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.

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.

Effect of H+ on the K+ activation of adenosine-5'-monophosphate aminohydrolase.

The activation of adenosine-5'-monophosphate aminohydrolase from rabbit skeletal muscle by H+ has been demonstrated. Evidence is presented which indicates that the binding of H+ and K+ is linked, in that the dissociation constant (KA) for K+ activation is reduced as the pH is lowered. Concomitantly, the pK of several enzyme functional groups is changed when K+ is added to a solution of enzyme. This change is pK results in an uptake or release of H+, depending on the pH, and shows that K+ interacts with the enzyme to achieve its effect. The uptake or release of H+ provides a simple method of following conformational changes in the enzyme following interaction of K+. The KD for K+ interaction monitored by following pH changes is the same within experimental error as that measured from kinetic data.

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.

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.

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.

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.