Reversible dissociation of enzymes at high dilutions and their inhibition by polyanions

Bernfeld, P.; Berkeley, Bronwyn; Bieber, Robert E.

doi:10.1016/0003-9861(65)90319-x

Cited by 57 publications

(16 citation statements)

References 24 publications

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“…0.16 jxg (protein)/ml. The effect was only shown with insoluble starch, and so, it would appear to be a different phenomenon to that reported by Bernfeld et al [2].…”

Section: Discussionmentioning

confidence: 35%

“…Triton X-100 and albumin, for instance, have been shown to protect ß-amylase, glucoamylase and amylase (B. subtilis) from inactivation at high dilutions [19], A decrease in the specific activity of a number of enzymes upon high dilution, e.g., aldo lase, LDH, porcine a-amylase and ß-amylase, has been reported [2]. In the case of a-amylase, the specific activity was reduced 2-fold at a concentration of 0.005 fxg enzyme/ml.…”

Section: Discussionmentioning

confidence: 99%

“…The inhi bition caused by the anionic detergent dodecylbenzene sodium sulphonate (table II) is probably due to enzyme dénaturation. Polyanions are known to cause enzyme inhibition [2], On the other hand, Walker and Hope [20] demonstrated that the amylolytic degradation of maize starch granules was enhanced (30%) by bile and deoxycholate. The effect was believed due to the disruption of the granule rather than to an effect on amylase itself.…”

Section: Discussionmentioning

confidence: 99%

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Activation of α-Amylase by Protein and Detergents1

O’Donnell¹,

McGeeney²

1974

Enzyme

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Optimal salt concentration for α-amylase hydrolysis of the PHADEBAS starch substrate was 50 mmol/1 NaCl, and 50 mmol/1 phosphate buffer (pH 7.0). Under these conditions, 7 μmol/l albumin increased the activity of the enzyme 1.3-fold. The degree of activation was shown to depend on pH. Activation was greatest in the more acid region. At pH 5.4 with 7 μmol/1 albumin, there was a 6-fold increase in activity over the control. The degree of activation fell to 2-fold at pH 6.0. A similar trend in activation versus pH was shown with the bile salt sodium taurodeoxycholate, the neutral detergent Triton X-100 and the polymer polyethylene glycol. The degree of activation, however, was less than with protein. With 1.25 mmol/1 bile salt, the activation was 2.5-fold at pH 5.4, falling to 1.4-fold at pH 6.0. Albumin caused an increase in Vmax accompanied by an apparent decrease in K(m). With taurodeoxycholate, V(max) was increased without change in the apparent K(m). The possible mechanism of activation is discussed.

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“…0.16 jxg (protein)/ml. The effect was only shown with insoluble starch, and so, it would appear to be a different phenomenon to that reported by Bernfeld et al [2].…”

Section: Discussionmentioning

confidence: 35%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Activation of α-Amylase by Protein and Detergents1

O’Donnell¹,

McGeeney²

1974

Enzyme

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“…The decrease at c < 4 pg/ml may be attributed to concentration-dependent deactivation [29,30] and/or insufficient time of reactivation. The final recovery of enzymic activity compared to the activity of native aldolase incubated under identical conditions (except for the acid treatment) is about 95% in the whole concentration range under concern.…”

Section: Concentration Dependence Of Reactivation and Renaturationmentioning

confidence: 99%

Equilibrium Studies on the Refolding and Reactivation of Rabbit‐Muscle Aldolase after Acid Dissociation

Engelhard

Rudolph

Jaenicke

1976

European Journal of Biochemistry

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Dissociation, denaturation, and deactivation of aldolase from rabbit muscle in the acid pH range have been investigated using sedimentation analysis, fluorescence, circular dichroism, and activity tests. Under comparable experimental conditions the pH-dependent profiles of deactivation and denaturation parallel the dissociation of the enzyme. In the range of dissociation at pH 4 -5 tetramers and monomers are in equilibrium. Intrinsic chromophores and far-ultraviolet circular dichroism suggest the transition to be a complex multistep process.At pH x 2.3 the enzyme is split into its fully inactive monomers which still contain some residual secondary structure. After reassociation under optimum conditions (0.2 M phosphate buffer pH 7.6, 1 mM EDTA, 0.1 mM dithiothreitol, 0 "C, enzyme concentration 0.4 -50 pg/ml) up to 95 % enzymic activity is recovered which belongs to a renatured tetrameric species indistinguishable from the native enzyme by all available biochemical and physicochemical criteria.There is well documented experimental evidence that oligomeric proteins, after deactivation and disruption of their three-dimensional structure under certain denaturing conditions, will spontaneously reassume an active conformation after removing the denaturant. Taking rabbit muscle aldolase as an example, reversible denaturation may be accomplished by a variety of denaturing and/or dissociating agents [l -171. As a result association has been considered to be a prerequisite of full enzymic activity [2,6,18]. On the other hand certain pieces of evidence point to active subunits [13,. This raises the question concerning the significance of enzyme subunit structure in cases where allosteric regulation does not seem to be of importance. The present renaturation experiments have been undertaken in order to elucidate the effect of refolding and quaternary structure formation on the catalytic properties of rabbit muscle aldolase. To compare the product of reactivation after acid dissociation with the native enzyme in its initial state a thorough analysis of the native, dissociated, and reassociated enzyme was performed. MATERIALS AND METHODSFructose-l,6-bisphosphate aldolase from rabbit muscle, fructose 1,6-bisphosphate, NADH, and the mixture of glycerol-3-phosphate dehydrogenase and triose phosphate isomerase for the coupled aldolase assay were purchased from Boehringer (Mannheim). Bovine serum albumin was a product of Serva (Heidelberg). Trypsin obtained from Serva and Boehringer was treated with L-( 1-tosylamido-2-pheny1)-ethyl chloromethyl ketone (Serva) to inhibit the proteolytic activity of chymotrypsin [22]. Dithiothreitol was purchased from Calbiochem (Lucerne). All other reagents were A-grade substances from Merck (Darmstadt). Quartz bidistilled water was used throughout.Stock solutions of the enzyme (c = 4 mg/ml) were prepared by repeated dialysis at 4 "C against potassium phosphate buffer pH 7.6, 0.2 M, containing 1 mM EDTA and 0.1 mM dithiothreitol. Aldolase activity was determined in 0.05 M triethanolamine buffer pH...

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“…The activation, however, was not thought to be due to the prevention of loss of activity during preincubation. O n the other hand, Bernfeld et al (37) found that various polycations activated rabbitmuscle aldolase presumably by preventing the dissociation of the enzyme (see Section I.B.3). These authors reported that a variety of polyanions (sulfated polymers) inhibited this enzyme.…”

Section: Rabbit-muscle Aldolasementioning

confidence: 99%

Interactions of Polynucleotides and Other Polyelectrolytes with Enzymes and Other Proteins

Elbein¹

1974

Advances in Enzymology - And Related Areas of Molecular Biology

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Reversible dissociation of enzymes at high dilutions and their inhibition by polyanions

Cited by 57 publications

References 24 publications

Activation of α-Amylase by Protein and Detergents1

Activation of α-Amylase by Protein and Detergents1

Equilibrium Studies on the Refolding and Reactivation of Rabbit‐Muscle Aldolase after Acid Dissociation

Interactions of Polynucleotides and Other Polyelectrolytes with Enzymes and Other Proteins

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