Growth, starvation and enzyme activity in white muscle of atlantic cod: at what point do muscle metabolic capacities change?

Muscle protein decreases only during prolonged starvation of Atlantic cod (Gadus morhua, Gadidae), but in the absence of protein renewal, muscle metabolic capacities may decrease before marked loss of muscle protein. This study aimed at elucidating the threshold at which decreases in growth and condition reduce muscle metabolic capacities, as well as identifying the indicators that best explain changes in metabolic capacities. To generate a wide spectrum of individual growth rates, condition factors and proximate compositions, cod showing different initial condition were fed or starved for different periods of time. The relationships between muscle proteins and metabolic enzyme activities (LDH and CCO) on one hand, and growth rate, condition factor, hepato- and gonadosomatic index and muscle and liver water and energy contents, on the other hand, were examined through linear regression models. Multiple linear regressions explained a large proportion of the observed variability in proteins and enzyme activities. Changes in LDH and CCO activities were not driven by changes in growth rate. Muscle water was the only significant correlate for both enzymes. Enzyme activities decreased as soon as muscle water began to rise. Increases in water content from 79 to 92% resulted in a 10-fold decrease in LDH and CCO activities.     

Seasonal changes in thermal environment and metabolic enzyme activity in the diamondback terrapin (Malaclemys terrapin)

Diamondback terrapins experience broad fluctuations in temperature on both a daily and seasonal basis in their estuarine environment. We measured metabolic enzyme activity in terrapin muscle tissue to assess thermal dependence and the role of temperature in seasonal metabolic downregulation in this species. Activity of lactate dehydrogenase (LDH), pyruvate kinase (PK), citrate synthase (CS), and cytochrome c oxidase (CCO) was assayed at 10, 20, 30, and 40 °C for tissue collected during summer and winter. The Q(10) for enzyme activity varied between 1.31 and 2.11 within the temperature range at which terrapins were active (20-40 °C). The Q(10) for LDH, CS, and CCO varied between 1.39 and 1.76 and between 10 and 20 °C, but PK exhibited heightened thermal sensitivity within this lower temperature range, with a Q(10) of 2.90 for summer-collected tissue and 5.55 for winter-collected tissue. There was no significant effect of season on activity of LDH or PK, but activity of CS and CCO was significantly lower in winter-collected tissue compared with summer-collected tissue. Results indicate that temperature effects contribute to seasonal metabolic downregulation and dormancy in terrapins, but other environmental factors (i.e. oxygen availability), as well as seasonal shifts in blood biochemistry and circulating hormones may also play an important role.     

Longitudinal and allometric variation in indicators of muscle metabolic capacities in atlantic cod (Gadus morrhua)

This study evaluated whether indicators of metabolic capacity of cod white muscle differ along the length of the body, whether this variation persists over a large range of body sizes, and whether the allometry of metabolic capacities is similar along the length of the body. We examined the maximal activities of two glycolytic enzymes, phosphofructokinase (PFK) and lactate dehydrogenase (LDH), a mitochondrial enzyme, cytochrome C oxidase (CCO), and the biosynthetic enzyme nucleotide diphosphate kinase (NDPK). All enzymes examined showed significant size dependence, which was generally apparent in all regions. The activity of glycolytic enzymes increased with size, whereas that of CCO and NDPK decreased with size. For PFK and LDH, the size dependence decreased caudally, whereas for CCO and NDPK it was strongest in the caudal sample. For each size range, the activities of PFK, LDH, and CCO were higher in the last third of the body than in the middle or just behind the head. In contrast, NDPK activity was higher just behind the head than at the middle or in the last third of the body, suggesting that nuclear proliferation is more rapid in this zone. The high capacity for adenosine triphosphate (ATP) generation in the caudal region suggests that increases in mass-specific ATP output are advantageous in this relatively thin section of the body.     

A nonlinear compartmental model of Sr metabolism. I. Non-steady-state kinetics and model building

A model of Sr metabolism was developed by using plasma and urinary Sr kinetic data obtained in groups of postmenopausal women who received four different oral doses of Sr and collected during the Sr administration period (25 days) and for 28 days after cessation of treatment. A nonlinear compartmental formalism that is appropriate for study of non-steady-state kinetics and allows dissociation of variables pertaining to Sr metabolism (system 1) from those indirectly operating on it (system 2) was used. At each stage of model development, the dose-dependent model response was fitted to the four sets of data considered simultaneously (1 set per dose). A seven-compartment model with internal Sr distribution and intestinal, urinary, and bone metabolic pathways was selected. It includes two kinds of nonlinearities: those accounting for saturable intestinal and bone processes, which behave as intrinsic nonlinearities because they are directly dependent on Sr, and extrinsic nonlinearities (dependent on system 2), which suggest the cooperative involvement of plasma Sr changes in modulating some intestinal and bone mineral metabolic pathways. With the set of identified parameter values, the initial steady-state model predictions are relevant to known physiology, and some peculiarities of model behavior for long-term Sr administration were simulated.     

Design of large metabolic responses. Constraints and sensitivity analysis

Metabolic control analysis (Kacser & Burns (1973). Symp. Soc. Exp. Biol.27, 65-104; Heinrich & Rapoport (1974). Eur. J. Biochem.42, 89-95) has been extensively used to describe the response of metabolic concentrations and fluxes to small (infinitesimal) changes in enzyme concentrations and effectors. Similarly, metabolic control design (Acerenza (1993). J. theor. Biol.165, 63-85) has been proposed to design small metabolic responses. These approaches have the limitation that they were not devised to deal with large (non-infinitesimal) responses. Here we develop a strategy to design large changes in the metabolic variables. The only assumption made is that, for all the parameter values under consideration, the system has a unique stable steady state. The procedure renders the kinetic parameters of the rate equations that when embedded in the metabolic network produce the pattern of large changes in the steady-state variables that we aim to design. Structural and kinetic constraints impose restrictions on the type of responses that could be designed. We show that these conditions can be transformed into the language of mean-sensitivity coefficients and, as a consequence, a sensitivity analysis of large metabolic responses can be performed after the system has been designed. The mean-sensitivity coefficients fulfil conservation and summation relationships that in the limit reduce to the well-known theorems for infinitesimal changes. Finally, it is shown that the same procedure that was used to design metabolic responses and analyse their sensitivity properties can also be used to determine the values of kinetic parameters of the rate laws operating "in situ".     

'Slave' metabolites and enzymes. A rapid way of delineating metabolic control.

Although control of fluxes and concentrations tends to be distributed rather than confined to a single rate-limiting enzyme, the extent of control can differ widely between enzymes in a metabolic network. In some cases, there are enzymes that lack control completely. This paper identifies one surprising origin of such lack of control: If, in a metabolic system, there is a metabolite that affects the catalytic rate of only one enzyme, the corresponding enzyme cannot control any metabolic variable other than the concentration of that metabolite. We call such enzymes 'slave enzymes', and the corresponding metabolites 'slave metabolites'. Implications of the existence of slave enzymes for the control properties of enzymes further down the metabolic pathway are discussed and examined for the glycolytic pathway of yeast. Inadvertent assumptions in metabolic models may cause the latter incorrectly to calculate absence of metabolic control. The phenomenon of slave enzymes may well be important in enhancing metabolic signal transduction.     

Carbon-13 NMR in conformational analysis of nucleic acid fragments. 4. The torsion angle distribution about the C3''-O3'' bond in DNA constituents.

Carbon-13 and proton NMR spectra of a series of oligodeoxynucleotides (d(CT), d(CC), d(TA), d(AT), d(CG), d(GC), d(AG), d(AAA), d(TATA) and d(GGTAAT] were measured at various temperatures. The three coupling constants that are related to the magnitude of backbone angle epsilon (J(C4'-P), J(C2'-P) and J(H3'-P] are analyzed in terms of a three-state equilibrium about this bond. Two epsilon (trans) angles occur, which differ in magnitude depending on the conformation (N or S) of the adjoining deoxyribose ring. The S-type deoxyribose ring is associated with a smaller epsilon (trans) angle: epsilon (t,S) = 192 degrees. The N-type deoxyribose ring is associated with a larger epsilon (trans) angle epsilon (t,N) = 212 degrees. The third rotamer participating in the conformational equilibrium, is a gauche(-) (epsilon (-] conformer and occurs exclusively in combination with the S-type sugar ring (epsilon (-,S) = 266 degrees). Within the limits of experimental error, the magnitude of these three angles appears to be independent of the particular base sequence, except in the case of d(CG) where a slightly larger epsilon (t,S) angle (197 degrees) is indicated. A simple equation is proposed which may be used to calculate the population of epsilon (t,S) conformer in cases where only J(H3'-P) is known.     

APS kinase from Penicillium chrysogenum. Dissociation and reassociation of subunits as the basis of the reversible heat inactivation

Adenosine-5'-phosphosulfate (APS) kinase from Penicillium chrysogenum, loses catalytic activity at temperatures greater than approximately 40 degrees C. When the heat-inactivated enzyme is cooled to 30 degrees C or lower, activity is regained in a time-dependent process. At an intermediary temperature (e.g. 36 degrees C) an equilibrium between active and inactive forms can be demonstrated. APS kinase from P. chrysogenum is a dimer (Mr = 57,000-60,000) composed of two apparently identical subunits. Three lines of evidence suggest that the reversible inactivation is a result of subunit dissociation and reassociation. (a) Inactivation is a first-order process. The half-time for inactivation at a given temperature is independent of the original enzyme concentration. Reactivation follows second-order kinetics. The half-time for reactivation is inversely proportional to the original enzyme concentration. (b) The equilibrium active/inactive ratio at 36 degrees C increases as the total initial enzyme concentration is increased. However, Keq,app at 5 mM MgATP and 36 degrees C calculated as [inactive sites]2/0.5 [active sites] is near-constant at about 1.7 X 10(-8) M over a 10-fold concentration range of enzyme. (c) At 46 degrees C, the inactive P. chrysogenum enzyme (assayed after reactivation) elutes from a calibrated gel filtration column at a position corresponding to Mr = 33,000. Substrates and products of the APS kinase reaction had no detectable effect on the rate of inactivation. However, MgATP and MgADP markedly stimulated the reactivation process (kapp = 3 X 10(5) M-1 X s-1 at 30 degrees C and 10 mM MgATP). The kapp for reactivation was a nearly linear function of MgATP up to about 20 mM suggesting that the monomer has a very low affinity for the nucleotide compared to that of the native dimer. Keq,app at 36 degrees C increases as the MgATP concentration is increased. The inactivation rate constant increased as the pH was decreased but no pK alpha could be determined. The reactivation rate constant increased as the pH was increased. An apparent pK alpha of 6.4 was estimated.     

The solution structure of the circular trinucleotide cr(GpGpGp) determined by NMR and molecular mechanics calculation.

The 3'-5' circular trinucleotide cr(GpGpGp) was studied by means of 1D and 2D high resolution NMR techniques and molecular mechanics calculations. Analysis of the J-couplings, obtained from the 1H and 13C-NMR spectra, allowed the determination of the conformation of the sugar rings and of the 'circular' phosphate backbone. In the course of the investigations it was found that the Karplus-equation most recently parametrized for the CCOP J-coupling constants could not account for the measured J(C4'P) of 11.1 Hz and a new parametrization for both HCOP and CCOP coupling constants is therefore presented. Subsequent analysis of the coupling constants yielded 'fixed' values for the torsion angles beta and delta (with beta = 178 degrees and delta = 139 degrees). The value of the latter angle corresponds to an S-type sugar conformation. The torsion angles gamma and epsilon are involved in a rapid equilibrium in which they are converted between the gauche(+) and trans and between the trans and gauche(-) domain respectively. We show that the occurrence of epsilon in the gauche(-) domain necessitates S-type sugar conformations. Given the aforementioned values for beta, gamma, delta and epsilon the ring closure constraints for the ring, formed by the phosphate backbone can only be fulfilled if alpha and zeta adopt some special values. After energy minimization with the CHARMm force field only two combinations of alpha and zeta result in energetically favourable structures, i.e. the combination alpha (t)/zeta(g-) in case gamma is in a gauche(+) and epsilon is in a trans conformation, and the combination alpha (t)/zeta (g+) for the combination gamma (t)/epsilon (g-). The results are discussed in relation to earlier findings obtained for cd(ApAp) and cr(GpGp), the latter molecule being a regulator of the synthesis of cellulose in Acetobacter xylinum.     

Time hierarchy, equilibrium and non-equilibrium in metabolic systems.

A metabolic system consists of cooperating biochemical reactions. The motion is described by differential equations in the metabolites. The right-hand sides of these equations are linear combinations of the velocities of the individual reactions. These velocities depend in a non-linear manner on the metabolite concentrations (according to the law of mass action). A characteristic "metabolic" time may be defined for the motion of the whole system. It scales the essential metabolic events whose evolution time is comparable to this metabolite time unit. The constituent reactions of the metabolic system have an individual characteristic time which need not coincide with the general metabolic time. The individual time characterises the approach to the individual equilibrium of the isolated undisturbed reaction. According to the ratio of these two time scales, a single reaction may be fast, or slow, or essential, as compared with the metabolic events. Characteristic time of a single reaction and its steady-state deviation from equilibrium are closely related. It can be shown that the relative deviation from equilibrium of a reaction within the metabolic network is of the same numerical order as the ratio between individual time to metabolic time. The interaction of many reactions with different characteristic times introduces a time hierarchy into the system. This can be made transparent by appropriate scaling and by linear transformation of the system. The subsystem of fast cooperating reactions (dehydrogenases, phosphotransferases) attains a state which is near to the individual equilibrium and reestablishes this state after perturbation. The equilibration is fast; an ultrarapid phase of cofactor equilibrium can be distinguished from the fast phase of substrate equilibrium (exchange of metabolic material between different pathways). During the slower metabolic phase these near-equilibria manifest themselves as stoichiometric linkage between unrelated metabolites. The latter cease to be independent variables and combine to metabolic pools. It can be strictly shown that the essential variables at the metabolic time scale are carrier pools and the degree of occupancy of these carriers by metabolic groups. Chemically different types of carrier pools may be functionally linked together by fast reactions. A consequence of such an arrangement of reactions are distance effects: Changes at one end of a metabolic map may be directly conveyed to other pathways via stoichiometric linkage brought about by fast equilibration of cofactor reactions.     

Specific metabolic rate, longevity and "Rubner constant" for birds

An assumption was made that age constituent alpha x(beta) of mortality of individuals in a population in Weibull equation mx = m0 + alpha x(beta) (Ricklefs, 2000) reflects change of specific metabolic rate of one individual with age. Based upon that hypothesis a formula was proposed for relationship of specific metabolic rate of an adult individual after cessation of growth, when mass W is attained, and age t: q(t) = q0(1-omega(beta) + 1t(beta)) where q0 = aW(-b) is value q(t) at the moment of growth cessation and omega = alpha(1/(beta + 1)) is "ageing rate", determined and estimated by R. Ricklefs. Maximum longevity of an individual was determined as [equation: see text], where qcrit is specific metabolic rate at the age tmax. Parameter beta and relationships omega(W) and (qcrit/q0)(W) were approximated for birds from data of Ricklefs. Statistical comparison of results of calculations of tmax was carried out on the basis of the above formula and other known formulas for groups of Passeriformes and non-Passeriformes. Rubner constant [equation: see text] was calculated assuming that body mass of an adult individual (W) is attained in the first year of life (tA = 0). Average values of 602.4 +/- 2.5 kcal g(-1) (n = 83) for non-Passeriformes and 963 +/- 6.3 kcal g(-1) (n = 41) for Passeriformes were obtained.     

Autophosphorylation-dependent inactivation of plant chimeric calcium/calmodulin-dependent protein kinase.

Chimeric calcium/calmodulin dependent protein kinase (CCaMK) is characterized by the presence of a visinin-like Ca(2+)-binding domain unlike other known calmodulin- dependent kinases. Ca(2+)-Binding to the visinin-like domain leads to autophosphorylation and changes in the affinity for calmodulin [Sathyanarayanan P.V., Cremo C.R. & Poovaiah B.W. (2000) J. Biol. Chem. 275, 30417-30422]. Here, we report that the Ca(2+)-stimulated autophosphorylation of CCaMK results in time-dependent loss of enzyme activity. This time-dependent loss of activity or self-inactivation due to autophosphorylation is also dependent on reaction pH and ATP concentration. Inactivation of the enzyme resulted in the formation of a sedimentable enzyme due to self-association. Specifically, autophosphorylation in the presence of 200 microm ATP at pH 7.5 resulted in the formation of a sedimentable enzyme with a 33% loss in enzyme activity. Under similar conditions at pH 6.5, the enzyme lost 67% of its activity and at pH 8.5, 84% enzyme activity was lost. Furthermore, autophosphorylation at either acidic or alkaline reaction pH lead to the formation of a sedimentable enzyme. Transmission electron microscopic studies on autophosphorylated kinase revealed particles that clustered into branched complexes. The autophosphorylation of wild-type kinase in the presence of AMP-PNP (an unhydrolyzable ATP analog) or the autophosphorylation-site mutant, T267A, did not show formation of branched complexes under the electron microscope. Autophosphorylation- dependent self-inactivation may be a mechanism of modulating the signal transduction pathway mediated by CCaMK.     

Control analysis of metabolic networks. 2. Total differentials and general formulation of the connectivity relations

The mathematical background of the connectivity relations of metabolic control theory is analysed. The connectivity relations are shown to reflect general properties of total differentials of reaction rate vi, flux J, and metabolite concentration Xj. Connectivity relations hold for any metabolic network in which all vi are homogeneous functions of enzyme concentration Ei. This notion allows established algebraic methods to be used for the formulation of connectivity relations for metabolic systems in which numerous constraints are imposed on metabolite concentrations. A general procedure to derive connectivity relations for such metabolic systems is given. To encourage a broader audience to apply control theory to physiological systems, an easy-to-use graphical procedure is derived for formulating connectivity relations for biochemical systems in which no metabolite is involved in more than one constraint.     

Growth,starvation and enzyme activity in white muscle of atlantic cod: at what point do muscle metabolic capacities change?

Muscle protein decreases only during prolonged starvation of Atlantic cod (Gadus morhua, Gadidae), but in the absence of protein renewal, muscle metabolic capacities may decrease before marked loss of muscle protein. This study aimed at elucidating the threshold at which decreases in growth and condition reduce muscle metabolic capacities, as well as identifying the indicators that best explain changes in metabolic capacities. To generate a wide spectrum of individual growth rates, condition factors and proximate compositions, cod showing different initial condition were fed or starved for different periods of time. The relationships between muscle proteins and metabolic enzyme activities (LDH and CCO) on one hand, and growth rate, condition factor, hepato- and gonadosomatic index and muscle and liver water and energy contents, on the other hand, were examined through linear regression models. Multiple linear regressions explained a large proportion of the observed variability in proteins and enzyme activities. Changes in LDH and CCO activities were not driven by changes in growth rate. Muscle water was the only significant correlate for both enzymes. Enzyme activities decreased as soon as muscle water began to rise. Increases in water content from 79 to 92% resulted in a 10-fold decrease in LDH and CCO activities.     

Effect of muscle action and metabolic strain on oxidative metabolic responses in human skeletal muscle.

A recent report suggests that differences in aerobic capacity exist between concentric and eccentric muscle action in human muscle (T. W. Ryschon, M. D. Fowler, R. E. Wysong, A. R. Anthony, and R. S. Balaban. J. Appl. Physiol. 83: 867-874, 1997). This study compared oxidative response, in the form of phosphocreatine (PCr) resynthesis rates, with matched levels of metabolic strain (i.e., changes in ADP concentration or the free energy of ATP hydrolysis) in tibialis anterior muscle exercised with either muscle action in vivo (n = 7 subjects). Exercise was controlled and metabolic strain measured by a dynamometer and (31)P-magnetic resonance spectroscopy, respectively. Metabolic strain was varied to bring cytosolic ADP concentration up to 55 microM or decrease the free energy of ATP hydrolysis to -55 kJ/mol with no change in cytoplasmic pH. PCr resynthesis rates after exercise ranged from 31.9 to 462.5 and from 21.4 to 405.4 micromol PCr/s for concentric and eccentric action, respectively. PCr resynthesis rates as a function of metabolic strain were not significantly different between muscle actions (P > 0.40), suggesting that oxidative capacity is dependent on metabolic strain, not muscle action. Pooled data were found to more closely conform to previous biochemical measurements when a term for increasing oxidative capacity with metabolic strain was added to models of respiratory control.     

INDISIM, an individual-based discrete simulation model to study bacterial cultures.

An individual-based model has been developed and designed to simulate the growth and behaviour of bacterial colonies. The simulator is called INDISIM, which stands for INDividual DIScrete SIMulations. INDISIM is discrete in space and time, and controls a group of bacterial cells at each time step, using a set of random, time-dependent variables for each bacterium. These variables are used to characterize its position in space, biomass, state in the cellular reproduction cycle as well as other individual properties. The space where the bacterial colony evolves is also discrete. A physical lattice is introduced, subject to the appropriate boundary conditions. The lattice is subdivided into spatial cells, also defined by a set of random, time-dependent variables. These variables may include concentrations of different types of particles, nutrients, reaction products and residual products. Random variables are used to characterize the individual bacterium and the individual particle, as well as the updating of individual rules. Thus, the simulations are stochastic rather than deterministic. The whole set of variables, those that characterize the bacterial population and the environment where they evolve, enables the simulator to study the behaviour of each microorganism-such as its motion, uptake, metabolism, and viability-according to given rules suited for the system under study. These rules require the input of only a few parameters. Once this information is inputted, INDISIM simulates the behaviour of the system providing insights into the global properties of the system from the assumptions made on the properties of the individual bacteria. The relation between microscopic and global properties of the bacterial colony is obtained by using statistical averaging. In this work INDISIM has been used to study (a) biomass distributions, (b) the relationship between the rate of growth of a bacterial colony and the nutrient concentration and temperature, and (c) metabolic oscillations in batch bacterial colonies. The simulation results are found to be in very good qualitative agreement with available experimental data, and provide useful insights into the mechanisms involved in each case.     

Sequential Activation of Metabolic Pathways: a Dynamic Optimization Approach

The regulation of cellular metabolism facilitates robust cellular operation in the face of changing external conditions. The cellular response to this varying environment may include the activation or inactivation of appropriate metabolic pathways. Experimental and numerical observations of sequential timing in pathway activation have been reported in the literature. It has been argued that such patterns can be rationalized by means of an underlying optimal metabolic design. In this paper we pose a dynamic optimization problem that accounts for time-resource minimization in pathway activation under constrained total enzyme abundance. The optimized variables are time-dependent enzyme concentrations that drive the pathway to a steady state characterized by a prescribed metabolic flux. The problem formulation addresses unbranched pathways with irreversible kinetics. Neither specific reaction kinetics nor fixed pathway length are assumed. In the optimal solution, each enzyme follows a switching profile between zero and maximum concentration, following a temporal sequence that matches the pathway topology. This result provides an analytic justification of the sequential activation previously described in the literature. In contrast with the existent numerical approaches, the activation sequence is proven to be optimal for a generic class of monomolecular kinetics. This class includes, but is not limited to, Mass Action, Michaelis–Menten, Hill, and some Power-law models. This suggests that sequential enzyme expression may be a common feature of metabolic regulation, as it is a robust property of optimal pathway activation.     

Kinetics of the reaction of streptokinase-plasmin complex with purified human and mouse alpha 2-macroglobulin. Implications for mechanism.

Streptokinase-human plasmin complex (Sk-hPm) reacted rapidly with purified mouse alpha 2-macroglobulin (m alpha 2M) in vitro at 37 degrees C. Approx. 98% of the plasmin in Sk-hPm bound covalently to at least one m alpha 2M subunit. Most of the streptokinase dissociated (95%). The rate of Sk-hPm inactivation clearly depended on the m alpha 2M concentration. With 1.2 microM-m alpha 2M, 50% of the Sk-hPm (0.02 microM) reacted in less than 50 s. A double-reciprocal plot comparing pseudo-first-order rate constants (kapp.) and m alpha 2M concentration yielded a second-order rate constant of 2.3 x 10(4) M-1.s-1 (r = 0.97). This value is an approximation, since Sk-hPm preparations are heterogeneous. Sk-hPm reacted with human alpha 2M (h alpha 2M), forming alpha 2M-plasmin complex (98% covalent). More than 99% of the streptokinase dissociated. The rate of reaction of Sk-hPm with h alpha 2M did not clearly depend on inhibitor concentration. The kapp. values determined with 0.6-1.2 microM-h alpha 2M were decreased 10-20-fold compared with m alpha 2M. In order to study the effect of Sk-hPm heterogeneity on the reaction with alpha 2M, the proteinase was incubated for various amounts of time at 37 degrees C before addition of inhibitor. The enzyme amidase activity was maximal within 5 min; however, reaction of Sk-hPm with m alpha 2M or h alpha 2M was most extensive after 20 min and 2 h respectively. After incubation for more than 1 h, Sk-hPm acquired fibrinogenolytic activity, suggesting plasmin dissociation. Therefore the enhanced reaction of h alpha 2M with 'older' Sk-hPm preparations may have resulted in part from dissociated plasmin or 'plasmin-like' species. By contrast, the reaction of Sk-hPm with m alpha 2M was most rapid when the proteinase preparation was free of plasmin, indicating direct reaction of Sk-hPm with m alpha 2M as the only major mechanism. Finally, streptokinase-cat plasminogen complex reacted more extensively with m alpha 2M than with h alpha 2M, suggesting that m alpha 2M may be a superior inhibitor with this class of plasminogen activators in general.     

Control analysis of transition times in metabolic systems.

The transition time, tau, of a metabolic system is defined as the ratio of the metabolite concentrations in the system, sigma, to the steady-state flux, J. Its value reflects a temporal characteristic of the system as it relaxes towards the steady state. Like other systemic properties, the value of tau will be a function of the enzyme activities in the system. The influence of a particular enzyme activity on tau can be quantified by a Control Coefficient, C tau ei. We show that it is possible to derive a Summation Theorem sigma ni = 1 C tau ei = -1 and a Connectivity Theorem sigma ni = 1 C tau ei.epsilon viSk = -Sk/sigma. We establish a 'sign rule' that predicts the order of positive and negative Control Coefficients in a sequence.