A simple method for calculating Km and V from a single enzyme reaction progress curve.

A method of calculating Km and V from a single reaction progress curve is presented. The integrated Michaelis-Menten equation Vt = So + Km 1n(So/S), may be rearranged to the form 1/v = 1/V + Km/VS, where v = (Si - Sj)/deltat = deltaS/deltat and S = (Si - Sj)/ln(Si/Sj) or S is approximated by (Si + Sj)/2.Si and Sj represent substrate concentrations at two points along a reaction progress curve separated by the time interval, deltat. The error resulting from the approximation depends on the magnitude of deltaSi/Si; when deltaSi/Si less than 0.3, the error is insignificant; when deltaSi/Si greater than 0.3, the error becomes significant. Procedures are presented to correct this error. Simulated data and application to the direct spectrophotometric assay of AMP aminohydrolase and the lactate dehydrogenase coupled assay of pyruvate kinase are provided. The method is recommended when routine Km and V values are desired. Compared to the initial rate method, it is faster, requires less substrate, and eliminates pipetting errors.

5'-AMP aminohydrolase activity in erythrocytes from normal and dystrophic individuals.

5'-AMP aminohydrolase activities in red blood cells from normal human adults and from patients with pseudohypertrophic dystrophy, myotonic dystrophy, limb girdle dystrophy, neuromuscular atrophy, Charcot-Marie Tooth Disease, and spinal muscular atrophy were examined. In contrast to the greatly decreased skeletal muscular levels of 5'-AMP aminohydrolase observed in Pseudohypertrophic muscular dystrophy, levels in red blood cells of all patients were not significantly different from normals. Pyruvate kinase and creatine kinase activities were determined for comparative purposes. The density distribution of normal and dystrophic erythrocytes were essentially identical.

Computer-interfaced rapid scanning molecular absorption and emission spectrophotometer for stopped-flow studies of enzyme reactions.

An improved stopped-flow system with a rapid-scanning spectrometer permits measurement of either light absorption or emission at scan speeds of up to 150 spectra per second. The entire flow system, including valves, syringes and quartz flow cell is contained in a thermostat bath and is leak-tight from 5 to 45 degrees C. Pneumatic valves control the flow through Teflon tubing. Quartz fiber-optic light guides are used to transmit light to and from the flow cell. Experimental data are given to demonstrate the absorbance and fluorescence modes. The growth and decay of the fluorescence spectrum of NADH was followed in a reaction catalyzed by lactate dehydrogenase (LDH). The kinetics of binding of 1-anilino-8-naphthalene sulfonate (ANS) to bovine serum albumin (BSA) was studied by both scanning and fixed-wavelength fluorescence emission. The fluorescence of BSA was completely quenched within two milliseconds accompanied by an abrupt increase in the fluorescence of ANS which was followed by a slower first-order growth.

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.

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.

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.

How to prevent losses of protein by adsorption to glass and plastic.

An improved procedure for reducing the loss of protein by adsorption to glass or plastic surfaces is reported. For working with proteins at the microgram level, the solvent is modified by adding glycerol (50% final concentration) or Triton X-100 (0.2 mM final concentration). Coating the plastic or glass surfaces with proteins such as bovine serum albumin or other materials is not as effective; adding proteins such as bovine serum albumin to the solvent is counterproductive.

Skeletal muscle lysosomes: comparison of lysosomes from normal and dystrophic avian pectoralis muscle as a function of age.

The properties of skeletal muscle lysosomes from normal and dystrophic chickens were studied to assess their involvement in the dystrophic process. A method is described for isolation of a three-to-sevenfold purified lysosome fraction with 29-33% yield. Lysosomal enzymes in crude homogenates and isolated lysosome-enriched fractions from dystrophic muscle exhibit decreased latency for N-acetyl beta-D-glucosaminidase, acid phosphatase, and cathepsin D. However, no differences in the fragility of lysosomes in isolated lysosome-enriched fractions from normal and dystrophic muscle were observed using shear, sonication and detergent stress. Lower percent recovery, enrichment factor and percent latency of acid phosphatase compared to N-acetyl-beta-D-glucosaminidase and cathepsin D were observed from both normal and dystrophic muscle. These results are consistent with the presence of a significant amount of nonlysosomal acid phosphatase activity in skeletal muscle.

The levels of creatine kinase and adenylate kinase in the plasma of dystrophic chickens reflect the rates of loss of these enzymes from the circulation.

The rates of loss of adenylate kinase and creatine kinase from the circulation after intravenous injection of homogenous chicken skeletal muscle enzymes were examined to determine the role of plasma clearance rates in determining the plasma levels of these enzymes in normal and dystrophic chickens. The rapid clearance of adenylate kinase activity (average half-life of 5 min) and the slower biphasic clearance of creatine kinase activity (average half-lives of 0.95 and 11 hr) are consistent with the elevation of creatine kinase but not adenylate kinase in the blood plasma of dystrophic chickens compared to normal chickens. The rates of clearance of these enzymes were similar in normal chickens compared to dystrophic chickens. Radioiodinated enzymes were cleared at similar, but slightly more rapid rates than the loss of enzyme activity. The loss of adenylate kinase activity from the circulation may be due in part to inactivation since adenylate kinase activity is rapidly inactivated in serum in vitro, and because no increase in adenylate kinase activity is observed in the most specific sites of clearance of the radioiodinated enzyme, the liver and spleen. The comparison of enzyme activities in press juices to the activities in high-ionic-strength homogenates of muscle tissue from normal and dystrophic muscle, indicates that adenylate kinase activity is not associated with intracellular structures to the extent that would prohibit release from dystrophic muscle tissue. These results, and those presented previously with regard to plasma levels and clearance rates of AMP aminohydrolase and pyruvate kinase in normal and dystrophic chickens (11) support our hypothesis that the rates of loss of muscle enzyme activities from the circulation are important in determining the circulating levels of muscle enzymes in dystrophic chickens. Furthermore, from the measurement of plasma levels and clearance rates of creatine kinase, it was estimated that the efflux rate of creatine kinase from dystrophic muscle tissue is 2.0% of the total breast muscle creatine kinase per day.