Percy J. Russell

Isoenzymes of adenylate kinase in human tissue

Abstract A study of the isoenzymes of adenylate kinases (ATP:AMP phosphotransferases EC 2.7.4.3) from several tissues of human and rabbit indicated that there is a minimum of two sets of isoenzymes within an individual. Isoenzyme sets were distinguished by differences in inhibitions by AgNO3 and an antiserum against rabbit muscle adenylate kinase. The adenylate kinases from muscle, erythrocytes and brain were similar and form one set of isoenzymes. The adenylate kinases of liver, kidney, spleen and heart were similar and form another set of isoenzymes.

Inhibition of Rabbit Muscle Isozymes by Vitamin C

Abstract The ability of vitamins C, E and K to inhibit enzymes directly has been investigated. It was found that vitamin E and some analogs and menadione (vitamin K3) inhibited several enzymes irreversibility at concentrations below one millimolar. Ascorbate inhibits rabbit muscle 6-phosphofructokinase (MPFK-1; EC 2.7.1.11), muscle type LDH (EC 1.1.1.27), and muscle AK (EC 2.7.4.3) at low concentrations that do not inhibit equivalent liver isozymes. Ascorbate Ki values for muscle-type LDH and heart-type LDH isozymes are 0.007 and 3 mM, respectively. The ascorbate Ki value for rabbit skeletal muscle PFK-1 is 0.16mM; liver PFK-1 is not inhibited by ascorbate. Dehydroascorbate does not inhibit any enzyme at ascorbate concentrations normally found in cells. All ascorbate inhibitions are completely reactivated or nearly so by L-ascorbate oxidase, CYS, GSH, or DTT. We propose a hypothesis that ascorbate facilitates glycogen storage in muscle by inhibiting glycolysis. The relationship between ascorbate metabolism and diabetes is discussed.

Aldolase and actin protect rabbit muscle lactate dehydrogenase from ascorbate inhibition.

Muscle-type LDH (LDH-m4) activity is critical for efficient anaerobic glycolysis. The results here show that rabbit LDH-M4 is inhibited by concentrations of ascorbate normally found in tissues. Aldolase and muscle G-actin were found to protect and to reverse inhibitions of LDH-m4 by ascorbate. G-actins showed some species specificity. Myosin, tropomyosin and troponin from rabbit muscle and muscle proteins from other animal sources had no affect on the inhibitions by ascorbate. The substrate inhibition of LDH-m4 by pyruvate is partially relieved by the presence of aldolase and lowers the Km without affecting the Vm. G-actin under similar conditions has no affect. It is believed that these studies reflect some of the resting properties of glycolytic enzymes that bind and unbind to contractile elements. It is proposed that ascorbate facilitates the storage of glycogen in muscle at rest by inhibiting glycolysis.

On kinetic treatments of enzyme-antienzyme reactions

Abstract Antibodies against enzymes usually bind tightly to their antigens, and under such conditions Michaelis-Menten or steady-state kinetics are not applicable. This paper points out some of the misinterpretations which arise when reversible kinetics are applied to tight-binding or essentially irreversible systems. The antiserum against yeast adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) was used for the purposes of illustration. Several criteria for evidence of a reversible tight-binding system are given. One of the most useful methods is a plot of initial velocities as a function of enzyme concentrations in the presence and absence of antienzyme.

Characteristics of rabbit muscle adenylate kinase inhibition by ascorbate

Earlier studies showed that of the glycolytic enzymes, the muscle isozymes PFK-1, LDH, and AK were inhibited by ascorbic acid. These studies on the characteristics of the inhibition of RMAK by ascorbate are part of a hypothesis [] that ascorbate facilitates the storage of skeletal muscle glycogen by inhibiting glycolysis when the muscle is at rest. These studies examine conditions for RMAK inhibition, prevention of inhibition, and reversal of ascorbate inhibition. We found that the concentration of RMAK was an important condition for inhibition. Above 200 nM RMAK, inhibition by ascorbate could not be demonstrated and below that concentration RMAK became increasingly sensitive to ascorbate inhibition. Associated with increased sensitivity to inhibition by ascorbate is a deviation from a linear to a concave relationship between low RMAK concentrations and enzyme activity. At low RMAK concentrations, the concave relationship becomes convex in the presence of muscle aldolase. In addition, aldolase reverses inhibitions by ascorbate. A comparison of inhibition of RMAK by ascorbate and inhibition of LDH-m4 [] is discussed. Other proteins prevent RMAK inhibition but do not reverse inhibition by ascorbate. The role of RMAK as a factor in the control of the rate of glycolysis is presented as is the role of compartmentalization with respect to the proposed role for ascorbate inhibition.

Some characteristics of rabbit muscle phosphofructokinase-1 inhibition by ascorbate.

These studies relate to a working hypothesis that glycogen storage is facilitated in resting muscle by inhibiting glycolysis via inhibition of LDH, AK, and PFK-1 by ascorbate; when muscle is active, these isozymes combine with muscle proteins and are released and protected from inhibition by ascorbate and glycolysis proceeds. Focus in these studies is on the ability of G-actin and aldolase to prevent PFK-1 inhibition by ascorbate. We found that inhibition by ascorbate was PFK-1 concentration dependent; ascorbate does not inhibit above 200 nM PFK-1. We conclude that ascorbate inhibits PFK-1 dimers (and perhaps monomers) but not PFK-1 tetramers. Separation of PFK-1 dimers from tetramers was achieved with centrifugal filter devices and differences in their sensitivity to ascorbate inhibition were demonstrated. Some comparisons are made with attributes of AK inhibitions by ascorbate that, like PFK-1, are also enzyme concentration dependent. Discussions relate findings to cellular infrastructure and the role of ascorbate in glycogen synthesis.

Histologic, ultrastructural, and enzymatic measurements of infarct size following coronary artery stenosis and occlusion.

Coronary artery narrowing (CAN), which reduced resting coronary blood flow (BF) by 50%, was induced in 10 conscious dogs and was maintained for 4 hours. Five additional dogs (group 1) with complete coronary artery occlusion were compared to the dogs with CAN. Serum isoenzymes of creatine phosphokinase (CK) and lactate dehydrogenase (LD) were monitored hourly in all groups. After 36 hours, samples were obtained for regional myocardial BF, quantitative histology, and quantitative ultrastructural (EM) morphology. Six dogs with CAN had small infarcts (MI) of less than 1 gm and persistent myocardial cell injury (group 2). The other four dogs with CAN has only persistent myocardial cell injury by ultrastructural criteria (group 3). Peak serum CK activities in groups 2 and 3 were similar, as well MI sizes calculated from serum CK and myocardial depletion. MB CK was of diagnostic value in group 1 but not in groups 2 and 3. The ratio of LD 1/LD 2 had diagnostic value in all three groups. MI size by enzyme estimates was consistently higher than planimetered MI size at autopsy in both groups 1 and 2. All three groups had significant amounts of ultrastructural damage outside of histologically demonstrated. MI. These findings suggest that (1) gross and histologic MI size determination of 36 hours after ischemia underestimate extent of damage, and (2) ultrastructural cell changes cause significant release of CK and LD in coronary disease (CAD).

Characteristics of Rabbit Muscle Adenylate Kinase Inhibition by Sulfur and Recovery by Dithiothreitol

Structure-function relationships of rabbit muscle adenylate kinase (RMAK) were studied by examining the characteristics of inhibitions by hydrophobic inhibitors and reactivations by sulfhydryl reagents. RMAK is inhibited by 1-butanol,N-ethylmaleimide (NEM) and elemental sulfur (S8) with increasing effectiveness in the order of increasing hydrophobicity. Characteristics of these hydrophobic inhibitors are compared with inhibitors forming covalent bonds or reversible complexes. A mechanism is proposed for hydrophobic inhibitors of RMAK that involves conformational changes promoted by interacting with hydrophobic regions. The reversal of RMAK inhibition by sulfhydryl compounds involves a conformational change that exposes hydrophobic regions and the inhibitor to water. Circular dichroism (CD) data show changes in the secondary structures of RMAK, indicating that the inhibitors and the sulfhydryl compounds promote conformational changes. The results of these studies show that the activity of a small enzyme can be controlled in a manner analogous to the allosteric control of larger enzymes.

Effect of lithium salts on lactate dehydrogenase, adenylate kinase, and 1-phosphofructokinase activities

Inhibitions of 30 nM rabbit muscle 1-phosphofructokinase (PFK-1) by lithium, potassium, and sodium salts showed inhibition or not depending upon the anion present. Generally, potassium salts were more potent inhibitors than sodium salts; the extent of inhibition by lithium salts also varied with the anion. Li2CO3 was a relatively potent inhibitor of PFK-1 but LiCl and lithium acetate were not. Our results suggest that extents of inhibition by monovalent salts were due to both cations and anions, and the latter needs to be considered before inhibition can be credited to the cation. An explanation for monovalent salt inhibitions is proffered involving interactions of both cations and anions at negative and positive sites of PFK-1 that affect enzyme activity. Our studies suggest that lithium cations per se are not inhibitors: the inhibitors are the lithium salts, and we suggest that in vitro studies involving the effects of monovalent salts on enzymes should involve more than one anion.

Effect of ammonium, sodium, and potassium ions on rabbit muscle phosphofructokinase-1 and adenylate kinase activities

This report shows that 30 nM PFK-1 and 30 nM AK were both affected by the presence of NH4+, Na+, and K+ salts but with opposite consequences. Low concentrations of PFK-1 lose about half of its activity as a result of dilution and become susceptible to further activity losses owing to the presence of monovalent salts. On the other hand low concentrations of AK lose about 75 percent of its activity but regains activity losses owing to the presence of monovalent salts. It was determined that regain of AK activity did not appear to be a reflection of a major effect on the Km value of either AMP or ATP. Dilution to 30 nM AK resulted in no increase Km values compared to Km values at 140 nM AK. Dilution caused major decreases in the maximum velocities, Vmax, when ATP or fructose 6-phosphate was the variable substrate. It was shown in earlier reports that these same low concentrations of PFK-1 and AK were susceptible inhibitions by ascorbate. These attributes are discussed as they may relate to the role of ascorbate facilitation glycogen synthesis in resting muscle and the role that the cytoskeleton infrastructure scaffold may play is also discussed.