William L. Meyer

Skeletal Muscle and Cardiovascular Adaptations to Exercise Conditioning in Older Coronary Patients

BACKGROUND Older coronary patients suffer from a low functional capacity and high rates of disability. Supervised exercise programs improve aerobic capacity in middle-aged coronary patients by improving both cardiac output and peripheral extraction of oxygen. Physiological adaptations to aerobic conditioning, however, have not been well studied in older coronary patients. METHODS AND RESULTS The effect of a 3-month and a 1-year program of intense aerobic exercise was studied in 60 older coronary patients (mean age, 68 +/- 5 years) beginning 8 +/- 5 weeks after myocardial infarction or coronary bypass surgery. Outcome measures included peak aerobic capacity, cardiac output, arterio-venous oxygen difference, hyperemic calf blood flow, and skeletal muscle fiber morphometry, oxidative enzyme activity, and capillarity. Training results were compared with a sedentary, age- and diagnosis-matched control group (n = 10). Peak aerobic capacity increased in the intervention group at 3 months and at 1 year by 16% and 20%, respectively (both P < .01). Peak exercise cardiac output, hyperemic calf blood flow, and vascular conductance were unaffected by the conditioning protocol. At 3 and 12 months, arteriovenous oxygen difference at peak exercise was increased in the exercise group but not in control subjects. Histochemical analysis of skeletal muscle documented a 34% increase in capillary density and a 23% increase in succinate dehydrogenase activity after 3 months of conditioning (both P < .02). At 12 months, individual fiber area increased by 29% compared with baseline (P < .01). CONCLUSIONS Older coronary patients successfully improve peak aerobic capacity after 3 and 12 months of supervised aerobic conditioning compared with control subjects. The mechanism of the increase in peak aerobic capacity is associated almost exclusively with peripheral skeletal muscle adaptations, with no discernible improvements in cardiac output or calf blood flow.

Decreased glycolytic enzyme activity in epiphyseal cartilage of cortisone-treated rats☆

Abstract Procedures are described for highly reproducible assays of protein content and the activity of hexokinase, phosphofructokinase, aldolase, pyruvate kinase, lactate dehydrogenase, NAD-linked glycerophosphate dehydrogenase, and glucose-6-phos-phate dehydrogenase in epiphyseal cartilage from rat tibiae. Glucokinase is not present. With cortisone treatment the four glycolytic enzymes, phosphofructokinase, aldolase, pyruvate kinase, and lactate dehydrogenase, exhibit a closely coordinated decrease in activity of over 50% per cell, or an average decrease of 34% in specific activity (Enzyme Units/milligram protein). Glycerophosphate dehydrogenase follows a generally similar pattern. Hexokinase and glucose-6-phosphate dehydrogenase are also decreased per cell, but only to the same extent as total protein so that there is little change in specific activity. Starvation brings about changes in the enzyme activity pattern similar to those of cortisone-treatment.

Ribonuclease-Inhibitor System Abnormality in Dystrophic Mouse Skeletal Muscle

Skeletal muscle extracts from mice with muscular dystrophy contain severalfold higher than normal levels of free alkaline ribonuclease II activity and none of the free ribonuclease inhibitor normally present. This abnormal pattern is not seen in heart or liver extracts from dystrophic mice.

The inductive effect of rickets on glycolytic enzymes of rat epiphyseal cartilage and its reversal by vitamin D and phosphate

Abstract In rickets produced in rats by low phosphate, vitamin D-free diets, epiphyseal cartilage increases in wet weight, and in deoxyribonucleic acid, glycogen, and protein content. Dietary phosphate reverses all of these effects. Vitamin D reverses all of these rachitic changes with the exception of that in deoxyribonucleic acid content. The effectiveness of vitamin D in reducing cartilage cell hypertrophy without decreasing cell numbers differentiates the mechanism of vitamin D action from that of phosphate. Glycolysis is doubled per cell in rachitic cartilage and there is a corresponding coordinate increase in specific activities (EU/mg protein) of phosphofructokinase, aldolase, pyruvate kinase, and lactate dehydrogenase. Both effects are reversed by dietary phosphate or vitamin D. It is suggested that increased glycolysis may be due to a specific increment in glycolytic enzyme levels supplemented by a general increase in tissue protein. A proportional relationship between cellular lactate production and glycolytic enzyme activity is demonstrated in nine different physiological states. The hypothesis is advanced that a regulatory mechanism operates in cartilage to coordinately control the levels of glycolytic enzymes and cellular metabolism.

The effect of pH and temperature on the kinetics of native and altered glycogen phosphorylase

Abstract Data on the effect of pH and temperature on the kinetics of rabbit muscle phosphorylases a and b and reduced phosphorylase b (α-1,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) with glycogen as the saturating and inorganic phosphate the variable substrate are presented. The kinetic profiles as a function of pH are similar for these enzyme species except that the positions of the pH-maximal velocity profiles for reduced phosphorylase b are relatively invariant in the 15 °–30 ° range, whereas the “native” phosphorylases exhibit a substantial shift of the lower pH limb of the profile toward the acid side when the temperature is lowered from 30 to 15 °C. It is proposed that a group with a pK near 6.0 at 30 °C determines the acid limb of maximal velocity profiles. The phosphoryl moiety of enzyme bound pyridoxal 5′-phosphate is suggested for this group. A conformational transition in the protein, which is somehow modified when the aldimine bond between protein and pyridoxal 5′-phosphate is reduced, is invoked to account for the large decrease of this acid side apparent pK for the ternary complex of native phosphorylases when the temperature is lowered. A group with a pK near 7.1 and a heat of ionization of about 8000 cal/mol determines the alkaline limb of maximal velocity profiles at 30 °C. An imidazoyl ring ionization of an enzyme histidyl group is proposed to account for this behavior. In the enzyme-glycogen binary complex, the apparent heat of ionization of this group has an anomalous value of about −10,000 cal/ mol. It is suggested that a neighboring amino or arginyl guanidinium group is able to interact with the imidazoyl ring in the absence of bound inorganic phosphate to cause this anomalous behavior. The effect of pH on Km for inorganic phosphate is simply explained by a group with a pK of 6.56 and low heat of ionization. The data are interpreted to indicate that the dianion of inorganic phosphate is the true substrate for all forms of phosphorylase. The kinetic results of this report are closely compared with other kinetic data in the literature on mammalian, plant, and bacterial α-glucan phosphorylases and general overall similarity is demonstrated. Various methods for analyzing pH-kinetic data for enzymes are briefly discussed, and the crucial difference in conclusions the choice of method can make is demonstrated with our data.

The effect of cortisone on the intermediary metabolism of epiphyseal cartilage from rats.

Abstract Total glucose stores per cell are reduced by half in tibial epiphyseal cartilage from rats after 3 days of cortisone treatment, and the ability to produce lactate in vitro without added substrate is abolished. Cortisone reduces lactate production from exogenous glucose to 30% of normal. The pentose shunt pathway and lactate formation from pyruvate also are decreased. In contrast, 14CO2 evolution from glucose-6-14C or labeled pyruvate is unaffected by cortisone treatment suggesting that the potential rate of pyruvate oxidation via the citric acid cycle is relatively insensitive to the hormone. High levels of pyruvate in vitro inhibit lactate formation from labeled glucose by over 90% in normal and cortisone-treated tissue while doubling 14CO2 evolution from glucose-1-14C. Total lactate production is depressed while glucose utilization is stimulated suggesting that pyruvate can inhibit glycolysis in cartilage tissue and divert glucose to alternate fates such as the pentose shunt and glycogen synthesis. The controlling influence of such hormonal and metabolic mechanisms on glycolysis may contribute significantly to the marked atrophic effect of glucocorticoids on growth cartilage as well as to the phenomenon of decreased peripheral glucose utilization induced by these hormones.

Cardiac and skeletal muscle adaptations to training in systemic hypertension and effect of beta blockade (metoprolol or propranolol)

Cardiovascular and peripheral adaptations to an aerobic conditioning program were studied in 30 hypertensive adults taking either placebo, beta 1-selective beta-adrenergic blocker (metoprolol) or beta 1-nonselective beta-adrenergic blocker (propranolol). The placebo group increased aerobic capacity (VO2max) 24% (p less than 0.002), largely explained by an increased peripheral arteriovenous (AV) oxygen difference with minimal changes in cardiac size and function. Resting blood pressure and total systemic resistance also decreased. The group taking a beta 1-selective beta blocker increased VO2max 8% (p less than 0.05), reduced resting blood pressure but had no significant change of AV oxygen difference or cardiac size or function. The group taking the beta 1-nonselective beta blocker propranolol had no increase in VO2max, no decrease in resting blood pressure and no cardiovascular or peripheral adaptations to the exercise program. Thus, beta 1-selective and beta 1-nonselective beta blockers attenuate conditioning in hypertensive patients to differing degrees, in each case by blocking peripheral mechanisms of conditioning.

Effects of cortisone, starvation, and rickets on oxidative enzyme activities of epiphyseal cartilage from rats.

Abstract Epiphyseal cartilage fractions from rats have been shown to have the enzymatic complement for oxidizing a wide variety of substrates though at relatively low rates compared to tissues such as liver and heart. In contrast to previous data for glycolytic enzymes, mitochondrial oxidative enzyme levels do not appear to be specifically affected by dietary rickets, starvation, or cortisone treatment and do not correlate with the oxidative activity of cartilage slices. These findings give added emphasis to our earlier suggestion that control of glycolytic enzyme levels plays a central role in regulation of cartilage cell economy. A marked difference in the relative distribution between supernatant and pellet fractions of glycerol-3- P oxidase compared to other typical mitochondrial enzymes including succinate dehydrogenase is interpreted as evidence for two classes of mitochondria in cartilage. According to this hypothesis, there is a class of more readily sedimented mitochondria which contain relatively much more glycerol-3- P oxidase. Although this enzyme is thought to play a role in regulation of glycolysis, the control of synthetic-degradative mechanisms for it does not appear to be coordinated with those for the glycolytic enzymes and glycerol-3- P dehydrogenase of the cartilage cytoplasm. It is suggested that the oxidase may have a special role in Ca 2+ accumulation by mitochondria.

A comparative study of nucleases exhibiting preference for single-stranded nucleic acid

Abstract We present here a comparative study of three ss-preferential nucleases of class EC 3.1.30.1 commonly used to probe nucleic acid secondary structure (S 1 nuclease, mung bean nuclease and Neurospora crassa endonuclease) and SC nuclease, an enzyme we have isolated from the fungus Schizophyllum commune . In the accompanying paper (Martin, S.A., Ullrich, R.C. and Meyer, W.L. (1986) Biochim. Biophys. Acta 867, 67–75) we report the sensitivity of SC nuclease activity to various anions and to zinc (required for activity). These characteristics have not been reported for other enzymes of this class. We find that these properties hold for commercial preparations of mung bean and Neurospora nucleases but not S 1 nuclease. In addition, we find that S 1 , mung bean and SC nucleases have a molecular weight of 34 000 and 35 000 whereas Neurospora nuclease has a molecular weight of 47 000. Finally, we have determined that mung bean nuclease has the highest preference for ssDNA (there is no measurable activity on dsDNA) followed by S 1 nuclease, SC nuclease and Neurospora nuclease. We suggest that SC nuclease may be the the best enzyme to use in studies of nucleic acid secondary structure where physiological conditions are required. SC nuclease and Neurospora nuclease are active at neutral (physiological) pH, whereas S 1 and mung bean nucleases are active at acid pH. Between SC and Neurospora nucleases SC nuclease would be the enzyme of choice, since it has a higher preference for ssDNA than Neurospora nuclease. In addition, SC nuclease activity can be inhibited by fluoride, which could be a more useful way to inhibit enzyme activity than the addition of chelators for studies of nucleic acid structure where metals are required.

Partial purification and properties of a nuclease from Schizophyllum commune with a preference toward single-stranded nucleic acid

Abstract A nuclease active at neutral pH and with a preference for single-stranded nucleic acid was purified from the Basidiomycete fungus, Schizophyllum commune (SC nuclease). Purification was up to 300-fold. The molecular weight of the native purified enzyme is estimated at 34 000 by gel filtration. This nuclease discriminates between single-stranded and double-stranded nucleic acid but does not discriminate between ribo- and deoxyribonucleic acids. The enzyme is a zinc or cobalt-metalloprotein with a mode of action that is endonucleolytic, generating dinucleotide products that terminate with 5′-phosphates. Although the above characteristics place SC nuclease in the same class (EC 3.1.30.1) as S 1 nuclease from Aspergillus oryzae , two additional useful features of SC nuclease not described for other nucleases of this class have been identified: sensitivity to anions and to zinc concentration. Anions such as chloride, phosphate, succinate, bromide, carbonate, oxalate, propionate and sulfate specifically activate the enzyme, whereas others such as fluoride, pyrophosphate and citrate inhibit activity. Enzyme activity was very sensitive to the concentration of zinc: 1.0 μM ZnCl 2 fully restored activity after chelators were added, whereas 50 mM ZnCl 2 restored only 50% of the activity. The characteristics determined for SC nuclease would make it a useful tool in research for the preferential hydrolysis of single-stranded nucleic acid. SC nuclease has the added advantages over S 1 and mung bean nucleases of functioning at physiological pH rather than at acid pH and of inactivation by fluoride.