scholarly journals pH-induced kinetic co-operativity of a thylakoid-bound polyphenol oxidase

1992 ◽  
Vol 286 (2) ◽  
pp. 623-626 ◽  
Author(s):  
E Valero ◽  
F García-Carmona
Keyword(s):  
Steady State ◽  
Polyphenol Oxidase ◽  
Hill Coefficient ◽  
Lag Phase ◽  
Transition Mechanism ◽  
Steady State Rate ◽  
State Rate ◽  
Lag Period ◽  
Ph Conditions ◽  
First Time

A study of the catecholase activity of a latent plant polyphenol oxidase, extracted and purified from the chloroplast membranes of grapes (Vitis vinifera cv. Airen), revealed for the first time a lag phase above pH 5.0, whereas a steady-state rate was reached immediately when pH values were lower, thus suggesting the hysteretic nature of the enzyme. During steady state, the enzyme showed negative co-operativity concomitant with the presence of the lag period, and followed classical Michaelis-Menten kinetics under more acid pH conditions. Statistical analysis of these data showed a minimal value for the extreme Hill coefficient of 0.54 at pH 6.0. This kinetic behaviour of polyphenol oxidase has been interpreted in terms of the pH-induced ‘slow’ transition mechanism reported by Ricard, Noat & Nari [(1984) Eur. J. Biochem. 145, 311-317] in which the conformational change does not affect the active site of the enzyme.

scholarly journals Calcium-induced dissociation of human plasma factor XIII and the appearance of catalytic activity

1974 ◽  
Vol 141 (3) ◽  
pp. 683-691 ◽  
Author(s):  
Rodney D. Cooke
Keyword(s):  
Steady State ◽  
Protein Concentration ◽  
Factor Xiiia ◽  
Lag Phase ◽  
Conformation Change ◽  
Platelet Factor ◽  
Plasma Enzyme ◽  
Plasma Factor ◽  
Steady State Rate ◽  
State Rate

1. The Ca2+dependence of the activity of plasma Factor XIIIa was studied by using the continuous assay based on the incorporation of dansylcadaverine into dephosphorylated acetylated β-casein (β-substrate). The Km for Ca2+is about 0.170mm. 2. At low concentrations of Ca2+there was a lag in attaining the steady-state rate. The size of the lag was decreased and eventually abolished if the enzyme was preincubated with a high concentration of Ca2+before assay. The concentration of Ca2+required to decrease the lag phase by 50% in 10min depended on the protein concentration: at 0.87mg of protein/ml it required 17mm-Ca2+and at 0.44mg/ml it needed 10mm-Ca2+. 3. The concentrations of Ca2+required either to abolish the lag phase in the appearance of enzyme activity or to activate the essential thiol for reaction with 5,5′-dithiobis-(2-nitrobenzoate) in 10min incubation were similar at the same protein concentration. This indicated that Ca2+induces a conformation change that is responsible for both phenomena. A model is proposed that links this conformation change to the dissociation of the tetrameric enzyme. 4. This was supported by the observation that the addition of excess of b chains to the Factor XIIIa (a′2b2) increased the concentration of Ca2+required to expose the reactive thiol, and inhibited the Ca2+-dependent aggregation of a′ chains. 5. Platelet Factor XIIIa (a′2) was inhibited by 5,5′-dithiobis-(2-nitrobenzoate) in the absence of Ca2+, and no lag phases were observed in attaining the steady-state rate at low Ca2+concentrations, thus confirming the model for the activation of the plasma enzyme. 6. The Ca2+dependence of platelet Factor XIIIa indicated that Ca2+has an additional role in the enzyme mechanism of the plasma enzyme, perhaps being involved in substrate binding. 7. The dependence of the stability of plasma Factor XIIIa on Ca2+and protein concentration indicates that the decay in activity is related to the tetramer dissociation. 8. β-Substrate decreased the Ca2+concentration required for (1) abolition of the lag phase and (2) enzyme inhibition by thiol reagents. The effect on the former is greater than on the latter. 9. The role of the b chains of the plasma Factor and the evolutionary significance of the plasma and platelet Factors are considered.

scholarly journals Functional Interaction between the Herpes Simplex Virus Type 1 Polymerase Processivity Factor and Origin-Binding Proteins: Enhancement of UL9 Helicase Activity

2003 ◽  
Vol 77 (23) ◽  
pp. 12646-12659 ◽  
Author(s):  
Kelly S. Trego ◽  
Deborah S. Parris
Keyword(s):  
Steady State ◽  
Herpes Simplex ◽  
Helicase Activity ◽  
Steady State Rate ◽  
State Rate ◽  
Lag Period ◽  
Processivity Factor ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The origin (ori)-binding protein of herpes simplex virus type 1 (HSV-1), encoded by the UL9 open reading frame, has been shown to physically interact with a number of cellular and viral proteins, including three HSV-1 proteins (ICP8, UL42, and UL8) essential for ori-dependent DNA replication. In this report, it is demonstrated for the first time that the DNA polymerase processivity factor, UL42 protein, provides accessory function to the UL9 protein by enhancing the 3′-to-5′ helicase activity of UL9 on partially duplex nonspecific DNA substrates. UL42 fails to enhance the unwinding activity of a noncognate helicase, suggesting that enhancement of unwinding requires the physical interaction between UL42 and UL9. UL42 increases the steady-state rate for unwinding a 23/38-mer by UL9, but only at limiting UL9 concentrations, consistent with a role in increasing the affinity of UL9 for DNA. Optimum enhancement of unwinding was observed at UL42/UL9 molecular ratios of 4:1, although enhancement was reduced when high UL42/DNA ratios were present. Under the assay conditions employed, UL42 did not alter the rate constant for dissociation of UL9 from the DNA substrate. UL42 also did not significantly reduce the lag period which was observed following the addition of UL9 to DNA, regardless of whether UL42 was added to DNA prior to or at the same time as UL9. Moreover, addition of UL42 to ongoing unwinding reactions increased the steady-state rate for unwinding, but only after a 10- to 15-min lag period. Thus, the increased affinity of UL9 for DNA most likely is the result of an increase in the rate constant for binding of UL9 to DNA, and it explains why helicase enhancement is observed only at subsaturating concentrations of UL9 with respect to DNA. In contrast, ICP8 enhances unwinding at both saturating and subsaturating UL9 concentrations and reduces or eliminates the lag period. The different means by which ICP8 and UL42 enhance the ability of UL9 to unwind DNA suggest that these two members of the presumed functional replisome may act synergistically on UL9 to effect initiation of HSV-1 DNA replication in vivo.

scholarly journals Transient kinetics of nicotinamide–adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria

1978 ◽  
Vol 171 (3) ◽  
pp. 743-750 ◽  
Author(s):  
K Dalziel ◽  
N McFerran ◽  
B Matthews ◽  
C H Reynolds
Keyword(s):  
Steady State ◽  
Isocitrate Dehydrogenase ◽  
Turnover Rate ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Lag Phase ◽  
Transient Kinetics ◽  
Dehydrogenase Reaction ◽  
Steady State Rate ◽  
State Rate ◽  
Kinetics Of

Pre-steady-state studies of the isocitrate dehydrogenase reaction show that the rate constant for the hydride-transfer step is above 990s-1, and that both subunits of the enzyme are simulataneously active. After the fast formation of NADPH in amounts equivalent to the enzyme subunit concentration, the rate of NADPH formation is equal to the steady-state rate if the enzyme has been preincubated with isocitrate and Mg2+. If the enzyme has been preincubated with NADP+ and Mg2+, in 0.05 M-triethanolamine chloride buffer, pH 7.0, with the addition of 0.1 M-NaCl, the amount of NADPH formed in the fast phase is only 60% of the enzyme subunit concentration, and the turnover rate is at first lower than the steady-state rate. In 0.05 M-triethanolamine chloride buffer, pH 7.0, if the enzyme is preincubated with NADP+ or NADPH, the turnover rate increases 3-fold to reach the steady-state rate after about 5 s. Preincubation of the enzyme with isocitrate and Mg2+ abolishes this lag phase, the steady-state rate being reached at once. It is suggested that the enzyme exists in at least two conformational forms with different activities, and that the lag phase represents the transition (k = 0.4s-1) from a form with low activity to the fully active enzyme, induced by the binding of isocitrate and Mg2+.

scholarly journals The role of Gi and the membrane-fluidizing agent benzyl alcohol in modulating the hysteretic activation of human platelet adenylate cyclase by guanylyl 5′-imidodiphosphate

1993 ◽  
Vol 291 (3) ◽  
pp. 945-949 ◽  
Author(s):  
S Spence ◽  
M D Houslay
Keyword(s):  
Steady State ◽  
Adenylate Cyclase ◽  
Benzyl Alcohol ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Steady State Rate ◽  
Fluidizing Agent ◽  
State Rate ◽  
Lag Period ◽  
Pre Treatment

The non-hydrolysable GTP analogue guanylyl 5′-imidodiphosphate (p[NH]ppG) elicited a profound increase in the adenylate cyclase activity of human platelets. This occurred after a well-defined lag period of around 6 min, whereupon an enhanced steady-state rate was evident. The duration of the lag period was unchanged over a range of concentrations of p[NH]ppG which gave very different steady-state rates of adenylate cyclase activity. Prior activation of the stimulatory G-protein Gs by cholera-toxin pre-treatment abolished the lag period and elicited a small increase in the steady-state rate. Manipulating function of the inhibitory G-protein Gi also led to profound changes in the lag periods. Thus marked decreases in the lag were seen (approximately 70-81%) when Gi function was ablated through pre-treatment of platelet membranes with pertussis toxin, or by using elevated (25 mM) Mg2+ levels in the assay, or when Mg2+ was replaced by 5 mM Mn2+ in the assay. In contrast with this, potentiation of Gi function led to an increase in the lag period, as seen under conditions of agonist occupancy of inhibitory alpha 2-adrenoceptors (increase approximately 74%) or with the addition of 100 mM NaCl to the assays (increase approximately 44%). The local anaesthetic and membrane-fluidizing agent benzyl alcohol elicited both a profound decrease (around 70% at 80 mM) in the p[NH]ppG-induced lag period and a marked augmentation (around 5-fold) in the steady-state adenylate cyclase activity. When adenylate cyclase assays were done at 35 degrees C instead of 25 degrees C, then the lag period for activation by p[NH]ppG was decreased by around 33% and the steady-state rate increased by around 3-fold. At 35 degrees C, the addition of benzyl alcohol led to the apparent abolition of the lag period for p[NH]ppG activation of adenylate cyclase and amplified the steady-state rate by only around 2.2-fold. It is shown that Gi plays a fundamental role in determining the rate of activation of Gs. The proposal is formulated that such an action may be mediated through the release of beta gamma-subunits. Thus beta gamma-subunit dissociation is proposed as providing the rate-limiting step in Gi activation.

Corticosterone secretion in rats as a function of ACTH input and adrenal blood flow

1965 ◽  
Vol 209 (4) ◽  
pp. 811-814 ◽  
Author(s):  
John C. Porter ◽  
M. S. Klaiber
Keyword(s):  
Blood Flow ◽  
Steady State ◽  
Steady Increase ◽  
Constant Input ◽  
Corticosterone Secretion ◽  
Steady State Rate ◽  
State Rate

The rate of secretion of corticosterone from the left adrenal of rats receiving a constant input of ACTH was determined for different flows of blood through the adrenal during the 2- to 3-hr interval following hypophysectomy. Two hours after hypophysectomy the secretion of corticosterone was low in all groups regardless of flow. An input of 0.26 mU ACTH/min caused a steady increase in secretion for 30–40 min before a steady-state rate was attained. The average steady-state rate of secretion was 1.1, 2.4, 3.5, 6.2, 7.2, 6.2, and 6.2 µg/5 min for flows of 0.005, 0.012, 0.023, 0.034, 0.039, 0.051, and 0.058 ml/min, respectively. Under the conditions of these experiments where the input of ACTH was 0.26 mU/min the secretion of corticosterone increased significantly with time of input of ACTH and with flow of blood through the adrenal.

scholarly journals The Natural Rate of Interest and the Optimal Rate of Interest in the Steady State

2021 ◽  
pp. 17-41
Author(s):  
Carl Christian von Weizsäcker ◽  
Hagen M. Krämer
Keyword(s):  
Steady State ◽  
Public Debt ◽  
Fundamental Equation ◽  
Optimal Rate ◽  
Natural Rate ◽  
Real Rate ◽  
Capital Theory ◽  
Steady State Rate ◽  
Natural Rate Of Interest ◽  
State Rate

AbstractThe “natural rate of interest” is the hypothetical, risk-free real rate of interest that would obtain in a closed economy, if net public debt were zero. It is considerably less than the optimal steady-state rate of interest, which is equal to the system’s growth rate. This holds for a very general “meta-model.” The fundamental equation of capital theory holds on the optimal steady-state path: T = Z − D, where T is the overall economic period of production, Z is the representative private “waiting period” of consumers and D is the public debt ratio. Prosperity is at least 30% lower at the natural rate of interest than at the optimal rate.

Kinetic Study Of Crystallisation In Amorphous Thin Lpcvd Si Films

1993 ◽  
Vol 321 ◽  
Author(s):  
B. Pieraggi ◽  
J. P. Guillemet ◽  
B. de Mauduit
Keyword(s):  
Experimental Data ◽  
Steady State ◽  
Isothermal Annealing ◽  
Nucleation Kinetics ◽  
Crystallisation Kinetics ◽  
Crystallisation Behaviour ◽  
Steady State Rate ◽  
State Rate ◽  
Si Films

ABSTRACTThe crystallisation behaviour of LPCVD silicon films has been investigated by TEM from in situ isothermal annealing of undoped a-Si films deposited from disilane (Si2H6) at temperatures 450,465 and 480 °C and at gas pressure of 200 MTorr. Nucleation kinetics, grain growth rates and crystallisation kinetics were determined for temperatures ranging from 600 to 675 °C. Nucleation kinetics have been experimentally determined in the early first stages of annealing : they do not show any steady-state rate and are fitted according to a power law. Experimental data for crystallisation kinetics are fitted by an Avrami law without introducing any incubation time.

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