The effect of inorganic phosphate on chick (Gallus domesticus) heart mitochondrial volume and cation content.

1. In the absence of exogenous Ca(II), Pi induces a swelling change that is kinetically first order with k = 1.08 +/- 0.1 min-1. The first-order rate constant is independent of [Pi] over the range of 0.5-45 mM. 2. In the presence of exogenous substrate, the volume change induced by Pi is monophasic and can be reversed by ADP. 3. The swelling process and the approach to steady state is accompanied by controlled losses of both K+ and Mg(II) from within the mitochondria. 4. The loss of K+ is biphasic as a function of time with ki = 14.1 +/- 1.6 and k2 = 4.4 +/- 0.34 nmol min-1 mg mitochondria-1. 5. The loss of Mg(II) is monophasic and the rate at which this cation is released decreases as a function of time. Ca(II) fluxes are not involved in the volume occurring secondary to Pi uptake. 6. In the absence of exogenous substrate, Pi induces a triphasic change in mitochondrial volume. 7. The sequence of volume changes corresponds to an initial first-order swelling secondary to the addition of Pi, a contraction apparently triggered by the loss of approximately 85% of total intra-mitochondrial Mg(II), and a second larger swelling phase that cannot be reversed with ADP. 8. The Pi-induced swelling of chick heart mitochondria is not inhibited by EGTA and does not depend on the provision of exogenous Ca(II). 9. The Ca(II) and Mg(II) ions released from within the mitochondria are responsible for activating divalent cation-dependent ATPases which cosediment with isolated chick heart mitochondria.

Respiratory control and ADP:O coupling ratios of isolated chick heart mitochondria.

Heart mitochondria isolated from 14- to 21-day-old chicks are highly coupled and often have respiratory control ratio (RCR) values exceeding 100. This paper presents data from a study of some of the properties of these mitochondria. The studies show that: (a) The ADP:O ratios and the state 4 rates of respiration are highly dependent upon the concentration of mitochondria at which these parameters are measured. (b) The mitochondrial isolate is contaminated with at least two divalent cation-stimulated ATPase, of which one is the F1F0-ATPase of broken mitochondria. (c) The oligomycin-sensitive component of state 4 respiration is completely inhibited by ethylene glycol bis(beta-amino-ethylether) N,N'-tetraacetic acid (EGTA). This inhibition is biphasic and attributable to the differential affinity of EGTA for Ca(II) and Mg(II). (d) Ca(II) and Mg(II) stimulate state 4 respiration, thereby depressing RCR values. These cations also decrease ADP:O ratios from greater than or equal to 3.25 to 3.0 for some NAD-linked substrates. (e) Uncoupled (i.e., oligomycin-insensitive) state 4 respiration can be abolished by treating the mitochondria with Nagarse and by preincubating mitochondria with exogenous substrate. (f) The ADP:O ratios obtained when these heart mitochondria oxidize pyruvate/malate, alpha-ketoglutarate, and beta-hydroxybutyrate are fractional and significantly greater than 3.0.

Theoretical analysis of compartmented coupling in linear enzyme systems.

Exact equations which describe the kinetic patterns of enzyme/enzyme complexes, when compartmented coupling occurs between them, are presented. Compartmented coupling refers to the creation of a local environment in which the concentration of an intermediate, shared by two enzymes, is higher than its solution concentration. This results in a higher coupling enzyme activity, a condition reflected in a shorter transition time for the system. In this paper, equations are presented which allow experimenters to quantitate the effect of compartmented coupling in terms of changes in the apparent Km and Vmax values. The equations presented in this paper are more exact than those previously derived since they do not incorporate first order assumptions before derivation.

Compartmented coupling of chicken heart mitochondrial creatine kinase to the nucleotide translocase requires the outer mitochondrial membrane.

The kinetic coupling of mitochondrial creatine kinase (MiMi-CK) to ADP/ATP translocase in chicken heart mitochondrial preparations is demonstrated. Measuring the MiMi-CK apparent Km value for MgATP2- (at saturating creatine) gives a value of 36 microM when MiMi-CK is coupled to oxidative phosphorylation. This Km value is threefold lower than the Km for enzyme bound to mitoplasts or free in solution. The nucleotide translocase Km value for ADP decreases from 20 to 10 microM in the presence of 50 mM creatine only with intact mitochondria. Similar experiments with mitoplasts do not give decreased Km values. The observed Km differences can be used to calculate the concentration of ATP and ADP under steady-state conditions showing that the observed differences in the kinetic constants accurately reflect the enzyme activities of MiMi-CK under the different conditions. The behavior of the Km values provides evidence for what we term compartmented coupling. Therefore, like the rabbit heart system (S. Erickson-Viitanen, P. Viitanen, P. J. Geiger, W. C. T. Yang, and S. P. Bessman (1982) J. Biol. Chem. 257, 14395-14404) compartmented coupling requires an intact outer mitochondrial membrane. The apparent Km values for normal or compartmentally coupled systems can be used to calculate steady-state values of ATP and ADP by coupling enzyme theory. Hence, the overall kinetic parameters accurately reflect the behavior of the enzymes whether free in solution or in the intermembrane space.

Homogeneous chicken heart mitochondrial creatine kinase purified by dye-ligand and transition-state analog-affinity chromatography.

A method for the preparation of homogeneous mitochondrial creatine kinase from chicken heart is presented. The two-column procedure, which can be completed in 2 days, uses Procion red dye and transition-state analog-affinity chromatography. The transition-state analog-affinity chromatographic system utilizes an ADP-hexane-agarose column in conjunction with the transition-state analog complex originally developed by E. J. Milner-White and D. C. Watts (1971, Biochem, J. 122, 727-740) composed of KNO3, MgCl2, creatine, and ADP. The enzyme is a dimer composed of 2 Mr 43,000 subunits. The sequence of the first N-terminal 20 amino acids shows that the enzyme is different from the cytosolic isozymes but similar to human mitochondrial creatine kinase. The enzyme has an extinction coefficient of epsilon 280 nm = 2.22 +/- 0.10 ml X mg-1 X cm-1 and a maximum velocity of 200 IU/ml at pH 7.0. The kinetic constants for the chicken heart mitochondrial isozyme are comparable to values for the canine and human heart isozyme.

Association of chicken mitochondrial creatine kinase with the inner mitochondrial membrane.

The stoichiometry and dissociation constant for the binding of homogeneous chicken heart mitochondrial creatine kinase (MiMi-CK) to mitoplasts was examined under a variety of conditions. Salts and substrates release MiMi-CK from mitoplasts in a manner that suggests an ionic interaction. The binding of MiMi-CK to mitoplasts is competitively inhibited by Adriamycin, suggesting that they compete for the same binding site. Fluorescence measurements also show that Adriamycin binds to MiMi-CK so that the effect of Adriamycin on the binding of MiMi-CK to mitoplasts is not simple. Titrating mitoplasts with homogeneous MiMi-CK at different pH values shows a pH-dependent equilibrium involving a group(s) on either the membrane or the enzyme with a pKa = 6. Extrapolating these titrations to infinite MiMi-CK concentration gives 14.6 IU bound/nmol cytochrome aa3 corresponding to 1.12 mol MiMi-CK/mol cytochrome aa3. Chicken heart mitochondria contain, after isolation, 2.86 +/- 0.42 IU/nmol cytochrome aa3. Titrating respiring mitoplasts with carboxyatractyloside gives at saturation 3.3 mol ADP/ATP translocase/mol cytochrome aa3. Therefore, chicken heart mitoplasts can maximally bind about 1 mol of MiMi-CK per 3 mol translocase; in normal chicken heart mitochondria about 1 mol of MiMi-CK is present per 13 mol translocase.

Quantitation of the efflux of acylcarnitines from rat heart, brain, and liver mitochondria.

The efflux of individual short-chain and medium-chain acylcarnitines from rat liver, heart, and brain mitochondria metabolizing several substrates has been measured. The acylcarnitine efflux profiles depend on the substrate, the source of mitochondria, and the incubation conditions. The largest amount of any acylcarnitine effluxing per mg of protein was acetylcarnitine produced by heart mitochondria from pyruvate. This efflux of acetylcarnitine from heart mitochondria is almost 5 times greater with 1 mM than 0.2 mM carnitine. Apparently the acetyl-CoA generated from pyruvate by pyruvate dehydrogenase is very accessible to carnitine acetyltransferase. Very little acetylcarnitine effluxes from heart mitochondria when octanoate is the substrate except in the presence of malonate. Acetylcarnitine production from some substrates peaks and then declines, indicating uptake and utilization. The unequivocal demonstration that considerable amounts of propionylcarnitine or isobutyrylcarnitine efflux from heart mitochondria metabolizing alpha-ketoisovalerate and alpha-keto-beta-methylvalerate provides evidence for a role (via removal of non-metabolizable propionyl-CoA or slowly metabolizable acyl-CoAs) for carnitine in tissues which have limited capacity to metabolize propionyl-CoA. These results also show propionyl-CoA must be formed during the metabolism of alpha-ketoisovalerate and that extra-mitochondrial free carnitine rapidly interacts with matrix short-chain aliphatic acyl-CoA generated from alpha-keto acids of branched-chain amino acids and pyruvate in the presence and absence of malate.

Lagtime: a program for calculating coupled enzyme assay parameters.

A program is described for calculating either (i) the time required for the observed rate to approximate the rate of the enzyme under study (the lagtime) when the concentrations of the auxiliary enzymes are known or (ii) the units of auxiliary enzymes needed to obtain a desired lagtime. The calculations for these coupled enzyme systems also apply to cases where one of the intermediates undergoes mutarotation; for example, equations for coupled reactions involving two enantiomers of glucose as intermediates incorporate the rate constants for mutarotation. When two auxiliary enzymes are used, the program also minimizes the total cost of the assay if the price per unit of the coupling enzymes are known. The equations used are those of S.P.J. Brooks et al. (Can. J. Biochem. Cell Biol. 62 (1984) 945-955; 956-963.

Estimating enzyme kinetic parameters: a computer program for linear regression and non-parametric analysis.

An IBM computer program, WILMAN4, is described which calculates the estimates, Km, V and Km/V from initial velocity measurements according to one of four statistical methods. Three of these methods involve linear regression analysis using weights given by assuming: (i) constant absolute error (G.N. Wilkinson, 1961, Biochem J., 80, 324-332), (ii) constant relative error (G. Johansen and R. Lumry, 1961, C.R. Trav. Lab. Carlsberg, 32, 185-214) and (iii) an error function in between the above two cases. (A. Cornish-Bowden, 1976, Principles of Enzyme Kinetics, Butterworths Inc, Boston, Mass., pp. 168-193). The fourth method is a non-parametric procedure derived by Eisenthal and Cornish-Bowden (Biochim. Biophys. Acta, 532 (1974) 268-272). Residuals are obtained by subtracting the experimental and the calculated velocities. Outliers, or residuals which are greater than two experimental standard deviations, can be identified and removed from the data set. If the sequence of positive and negative signs of the residuals is random as determined by a statistical probability calculation, the data set is assumed to obey the Michaelis-Menten equation.

Muscle cell cultures from chicken breast muscle have increased specific activities of creatine kinase when incubated at 41 degrees C compared with 37 degrees C.

Chicken muscle cell cultures were incubated at 41 degrees C, the physiological chicken body temperature, and compared with cultures incubated at 37 degrees C, the typical cell culture incubation temperature. The cultures incubated at 41 degrees C show not only an increase in creatine kinase (CK)-specific activity but also a marked increase in the percentage of adult muscle CK isozyme (MM-CK) in 7-day muscle cultures. Muscle cell cultures incubated in the presence of cytosine arabinoside (ara-C), a cell proliferation inhibitor, do not have the mononucleated cell overgrowth seen at 41 degrees C and thus exhibit a further increase in creatine kinase-specific activity compared with cultures incubated at 41 degrees C in the absence of ara-C. These results suggest that muscle cell cultures incubated at 41 degrees C are more highly differentiated than those incubated at 37 degrees C.

Decreased mitochondrial creatine kinase activity in dystrophic chicken breast muscle alters creatine-linked respiratory coupling.

Dystrophic chicken breast muscle mitochondria contain significantly less mitochondrial creatine kinase than normal breast muscle mitochondria. Breast muscle mitochondria from normal 16- to 40-day-old chickens contain approximately 80 units of mitochondrial creatine kinase per unit of succinate:INT (p-iodonitrotetrazolium violet) reductase, a mitochondrial marker, while dystrophic chicken breast muscle mitochondria contain 36-44 units. Normal chicken heart muscle mitochondria contain about 10% of the mitochondrial creatine kinase per unit of succinate:INT reductase as normal breast muscle mitochondria. The levels in heart muscle mitochondria from dystrophic chickens are not affected significantly. Evidence is presented which shows that the reduced level of mitochondrial creatine kinase in dystrophic breast muscle mitochondria is responsible for an altered creatine linked respiration. First, both normal and dystrophic breast muscle mitochondria respire with the same state 3 and state 4 respiration. Second, the post-ADP state 4 rate of respiration of normal breast muscle mitochondria in the presence of 20 mM creatine continues at the state 3 rate. However, the state 4 rate of dystrophic breast muscle mitochondria and mitochondria from other muscle types with a low level of mitochondrial creatine kinase, such as heart muscle and 5-day-old chicken breast muscle, is slower than the state 3 rate. Third, dystrophic breast mitochondria synthesize ATP at the same rate as normal breast muscle mitochondria but rates of creatine phosphate synthesis in 20-50 mM Pi are reduced significantly. Finally, increasing concentrations of Pi displace mitochondrial creatine kinase from mitoplasts of normal and dystrophic breast muscle mitochondria with the same apparent KD, indicating that the outer surface of the inner mitochondrial membrane and the mitochondrial creatine kinase from dystrophic muscle are not altered.

Resolution of the low-molecular-weight acid phosphatase in avian pectoral muscle into two distinct enzyme forms.

Three distinct acid phosphatases were recently reported in avian breast muscle [J. H. Baxter and C. H. Suelter (1984) Arch. Biochem. Biophys. 228, 397-406]. Of the increased acid phosphatase activity in dystrophic muscle compared to normal muscle, 84% can be accounted for as a low-molecular-weight, cytosolic form. This low-molecular-weight form has now been purified and resolved into two distinct forms, A and B, differing in isoelectric point, apparent molecular weight, substrate specificity, and activation by guanosine. One of the two enzymes exhibits substrate inhibition with 4-methylumbelliferyl phosphate, indicating a further difference. The evidence suggests that both enzymes are Class IV acid phosphatases. Their concentrations are highest in tissues with a high catabolic activity.

Theory and practical application of coupled enzyme reactions: one and two auxiliary enzymes.

An extended and practical set of equations which describe coupled enzyme reactions is presented. The mathematical treatment relies on two assumptions: (a) the rate of the primary enzyme reaction is constant and (b) the reverse reactions are negligible. The treatment leads to the development of new equations which relate the time required for the concentration of a reaction intermediate to reach a defined fraction of its steady-state concentration to the kinetic parameters of the enzymes when mutarotation of one of the intermediates does not occur. The new equations reduce to those previously derived when the steady-state concentration of the intermediate is small compared with its Km value. A method for minimizing the cost of the two auxiliary enzyme system is also provided.

Theory and practical application of coupled enzyme systems: one and two coupling enzymes with mutarotation of an intermediate.

This paper provides equations to calculate the elapsed time before the concentration of the final intermediate, in a sequence of coupled enzymatic reactions, achieves a defined fraction of its steady-state concentration when one of the intermediates undergoes mutarotation. The equations can be used to predict lag times for systems involving one coupling enzyme, as is the case when hexokinase or phosphoglucomutase activity is monitored using glucose-6-phosphate dehydrogenase as the auxiliary enzyme, or for systems of two coupling enzymes, as is the case when the activities of enzymes producing ATP (such as creatine kinase) are monitored by coupling the production of ATP to hexokinase and glucose-6-phosphate dehydrogenase. The theoretical aspects of the assay have been verified using hexokinase (as the primary enzyme) and glucose-6-phosphate dehydrogenase (as the coupling enzyme). A method of cost minimization, based on the above relationships, is also provided.

Circulatory clearance, internalization, and degradation of muscle AMP aminohydrolase.

Chicken muscle AMP aminohydrolase is cleared rapidly from the circulation of chickens after intravenous injection of the purified enzyme (Husic, H. D., and Suelter, C. H. (1980) Biochem. Biophys. Res. Commun. 95, 228-235). After the intravenous injection of unlabeled, 125I-labeled, or [14C]sucrose-labeled AMP aminohydrolase, enzyme activity and radioactivity are cleared at the same rates and concentrate in the liver and spleen. After injection of the [14C]sucrose-labeled enzyme, 14C is retained in the liver and spleen and low molecular weight 14C is recovered primarily in a fraction which cosediments with lysosomes when tissue homogenates are sedimented on sucrose density gradients. When liver cells are fractionated after clearance of [14C]sucrose-labeled enzyme, 14C is recovered primarily in the parenchymal cells. The circulatory clearance of AMP aminohydrolase is inhibited by heparin and other sulfated polysaccharides, but not by compounds which inhibit previously described carbohydrate-mediated systems for the uptake of circulatory glycoproteins. Heparin injection after the clearance of AMP aminohydrolase causes the release of the enzyme from the liver and spleen into the circulation. When heparin is injected at various times after clearance, decreasing amounts of enzyme are released with time; these results show that the enzyme is internalized with a half-life of 0.98 h.

Binding, internalization, and degradation of AMP aminohydrolase by avian hepatocyte monolayers.

Chicken muscle AMP aminohydrolase is cleared from the circulation of chickens after intravenous injection of the purified enzyme with a half-life of 3-5 min (Husic, H.D., and Suelter, C.H. (1980) Biochem. Biophys. Res. Commun. 95, 228-235). The enzyme is not inactivated before clearance, the clearance is inhibited by sulfated polysaccharides, and the enzyme is cleared primarily by the spleen and the parenchymal cells of the liver where it is internalized and degraded in lysosomes (Husic, H.D., and Suelter, C.H. (1984) J. Biol. Chem. 259, 4359-4364). The binding of AMP aminohydrolase to hepatocyte monolayers in vitro at 4 degrees C is saturable with a dissociation constant of 11.3 X 10(-8) M; there are 2.6 X 10(6) AMP aminohydrolase binding sites/hepatocyte. The interaction of the enzyme with hepatocyte monolayers is inhibited by sulfated polysaccharides, effectors of its enzymatic activity and high salt concentrations; various monosaccharides had little effect on the binding of the enzyme to hepatocyte monolayers. Heparitinase treatment of hepatocyte monolayers abolished 77% of the binding of the enzyme. Heparin promotes the dissociation of 125I-labeled or [14C]sucrose-labeled enzyme bound to the cell surface; radioactivity which is not dissociated by heparin is assumed to be internalized at 37 degrees C. Low molecular weight 125I-labeled degradation products are released into the media with time when the 125I-labeled enzyme, bound to hepatocytes at 4 degrees C, is incubated at 37 degrees C; when [14C]sucrose-labeled enzyme is incubated with hepatocytes at 37 degrees C, low molecular weight 14C-labeled degradation products are not released into the media but instead accumulate in the cells. The half-life for internalization of the bound enzyme based on this rate of accumulation is 0.77 h. These results suggest that glycosaminoglycans are involved in the binding of AMP aminohydrolase to the hepatocyte cell surface and that the bound enzyme is internalized and degraded.

Multiple acid phosphatases in avian pectoral muscle--the postmicrosomal supernatant acid phosphatase is elevated in avian dystrophic muscle.

There are at least three forms of acid phosphatase in avian pectoralis muscle differing in molecular weight, subcellular location, and response to various substrates and inhibitors. These enzymes are separated by differential sedimentation into postmicrosomal supernatant, lysosomal, and microsomal activities with apparent molecular weights in Triton X-100 of 68,000, 198,000, and 365,000, respectively. All of the enzymes show acid pH optima (pH approximately 5), but the postmicrosomal supernatant form is distinctly different from the other two forms in its resistance to most common phosphatase inhibitors and in its reduced activity against several organic phosphates. Quantitation of these three forms of acid phosphatase in normal and dystrophic avian pectoralis muscle shows that the postmicrosomal supernatant form is significantly elevated in dystrophic muscle; at 33 days ex ovo, 84% of the increased acid phosphatase activity in dystrophic muscle can be attributed to the postmicrosomal supernatant form. The microsomal form is only slightly elevated; the level of the lysosomal form is not altered.