scholarly journals Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver

1980 ◽  
Vol 187 (3) ◽  
pp. 623-636 ◽  
Author(s):  
K S Ramesh ◽  
N Appaji Rao
Keyword(s):  
Liver Enzyme ◽  
Thermal Inactivation ◽  
Heat Stability ◽  
Serine Hydroxymethyltransferase ◽  
Heat Denaturation ◽  
Monkey Liver ◽  
Catalytically Active ◽  
Antibody Complex ◽  
Bacterial Sources ◽  
Blue Sepharose

The homogeneous serine hydroxymethyltransferase purified from monkey liver, by the use of Blue Sepharose affinity chromatography, exhibited positive homotropic co-operative interactions (h = 2.5) with tetrahydrofolate and heterotropic interactions with L-serine and nicotinamide nucleotides. The enzyme had an unusually high temperature optimum of 60 degrees C and was protected against thermal inactivation by L-serine. The allosteric effects were abolished when the monkey liver enzyme was purified by using a heat-denaturation step in the presence of L-serine, a procedure adopted by earlier workers for the purification of this enzyme from mammalian and bacterial sources. The enzyme activity was inhibited completely by N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, dichloromethotrexate, aminopterin and D-cycloserine, whereas methotrexate and dihydrofolate were partial inhibitors. The insoluble monkey liver enzyme-antibody complex was catalytically active and failed to show positive homotropic co-operative interactions with tetrahydrofolate (h = 1) and heterotropic interactions with NAD+. The enzyme showed a higher heat-stability in a complex with its antibody than as the free enzyme. These results highlight the pitfalls in using a heat-denaturation step in the purification of allosteric enzymes.

scholarly journals Inhibition of monkey liver serine hydroxymethyltransferase by Cibacron Blue 3G-A

1980 ◽  
Vol 187 (1) ◽  
pp. 249-252 ◽  
Author(s):  
K S Ramesh ◽  
N Appaji Rao
Keyword(s):  
Absorption Spectrum ◽  
Binding Domain ◽  
Serine Hydroxymethyltransferase ◽  
Free Enzyme ◽  
Cibacron Blue ◽  
Monkey Liver ◽  
Dye Absorption ◽  
Blue Sepharose

Cibacron Blue 3G-A inhibited monkey liver serine hydroxymethyltransferase competitively with respect to tetrahydrofolate and non-competitively with respect to L-serine. NADH, a positive heterotropic effector, failed to protect the enzymes against inhibition by the dye and was unable to desorb the enzyme from Blue Sepharose CL-6B gel matrix. The binding of the dye to the free enzyme was confirmed by changes in the dye absorption spectrum. The results indicate that the dye probably binds at the tetrahydrofolate-binding domain of the enzyme, rather than at the ‘dinucleotide fold’.

scholarly journals Thermal inactivation and chaperonin-mediated renaturation of mitochondrial aspartate aminotransferase

1998 ◽  
Vol 334 (1) ◽  
pp. 219-224 ◽  
Author(s):  
James M. LAWTON ◽  
Shawn DOONAN
Keyword(s):  
Escherichia Coli ◽  
Aspartate Aminotransferase ◽  
Thermal Inactivation ◽  
Active Form ◽  
Active Conformation ◽  
E Coli ◽  
Catalytically Active ◽  
Mitochondrial Aspartate Aminotransferase ◽  
Chaperonin 10 ◽  
Chaperonin 60

Mitochondrial aspartate aminotransferase is inactivated irreversibly on heating. The inactivated protein aggregates, but aggregation is prevented by the presence of the chaperonin 60 from Escherichia coli (GroEL). The chaperonin increases the rate of thermal inactivation in the temperature range 55–65 °C but not at lower temperatures. It has previously been shown [Twomey and Doonan (1997) Biochim. Biophys. Acta 1342, 37–44] that the enzyme switches to a modified, but catalytically active, conformation at approx. 55–60 °C and the present results show that this conformation is recognized by and binds to GroEL. The thermally inactivated protein can be released from GroEL in an active form by the addition of chaperonin 10 from E. coli (GroES)/ATP, showing that inactivation is not the result of irreversible chemical changes. These results suggest that the irreversibility of thermal inactivation is due to the formation of an altered conformation with a high kinetic barrier to refolding rather than to any covalent changes. In the absence of chaperonin the unfolded molecules aggregate but this is a consequence, rather than the cause, of irreversible inactivation.

scholarly journals Preparation of Whey Protein Concentrates with Good Heat Stability Using Heat Denaturation

2010 ◽  
Vol 11 (4) ◽  
pp. 215-219
Author(s):  
Yuzoh ASANO ◽  
Manabu SUZUKI ◽  
Hiroshi ARASE ◽  
Naoki YUDA ◽  
Hitoshi SAITO ◽  
...  
Keyword(s):  
Whey Protein ◽  
Heat Stability ◽  
Heat Denaturation ◽  
Protein Concentrates ◽  
Whey Protein Concentrates ◽  
Good Heat

scholarly journals Endolysins from Antarctic Pseudomonas Display Lysozyme Activity at Low Temperature

Marine Drugs ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. 579
Author(s):  
Marco Orlando ◽  
Sandra Pucciarelli ◽  
Marina Lotti
Keyword(s):  
Low Temperature ◽  
Thermal Inactivation ◽  
Residual Activity ◽  
Heat Stability ◽  
Evolutionary Analysis ◽  
Glycosyl Hydrolases ◽  
Bioinformatics Analyses ◽  
Cold Environments ◽  
Local Flexibility ◽  
Cold Activity

Organisms specialized to thrive in cold environments (so-called psychrophiles) produce enzymes with the remarkable ability to catalyze chemical reactions at low temperature. Cold activity relies on adaptive changes in the proteins’ sequence and structural organization that result in high conformational flexibility. As a consequence of flexibility, several such enzymes are inherently heat sensitive. Cold-active enzymes are of interest for application in a number of bioprocesses, where cold activity coupled with easy thermal inactivation can be of advantage. We describe the biochemical and functional properties of two glycosyl hydrolases (named LYS177 and LYS188) of family 19 (GH19), identified in the genome of an Antarctic marine Pseudomonas. Molecular evolutionary analysis placed them in a group of characterized GH19 endolysins active on lysozyme substrates, such as peptidoglycan. Enzyme activity peaks at about 25–35 °C and 40% residual activity is retained at 5 °C. LYS177 and LYS188 are thermolabile, with Tm of 52 and 45 °C and half-lives of 48 and 12 h at 37 °C, respectively. Bioinformatics analyses suggest that low heat stability may be associated to temperature-driven increases in local flexibility occurring mainly in a specific region of the polypeptide that is predicted to contain hot spots for aggregation.

scholarly journals Regulation of monkey liver serine hydroxymethyltransferase by nicotinamide nucleotide

1978 ◽  
Vol 174 (3) ◽  
pp. 1055-1058 ◽  
Author(s):  
K S Ramesh ◽  
N A Rao
Keyword(s):  
Serine Hydroxymethyltransferase ◽  
Monkey Liver ◽  
First Time ◽  
Metabolic Interconversion ◽  
Time Lead

The positive homotropic binding of tetrahydrofolate to monkey liver serine hydroxymethyltransferase was abolished on preincubating the enzyme with NADH and NADPH. NAD+ was a negative heterotropic effector, whereas NADP+ was without effect. The allosteric effects of nicotinamide nucleotides on the serine hydroxymethyltransferase, reported for the first time, lead to a better understanding of the regulation of the metabolic interconversion of folate coenzymes.

Heat stability improvement of antimicrobial whey proteins fromyak milk and colostrum at various acidic conditions

2016 ◽  
Vol 35 (3) ◽  
Author(s):  
Shimo Peter Shimo ◽  
W U Xiaoyun ◽  
Ding Xuezhi ◽  
Yan Ping
Keyword(s):  
Whey Proteins ◽  
Sodium Dodecyl ◽  
Heat Stability ◽  
Heat Denaturation ◽  
Percentage Reduction ◽  
Processing Conditions ◽  
Yak Milk ◽  
Acidic Conditions ◽  
The Mean ◽  
Mean Concentration

The aim of this study was to characterize the effect of heat denaturation on Immunoglobulin G (IgG) and Lactoferrin (LF) in yak milk and colostral liquid whey at medium acidic conditions in presence of protectants (CaCl2; Glycerol and Sodium Dodecyl Sulphate - SDS). Results indicated significant (P less than 0.05) heat stability improvement of IgG and LF in liquid whey samples at medium acidic and temperature (72 - 90oC) with less precipitates formation regardless of type of protectants added especially at pH 3.5 and 4.6. The mean concentration values for IgG percentage reduction (72 – 90oC; pH 3.5 – 5.5; protectants) ranged from 6 to 26% and 15 to 31% in yak milk and colostral liquid whey, respectively. The percentage of heat denaturation effect for LF ranged from 11 to 32%t and 14 to 38% in yak milk and colostral liquid whey, respectively. Application of glycerol in both environmental processing conditions was the most effective in heat stability improvement followed by SDS and CaCl2.

scholarly journals Serological and fluorescence studies of the heat stability of bovine lactoferrin

1979 ◽  
Vol 46 (1) ◽  
pp. 83-93 ◽  
Author(s):  
Augustin Baer ◽  
Marko Oroz ◽  
Bernard Blanc
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Complement Fixation ◽  
Heat Stability ◽  
Heat Denaturation ◽  
Bovine Lactoferrin ◽  
Iron Binding ◽  
Binding Ability ◽  
Fluorescence Studies ◽  
Tryptophan Residues

SUMMARYThe heat denaturation of Fe-saturated lactoferrin (If) and Fe-free lactoferrin (apo-lf) was studied using the methods of micro-complement fixation and fluorescence. It was established that the change in conformation of apo-lf, induced by iron binding, conferred a higher heat stability to the molecule: the changes were observed at temperatures above 40 °C for apo-lf and above 60 °C for If. The Fe-binding ability of the protein was partially independent of the degree of denaturation. Fluorescence analyses indicated that tryptophan residues were probably not directly involved in the metal binding. There was no evidence of antibodies interfering with the binding sites.

Factors affecting the action of rennin in heated milk

1972 ◽  
Vol 39 (3) ◽  
pp. 413-419 ◽  
Author(s):  
G. A. Wilson ◽  
J. V. Wheelock
Keyword(s):  
Complex Formation ◽  
Heat Stability ◽  
Primary Phase ◽  
Effect Of Temperature ◽  
Heat Denaturation ◽  
Clotting Time ◽  
Factors Affecting ◽  
Heated Milk ◽  
Whole Milk ◽  
Β Lactoglobulin

SummaryThe effect of temperature and time of heating whole milk on the renninclotting time, the primary phase of rennin action and the protein (mainly β-lactoglobulin) soluble in 2% trichloroacetic acid (TCA), have been studied. Considerable changes in these parameters occurred above 60°C. The primary phase was inhibited (the degree of inhibition being both temperature and time-dependent), the clotting time was increased, and the protein soluble in 2% TCA decreased considerably.It is suggested that the inhibition of the primary phase was due to complex formation between κ-casein and β-lactoglobulin, the increase in clotting time to a combination of complex formation and a change in the distribution of Ca, and the decrease in β-lactoglobulin to both its interaction with κ-casein and its heat denaturation. The relevance of such changes to the heat stability of milk is discussed.

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