Remi E. Amelunxen

Crystallization of thermostable glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

Abstract 1. 1. A procedure for the crystallization of homogeneous glyceraldehyde-3-phosphate dehydrogenase ( D -glyceraldehyde-3-phosphate: DPN + oxidoreductase (phosphorylating) (EC 1.2.1.12)) from the thermophile Bacillus stearothermophilus is described. 2. 2. Data are presented demonstrating the homogeneity of this enzyme and its remarkable thermostability characteristics.

Comparative thermostability of enzymes from Bacillus stearothermophilus and Bacillus cereus.

Abstract The activity and thermostability of eleven enzymes from Bacillus stearothermophilus and Bacillus cereus were compared. The thermophilic enzymes with two exceptions showed much greater thermostability than their mesophilic counterparts. Thermophilic protein showed greater resistance to precipitation than mesophilic protein at various concentrations and temperatures. Data obtained from mixtures of thermophilic and mesophilic protein suggested the absence of stabilizing factors in the cellular environment of the thermophile.

Some chemical and physical properties of thermostable glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus

Abstract 1. 1. Data concerning pH optima, pH inactivation curves, general enzyme kinetics and optimal temperature for assay are presented for crystalline glyceraldehyde-3-phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate: DPN+ oxido-reductase (phosphorylating), EC 1.2.1.12) from Bacillus stearothermophilus and from rabbit muscle. 2. 2. Using column density-gradient procedures, the partial specific volume was found to be 0.754 ml/g. The molecular weight for the thermophilic enzyme was calculated as 130 000 by the approach to equilibrium method. 3. 3. Sulfhydryl titration of the thermophilic enzyme revealed 2–3 titratable groups in contrast to 13–14 for the muscle enzyme. p-Hydroxymecuribenzoate inhibition of the thermophilic enzyme was completely reversible by cystein, whereas the muscle enzyme showed only partial reversibility. 4. 4. When dissolved in 8.0 M urea, the thermophilic enzyme showed no change in optical rotation from the native state and under the assay conditions used, there was only a slight loss of enzymic activity; in contrast, the muscle enzyme showed a large negative increase in optical rotation and complete loss of enzymic activity. 5. 5. The thermophilic enzyme is extensively inactivated by DPNH; incubation with DPNH at 60° is necessary to attain the extensive inactivation obtained with the muscle enzyme at 37°.

The crystallization and properties of glyceraldehyde-3-phosphate dehydrogenase isolated from rabbit muscle by a simplified procedure

1. 1. A simplified procedure for the crystallization of glyceraldehyde-3-phosphate dehydrogenase (dglyceraldehyde-3-phosphate:DPN+ oxidoreductase (phosphorylating), EC 1.2.1.12) from rabbit muscle is described. 2. 2. The inactivation of this enzyme by reduced diphosphopyridine nucleotide has been studied with this preparation.

A proposed mechanism of thermophily in facultative thermophiles

Glyceraldehyde-3-phosphate dehydrogenase in crude extracts of Bacillus coagulans KU, a facultative thermophile, showed marked thermolability whether the cells were grown at mesophilic or thermophilic temperature. When extracts were brought to a given ionic strength by the addition of salt and then heat treated, it was possible to confer heat resistance well in excess of the thermophilic growth temperature. Disc electrophoresis is indicative that portions of the profiles are quite different between extracts of cells grown at 37° and 55°. Based on the data, a mechanism of thermophily is postulated that is different from any thus far proposed for thermophilic microorganisms.

Studies on the subunit structure of thermostable glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus☆

Abstract Sedimentation equilibrium studies of the thermophilic glyceraldehyde-3-phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate: NAD oxidoreductase (phosphorylating), EC 1.2.1.12) in 5.0 m guanidine-HCl showed that the enzyme is dissociated into monodisperse subunits having a molecular weight of 36, 000 ± 1,200. Based on the molecular weight of the undissociated enzyme, it is concluded that it exists as a tetramer in the native state. C-terminal amino acid analyses revealed a single-type amino acid, leucine, suggesting identical subunits. In kinetic studies, the thermophilic enzyme showed marked resistance to enzymic inactivation by 8.0 m urea at mesophilic temperatures, but is rapidly inactivated in this solvent at thermophilic temperatures; some reversibility is detectable at all temperatures. However, in guanidine-HCl, the thermophilic enzyme is rapidly and irreversibly inactivated at all temperatures studied. Optical rotatory dispersion studies showed that the thermophilic enzyme is extensively unfolded in these denaturing solvents at 30 ° and above. In contrast, heating the enzyme in water resulted in an alteration in secondary structure that was concomitant with an increase in enzymic activity. The possible mechanism of inactivation by urea and guanidine-HCl is discussed.

Coenzyme binding to glyceraldehyde-3-phosphate dehydrogenase after modification by substrate and coenzyme

Summary 1. The coenzyme-binding characteristics of glyceraldehyde-3-phosphate dehydrogenase ( D -glyceraldehyde-3-phosphate:DPN+ oxidoreductase(phosphorylating), EC 1.2.1.12) are altered when the enzyme is incubated with DPNH or DPNH and glyceraldehyde-3-phosphate. Extensive modification occurs only in the presence of oxygen and parallels the loss of enzymic activity. 2. The modified enzyme binds the same mole-ratio of DPNH as does enzyme which has had the DPN+ removed by mixing with charcoal, i.e. 3 moles per mole of protein. However, this binding does not quench, but rather enhances DPNH fluorescence as is the case for all other pyridine nucleotide-linked dehydrogenases thus far tested.

Thermophilic Glyceraldehyde-3-P Dehydrogenase

D-Glyceraldehyde-3-P:NAD oxidoreductase (phosphorylating) (E.C. 1.2.1.12) (abbr. GPDH) is a ubiquitous enzyme, found in most living systems, where it functions in the glycolytic pathway. There were several reasons for studying the enzyme in thermophilic bacteria. First, at the time this enzyme was characterized in the obligate thermophile Bacillus stearothermophilus (1503), no intracellular enzymes from thermophilic bacteria had been isolated in highly purified form. Second, the enzyme had been well characterized in many other systems, thereby providing a sound basis for comparison with the thermophilic enzyme. Third, GPDH is almost always present within cells at very high concentrations, thereby providing a large quantity of potential material necessary for extensive physicochemical analyses. Finally, was the demonstration by Amelunxen and Lins (1), that the enzyme in crude extracts from B. stearothermophilus was indeed remarkably thermostable, as seen in Table I.

Thermostable glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus I. Immunochemical studies

Abstract 1. 1. In this investigation, some immunological properties of thermophilic glyceraldehyde-3-phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate:NAD + oxido-reductase (phosphorylating), EC 1.2.1.12) were compared with those of its counterparts from yeast and rabbit muscle. 2. 2. The maximum inhibition of enzymic activity by homologous antiserum was 94% for the yeast enzyme, 96% for the thermophilic enzyme and 50% for the rabbit muscle enzyme. No cross inhibition could be demonstrated with any of the enzymes and their heterologous antisera. 3. 3. Preincubation of the thermophilic enzyme with NAD + had no effect on inhibition by homologous antiserum, in contrast to decreased inhibition observed with the yeast and rabbit muscle enzyme. 4. 4. Based on quantitative precipitin tests, 1.3 times the amount of homologous antibody is precipitated by the thermophilic enzyme compared to the yeast enzyme. By contrast, 7 times the amount of antibody is required for maximum inhibition of the thermophilic enzyme. 5. 5. No cross reaction could be demonstrated with any of the enzymes and their heterologous antisera using the quantitative precipitin test, immunodiffusion or immunoelectrophoresis. 6. 6. Data involving the effect of denaturants demonstrated that there are marked differences between the thermophilic enzyme and the yeast enzyme in terms of enzymic activity and immunological reactivity.

Absence of ACTH-Like Activity in Nocardia Asteroides

Abstract Seventeen-hydroxycorticosteroid determinations were done on adrenal effluent blood from intact and hypophysectomized dogs before and after injection of ACTH, Nocardia asteroides lysate, and supernatent culture medium. The lysate and culture medium caused an increase in secretion of 17-OHCS in the intact animals but not in the hypophysectomized animals. This indicates that the organism does not form an ACTH-like material previously suggested as an explanation for the simultaneous occurrence of nocardiosis and Cushings syndrome.