the disease and being financially supported were expectations at the national level, while closing borders to protect cases from overflowing into their villages was an international-level expectation. Conclusion Although hill tribes reside in very remote rural areas, they experience both positive and negative effects of the disease prevention and control measures implemented by organizations. Their expectations are formally and informally voiced to policy makers at the local, national and international levels.

Prothrombin Time Standardization: Report of the Expert Panel on Oral Anticoagulant Control

1979 ◽  
Vol 42 (04) ◽  
pp. 1073-1114 ◽  
Keyword(s):  
Prothrombin Time ◽  
Expert Panel ◽  
The Other ◽  
Rabbit Brain ◽  
Calibration Constant ◽  
Freeze Dried ◽  
The Mean ◽  
Point Of Intersection ◽  
And Control ◽  
Standardization Report

SummaryIn collaborative experiments in 199 laboratories, nine commercial thromboplastins, four thromboplastins held by the National Institute for Biological Standards and Control (NIBS & C), London and the British Comparative Thromboplastin were tested on fresh normal and coumarin plasmas, and on three series of freeze-dried plasmas. One of these was made from coumarin plasmas and the other two were prepared from normal plasmas; in each series, one plasma was normal and the other two represented different degrees of coumarin defect.Each thromboplastin was calibrated against NIBS&C rabbit brain 70/178, from the slope of the line joining the origin to the point of intersection of the mean ratios of coumarin/normal prothrombin times when the ratios obtained with the two thromboplastins on the same fresh plasmas were plotted against each other. From previous evidence, the slopes were calculated which would have been obtained against the NIBS&C “research standard” thromboplastin 67/40, and termed the “calibration constant” of each thromboplastin. Values obtained from the freeze-dried coumarin plasmas gave generally similar results to those from fresh plasmas for all thromboplastins, whereas values from the artificial plasmas agreed with those from fresh plasmas only when similar thromboplastins were being compared.Taking into account the slopes of the calibration lines and the variation between laboratories, precision in obtaining a patient’s prothrombin time was similar for all thromboplastins.

Dry chip feedrate control using online chip moisture

TAPPI Journal ◽  
2018 ◽  
Vol 17 (05) ◽  
pp. 295-305
Author(s):  
Wesley Gilbert ◽  
Ivan Trush ◽  
Bruce Allison ◽  
Randy Reimer ◽  
Howard Mason
Keyword(s):  
Production Rate ◽  
Control Strategy ◽  
Rate Control ◽  
Near Infrared ◽  
Dry Mass ◽  
The Other ◽  
Process Variability ◽  
Moisture Sensor ◽  
Feedback Control Strategy ◽  
And Control

Normal practice in continuous digester operation is to set the production rate through the chip meter speed. This speed is seldom, if ever, adjusted except to change production, and most of the other digester inputs are ratioed to it. The inherent assumption is that constant chip meter speed equates to constant dry mass flow of chips. This is seldom, if ever, true. As a result, the actual production rate, effective alkali (EA)-to-wood and liquor-to-wood ratios may vary substantially from assumed values. This increases process variability and decreases profits. In this report, a new continuous digester production rate control strategy is developed that addresses this shortcoming. A new noncontacting near infrared–based chip moisture sensor is combined with the existing weightometer signal to estimate the actual dry chip mass feedrate entering the digester. The estimated feedrate is then used to implement a novel feedback control strategy that adjusts the chip meter speed to maintain the dry chip feedrate at the target value. The report details the results of applying the new measurements and control strategy to a dual vessel continuous digester.

From Container to Claustrum: Projective Identification in Couples

2014 ◽  
Vol 4 (2) ◽  
Author(s):  
Tamara Feldman
Keyword(s):  
Psychological Health ◽  
Projective Identification ◽  
The Self ◽  
The Other ◽  
Intimate Partners ◽  
And Control ◽  
The Relationship ◽  
As If

This paper is a contribution to the growing literature on the role of projective identification in understanding couples' dynamics. Projective identification as a defence is well suited to couples, as intimate partners provide an ideal location to deposit unwanted parts of the self. This paper illustrates how projective identification functions differently depending on the psychological health of the couple. It elucidates how healthier couples use projective identification more as a form of communication, whereas disturbed couples are inclined to employ it to invade and control the other, as captured by Meltzer's concept of "intrusive identification". These different uses of projective identification affect couples' capacities to provide what Bion called "containment". In disturbed couples, partners serve as what Meltzer termed "claustrums" whereby projections are not contained, but imprisoned or entombed in the other. Applying the concept of claustrum helps illuminate common feelings these couples express, such as feeling suffocated, stifled, trapped, held hostage, or feeling as if the relationship is killing them. Finally, this paper presents treatment challenges in working with more disturbed couples.

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