Aging and accuracy of protein synthesis in man: search for inactive enzymatic cross-reacting material in granulocytes of aged people

Enzyme activities and immunological reactivities of seven different enzymes have been assayed in the polymorphonuclear leukocytes from 10 newborns, 10 aged (over 80 years old) and 10 young adults (20-30 years old). Polymorphonuclears have been chosen because both in the newborns, and in the aged people, they are young cells whose life span is a few hours only. Five cytoplasmic enzymes have been immunologically studied by means of monospecific antienzyme sera: leukocyte pyruvate kinase by immunoinactivation, glucose-6-phosphate dehydrogenase and glucose phosphate-isomerase by electroimmunodiffusion, lactic dehydrogenase (muscle-type) by radial immunodiffusion. Two lysosomal enzymes (alpha-mannosidase and beta-glycuronidase) have been titrated immunologically with the same polyvalent antihuman leukocyte rabbit serum. Small but significant differences in activity were observed for several of the enzymes. By contrast, no significant difference was found, for any of the seven enzymes, as regards the ratio enzyme activity/immunological reactivity, between newborns, young adults and aged people. These results of not support the theory that a decreased accuracy of the protein synthesis machinery is responsible for aging and death. By contrast, the authors emphasize that the enzyme abnormalities reported by various authors in old animals or in old fibroblast cultures closely resemble those demonstrated to be "post-translational modifications" in the case of human glucose-6-phosphate dehydrogenase.     

Altered nuclear deoxyribonucleic acid alpha-polymerases in senescent cultured chick embryo fibroblasts

DNA alpha-polymerase has been partially purified from nuclei of cultured chic, fibroblasts and separated on phosphocellulose columns into two distinct activities designated DNA polymerases alpha(a) and alpha(b), respectively. The enzyme preparations were devoid of activities of DNA beta,gamma-polymerases terminal deoxyribonucleoside transferase, DNase, DNA-dependent RNA polymerase, and phosphatase. DNA polymerases alpha(a) and alpha(b) both having molecular weights of 160 000, constitute 35-50 and 65-50%, respectively, of the activity of alpha-polymerase in the nucleus. These enzymes differ in their requirements for maximal activity, their relative ability to copy oligo(dG)-poly(dC), their response to ribonucleoside triphosphates, and their kinetics of heat inactivation. When the properties of alpha polymerases derived from early or late passage cultures have been compared, no difference could be detected as a function of cell age in the specific activities of the polymerases in crude cell extracts, their chromatographic behavior on diethylaminoethylcellulose and phosphocellulose columns, and their relative abilities to utilize single deoxyribonucleoside triphosphates with activated DNA template. On the other hand, both enzymes become partially heat labile in aging cells. Also, the activity of DNA polymerase alpha(a) from young cells was stimulated by 2--10 mM adenosine or cytidine triphosphates, whereas the same enzyme from old cultures was inhibited by these agents. Conversely, these ribonucleoside triphosphates inhibited the activity of polymerase alpha(b) in young cells but slightly stimulated this enzyme derived from senescent fibroblasts. In addition, the relative ability of DNA polymerase alpha(a) to copy oligo(dG)-poly(dC) decreased in aged cells, whereas that of DNA polymerase alpha(b) increased. We have also observed significant differences in the effects of potassium chloride and N-ethylmaleimide on the activity of DNA polymerase alpha(a) from old cells as compared to young cells. These age-related alterations in the properties of the two avian DNA polymerases may reflect structural or conformational changes in these enzymes.     

GM1 increases the content and mRNA of NGF in the brain of aged rats

Aged (20-22 months old) and young (3 months old) Sprague-Dawley rats were treated with GM1 ganglioside, 30 mg/kg i.p. for 30 days, and the content of nerve growth factor (NGF) and the high-affinity tyrosine receptor kinase (Trk) examined. NGF, estimated by a two-site enzyme immunoassay, was found moderately decreased in the frontal cortex and hippocampus, but not in the striatum of aged animals compared with young animals. The NGF decrease was accompanied by a reduction of NGF mRNA, evaluated by northern blot. Trk protein, determined by western blot with a pan-Trk antibody, was not altered in any region studied in the aged brain. GM1 treatment partially restored NGF and NGF mRNA in frontal cortex and hippocampus in the aged brain, but treatment had no effect on Trk protein. GM1 did not modify any of the parameters investigated in young animals.     

Reduction of fecal contamination of street-vended beverages in Guatemala by a simple system for water purification and storage, handwashing, and beverage storage

CD4+ cells from young (3 months) and old (19 months) mice were stimulated by plate-bound anti-CD3 monoclonal antibody (mAb) alone or also by soluble anti-CD28 mAb. Supernatants were analysed by enzyme-linked immunosorbent assay (ELISA) to determine cytokine concentrations. Total RNA was extracted from cells, reverse transcribed and the cDNA amplified by polymerase chain reaction (PCR) to evaluate the amount of specific mRNA. The results indicate that anti-CD3 alone is not sufficient to induce interleukin-2 (IL-2) production in CD4+ cells from both young and old mice. However, anti-CD28, together with anti-CD3 mAb, induces a much higher production of IL-2 in CD4+ cells from young as compared with old mice. Conversely, interferon-gamma (IFN-gamma) production is also induced by anti-CD3 alone and is higher in CD4+ cells from old as compared with young mice. Upon addition of anti-CD28 mAb, IFN-gamma production increases in both groups, but it remains much higher in old than in young mice. Also the production of IL-4 and IL-10 is induced by anti-CD3 mAb but it is increased by the addition of anti-CD28 mAb. CD4+ cells from old mice produce more IL-4 and IL-10 as compared with cells from young mice. The amounts of cytokine specific mRNA in CD4+ cells from young and old mice parallel the cytokine levels in culture supernatants. Results on the mRNA turnover indicate that when CD4+ cells are stimulated by anti-CD3 or costimulated also by anti-CD28 mAb, the IFN-gamma, IL-4 and IL-10 specific mRNAs are more stable in old than in young mice, suggesting that mRNA stability has a relevant role in the different patterns of cytokine production.     

Effect of swimming training on antioxidant enzymes in kidney of young and old mice

The current study was undertaken to investigate the effect of swimming training on the antioxidant enzyme system in kidney of young and old mice. Both young and old mice, aged 2 and 26 months old, respectively, were divided into the sedentary and swimming-trained groups. The trained mice underwent a 6-week swimming program (1 h/day, 5 days/week) in water at 35-36 degrees C. Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activity was significantly decreased with aging but was not influenced by swimming training, such changes being similar to those noted for catalase activity rather than for glutathione peroxidase activity. After swimming training Mn-SOD activity increased significantly only in old mice but was unaffected by aging. Although neither aging nor swimming training had overt effect on the expression of Cu,Zn-SOD mRNA, the immunoreactive Cu,Zn-SOD content in young mice decreased significantly after the training. Meanwhile, Mn-SOD mRNA expression in old mice was reduced by half after swimming training, accompanied by a significant decrease in its immunoreactive content; unexpectedly, however, Mn-SOD content in young mice did not parallel its mRNA expression. These findings suggest that the antioxidant enzyme system in mouse kidney trends to be down-regulated with aging, and that swimming training fails to attenuate such reduced levels of the antioxidant enzymes.     

Mutations in the activation loop tyrosine of the oncoprotein v-Fps

Mutations were made in the activation loop tyrosine of the kinase domain of the oncoprotein v-Fps to assess the role of autophosphorylation in catalysis. Three mutant proteins, Y1073E, Y1073Q, and Y1073F, were expressed and purified as fusion proteins of glutathione-S-transferase from Escherichia coli and their catalytic properties were evaluated. Y1073E, Y1073Q, and Y1073F have k(cat) values that are reduced by 5-, 35-, and 40-fold relative to the wild-type enzyme, respectively. For all mutant enzymes, the Km values for ATP and a peptide substrate, EAEIYEAIE, are changed by 0.4-2-fold compared to the wild-type enzyme. The slopes for the plots of relative turnover versus solvent viscosity [(k(cat))eta] are 0.71 +/- 0.08, 0.10 +/- 0.06, and approximately 0 for wild type, Y1073Q, and Y1073E, respectively. These results imply that the phosphoryl transfer rate constant is reduced by 19- and 130-fold for Y1073E and Y1073Q compared to the wild-type enzyme. The dissociation constant of the substrate peptide is 1.5-2.5-fold lower for the mutants compared to wild type. The inhibition constant for EAEIFEAIE, a competitive inhibitor, is unaffected for Y1073E and raised 3-fold for Y1073Q compared to wild type. Y1073E and Y1073Q are strongly activated by free magnesium to the same extent and the apparent affinity constant for the metal is similar to that for the wild-type enzyme. The data indicate that the major role of autophosphorylation in the tyrosine kinase domain of v-Fps is to increase the rate of phosphoryl transfer without greatly affecting active-site accessibility or the local environment of the activating metal. Finally, the similar rate enhancements for phosphoryl transfer in v-Fps compared to protein kinase A [Adams et al. (1995) Biochemistry 34, 2447-2454] upon autophosphorylation suggest a conserved mechanism for communication between the activation loop and the catalytic residues of these two enzymes.     

Thermosensitivity of nuclear RNA polymerase during the in vitro senescence of mouse fibroblasts

Embryonic mouse fibroblasts divide approximately twelve times in vitro prior to cessation of mitotic activity. During this period of cellular senescence the thermosensitivity of the RNA polymerase activity of isolated nuclei has been examined as a means of detecting the possible accumulation of defective enzyme molecules, as has been found by other workers for several cytoplasmic enzymes during the ageing of human fibroblasts in vitro. The total RNA polymerase activity of nuclei isolated from old (10th generation) cells is more thermoresistant than that of young (2nd generation) cells. However, the net RNA polymerase activity of nuclei from non-dividing (confluent) cells is more thermoresistant than that of exponentially growing cells of the same age. When allowance is made for the state of growth of the cultures, little difference is seens in the thermosensitivity of the activities of nuclei from old and young cells. Neither is there any difference between the thermosensitivity of the net activity of an established line of murine fibroblasts (L-cells) and cells in primary culture. Preheating nuclei increases the inhibition of their total RNA polymerase activity by alpha-amanitin, indicating that RNA polymerase II is the most heat resistance species present. There appears to be no difference between the thermosensitivity of the alpha-amanitin sensitive and resistance species of the enzyme in the nuclei of old and young cells. It is concluded that old cells resemble non-dividing young cells in containing a higher proportion of RNA polymerase II in their nuclei, resulting in greater thermoresistance of the total RNA polymerase activity over that of exponentially growing cells. However, there appears to be no increase in thermosensitivity of the enzymes with age.     

The A and the extended V N-terminal regions of streptococcal protein I/IIf mediate the production of tumour necrosis factor alpha in the monocyte cell line THP-1

Signal transduction is ubiquitously involved in the initiation of physiological signals that lead to growth and proliferation of cells. The signaling cascade mediated by the mitogen-activated protein kinase (MAPK) is considered essential for T cell growth and function. Therefore, it was of interest to determine the influence of age on the induction of MAPK in mitogen-activated T cells. T cells from young (4-6 months) and old (24-26 months) rats responded to concanavalin A (Con A) stimulation by increasing MAPK, c-jun amino terminal kinase (JNK), and p21ras activities. The time course of induction of MAPK/JNK and p21ras activities was similar in T cells isolated from young and old rats. The induction of JNK activity did not change significantly with age; however, the induction of MAPK and p21ras activities was significantly less (50 to 65%) in T cells from old rats than in T cells from young rats. Although the relative protein levels of p42 and p44 MAPK did not change with age, the proportion of the phosphorylated p44 MAPK decreased with age. In addition, it was found that the in vitro kinase activities of the T cell receptor-associated protein tyrosine kinase Lck (p56Lck) and ZAP-70 but not Fyn (p59Fyn) were lower in T cells from old rats than in T cells from young rats. The decline in activities of these signaling molecules with age was not associated with changes in their corresponding protein levels. Thus, our results demonstrate that aging alters the activation of the signal transduction cascade that leads to T cell activation.     

Impaired glutathione peroxidase activity accounts for the age-related accumulation of hydrogen peroxide in activated human neutrophils

BACKGROUND: As assessed by flow cytometry, the increase in hydrogen peroxide in individual neutrophils from old volunteers was significantly greater than in neutrophils from young volunteers. To explain the discrepancy in previous reports that showed reduced superoxide generation with age and our finding, we measured the kinetics of antioxidative enzymes. METHODS: Neutrophils were obtained from young (ages 21-34) and old (ages over 65) volunteers. The increase in hydrogen peroxide following stimulation with formyl peptide in individual neutrophils was assessed by flow cytometry by using dihydrorhodamine 123. The enzyme kinetics was determined from the best fit curve using Michaelis-Menten equations. RESULTS: Aging was associated with a significant reduction in the Vmax for glutathione peroxidase. The decreased activity was not due to selenium deficiency as the serum and neutrophil concentrations were identical with age. Following activation, a significant increase in the Km was noted in neutrophils from young but not from old volunteers. CONCLUSIONS: These results account for the increased intracellular accumulation of hydrogen peroxide as a function of age in stimulated neutrophils. These results provide evidence in humans of an age-related impairment in antioxidative defense mechanisms that support the free radical theory of aging.     

Toxicological effects of beryllium on platelets and vascular endothelium

The crystal structure of rabbit muscle pyruvate kinase complexed with Mn2+, K+, and pyruvate revealed a binding site of K+ [T. M. Larsen, L. T. Laughlin, H. M. Holden, I. Rayment, and G. H. Reed (1994) Biochemistry 33, 6301-6309]. Sequence comparisons of rabbit muscle pyruvate kinase and pyruvate kinases from Corynebacterium glutamicum and Escherichia coli, which do not exhibit a requirement for activation by monovalent cations, indicate that the only substitutions in the K+ binding site are conservative. Glu 117 in the rabbit muscle enzyme, which is close to the K+ site, is, however, replaced by Lys in these two bacterial pyruvate kinases. The proximity of Glu 117 to K+ in the structure of the rabbit enzyme and conservation of the binding site in the bacterial enzymes which lack a dependence on monovalent cations suggested that a protonated epsilon-amino group of Lys 117 in these bacterial enzymes may provide an "internal monovalent cation." Site-specific mutant forms of the rabbit enzyme corresponding to E117K, E117A, E117D, and E117K/K114Q pyruvate kinase were examined to test this hypothesis. The E117K pyruvate kinase exhibits 12% of the activity of the fully activated wild-type enzyme but is > 200-fold more active than the wild-type enzyme in the absence of activating monovalent cations. Moreover, the activity of E117K pyruvate kinase exhibits no stimulation by monovalent cations in the assay mixtures. Both E117A and E117D pyruvate kinases retain activation by monovalent cations but have reduced activities relative to wild type. The results are consistent with the hypothesis that pyruvate kinases that do not require activation by monovalent cations supply an internal monovalent cation in the form of a protonated epsilon-amino group of Lys. The results also support the assignment of the monovalent cation in the active site of pyruvate kinase.     

Grafting of enzymes on collagen films using Woodward''s reagent "K" and a water-soluble carbodiimide derivative

Two new methods of activation were developed to graft enzymes on collegen films. They involved chemical modifications of surface groups of collagen either by Woodward's reagent "K" or by EDC, a water-soluble derivative of carbodiimide. EDC was a better coupling agent and a detailed study was conducted with this agent. It could be used either in a global method of activation and coupling, or in a two-step procedure of activation of collagen, followed by spontaneous coupling of enzyme. All enzymes tested were successfully bound: malate dehydrogenase, lactate dehydrogenase, aspartate aminotransferase, urease, creatine kinase, hexokinase. The influence on the yield of grafted enzyme, of pretreatment of films, time and temperature of EDC activation, concentration of EDC and enzyme, protecting agents was studied. Stability of enzyme activity on storage was greatly increased after grafting. A co-grafted dual system creatine kinase/heoxkinase, was achieved which exhibited a good efficiency. A striking renaturing process at 0-4degreesC after thermal denaturation, was observed with hexokinase.     

Hospital staff reactions to accounts by survivors of childhood abuse

Two groups of male Fisher 344 rats (young: 4 months old; aged: 25 months old) underwent either 70% distal small bowel resection or sham operation (small bowel transection). Rats from each treatment group of each age were sacrificed on the 10th (N = 15: young rats; N = 13: aged rats) or 20th (N = 15: young; N = 13: aged) postoperative day (POD), and the duodenal mucosa was weighed and assayed for DNA, RNA, and protein contents, as well as for specific activities of the disaccharidase, sucrase, maltase, and lactase. Compared to the sham operation, distal small bowel resection significantly increased DNA by 48%, RNA by 122%, and protein by 75% in young rats and DNA by 40%, RNA by 92%, and protein by 71% in aged rats on the 20th POD. Both young and aged rats showed similar adaptive hyperplasia on the 10th POD. On the 20th POD after distal small bowel resection, specific activities of all tested enzymes were significantly increased in young rats (sucrase +86%, maltase +110% and lactase +64%), but showed no significant changes in aged rats. These findings suggest that the duodenum of aged rats may have sufficient proliferative potential to respond to distal small bowel resection, but that the mechanisms governing return of function in response to distal small bowel resection are inhibited in aged rats, compared to those mechanisms in the young.     

Assay of total protein kinase activity in mouse brain cortex

In previous studies we demonstrated that the adrenergic system is impaired in old animals and that the main alterations were observed at the level of receptor density and adenylate-cyclase activity. The decreased ability to produce cAMP could influence the activity of the cAMP dependent protein kinase (PKA), one of the enzymes responsible for the phosphorylation of protein substrates. Since protein phosphorylation is one of the most common and important mechanisms through which a cell regulates its activity, the characteristics of the phosphorus incorporation reaction were studied. Kinase activity was measured in homogenate of young mouse brain cortex prepared avoiding gross manipulations in order to maintain conditions as close to those present in the living animal as possible. Results show that phosphate incorporation is proportional to protein content and strictly dependent on ATP availability. Increasing the ATP concentration from 10 to 500 mumol/l, the length of incorporation phase increases, suggesting that the limiting point of the reaction is better represented by energy availability than by enzyme or protein substrate concentrations.     

Pathophysiology and treatment of cardioembolic stroke

To elucidate the pathophysiologic mechanism of cardioembolic stroke in elderly people and to devise therapeutic strategies for it, was analyzed 120 consecutive patients (77 men and 43 women aged 65 +/- 13 years) with acute cardioembolic stroke who were admitted within 7 days of the stroke onset. We compared underlying heart diseases. NIH stroke scale on admission, lesion size on computed tomography (CT), the relation between anticoagulant therapy and recurrence, complications during admission. ADL at discharge, recurrence, and death during the follow up period in three groups: patients aged less than 65 years (the young group), those aged from 65 to 74 years (the "non-old" group), and those aged more than 75 years (the "old old" group). In the "old old" group, non valvular atrial fibrillation (75.8%) was the most common underlying heart disease and so was rheumatic heart disease (33.3%) in the "non-old" group. NIH stroke scale score (median, 11) and the proportion of patients with a large lesion (> 3 cm) of CT were higher in the "old old" group than in the other two groups. Immediate anticoagulation (A/C) within 14 days of onset was performed in more than 70% of the "non-old" and the "young old" groups but in only 57.6% of the "old old" group. Stroke recurred more often in 34 patients who did not receive immediate A/C than in the 86 who did (11.8% v.s. 2.3%. Chi square test, p = 0.053). Hemorrhage during immediate A/C and other complications (infection and pulmonary embolism) were seen in 2 and 14 patients, respectively, in both the "young old" groups, but not in the "non-old" group. Good outcomes (able to walk with or without cane) were more common in the "non-old" group (78.9%) than the other groups (57.1%, Chi square test, p < (0.01). A/C after the acute stage was done in more than 80% of those in the "non-old" and the "young old" groups, but in less than 30% of those in the "old old" group (Chi square test, p = 0.0514). Survival without recurrence during the observation period (605 +/- 550 days) was significantly lower in the "old old" group than in the other two groups (log-rank test, p = 0.0091). Cardioembolic stroke in the elderly may be characterized as follows: (1) non valvular atrial fibrillation is the most common, (2) severe neurologic deficits on admission and large lesions on CT are noted, (3) complications (infection and pulmonary embolism) often occur, (4) A/C in both acute and chronic stages are done infrequently. Therefore, the indication and intensity of A/C for primary and secondary prevention and prevention of complications are important in management of cardioembolic stroke in the elderly.     

Age effects and health appraisal: a meta-analysis

OBJECTIVE: The purpose was to clarify the effects of methodological variables in the research on age differences in self-rated health and specifically the effects of: (a) the item type used to solicit the appraisal, (b) the approach to sampling, and (c) the criteria for assembling the age groups for study. METHODS: Studies differing on these dimensions were compared using meta-analytic techniques. Seventeen usable samples were grouped according to the age comparisons--seven compared "young" and "old" samples while ten compared "young-old" and "old-old" groups. RESULTS: The results failed to support the frequently reported health optimism of old-old samples when compared with young-old. Further, the findings suggest that the item type used in eliciting the rating of health may be instrumental in the respondent's appraisal process. For instance, use of the age-comparative item tends to favor "old" groups when compared to young, whereas the global health item has an effect in the other direction. Effect size was also related to a study's sampling procedure and to the method used in assembling groups (i.e., age ranges used to represent young and old). DISCUSSION: Practical implications and areas of needed research are discussed.     

Differential effect of morphine on dopaminergic neurons in frontal cortex and striatum of the rat

Lactate dehydrogenase-5 and creatine kinase from rabbit muscle were labeled by coupling with N-hydroxysuccinimidyl 3-(4'-hydroxy-[3',5'-125I]diiodophenyl)propionate. After purification, the analytical recovery of catalytically-active labeled enzyme averaged 90% for lactate dehydrogenase, 81% for creatine kinase. The labeled enzymes were injected intravenously into rabbits and disappearance from plasma of catalytic activity and radioactivity was measured. The disappearance curves for lactate dehydrogenase-5 differed considerably from those observed with the enzyme labeled by direct iodination. The discrepancy was due to rapid hydrolysis in vivo of the labeled amide-enzyme linkage, because about 50% of the injected radioactivity appeared in the urine as 125I-labeled 3-(4'-hydroxy-3',5'-diiodophenyl)propionic acid within 4-8 h of injection. Similar outputs were observed after administration of this acid to rabbits. The free acid was also detected in the urines of rabbits within 4-8 h of the intravenous injection of creatine kinase labeled similarly. We conclude that this method of labeling is unsuitable for preparing radioactive enzymes for study of their catabolism.     

Glutamate-459 is important for Escherichia coli branching enzyme activity

The branching enzyme belongs to the amylolytic family, a group of enzymes that cleave and/or transfer chains of glucan. The amylolytic enzymes are homologous and all contain four conserved regions, proposed to contain the active site. By primary structure analysis, a conserved position unique to branching enzymes has been identified. This residue, which is either Asp or Glu, depending on the species, is located immediately after the putative catalytic Glu-458 (Escherichia coli numbering). Branching enzymes differ from other amylolytic enzymes in having this acid pair, and we asked if this motif could be essential for branching enzyme action. We used site-directed mutagenesis of the Glu-459 residue in the E. coli branching enzyme in order to determine the significance of the conserved Asp/Glu in branching enzymes. A substitution of Glu-459 to Asp resulted in increased specific activity compared to wild-type, suggesting that the mutation had created a more efficient enzyme. Changing Glu-459 to Ala, Lys, or Gln lowered the specific activities and altered the preferred substrate from amylose to amylopectin.     

Two calcium/calmodulin-dependent protein kinases, which are highly concentrated in brain, phosphorylate protein I at distinct sites

Two calcium-stimulated protein kinase activities (ATP:protein phosphotransferase, EC 2.7.1.37) that phosphorylate protein I, a specific synaptic protein, have been identified in homogenates of rat brain. One of these is found in both the particulate and cytosolic fractions and phosphorylates a region of protein I that is phosphorylated in intact synaptosomes in response to calcium but not to cyclic AMP. The stimulation by calcium of the particulate enzyme and of the partially purified cytosolic enzyme requires the addition of calmodulin. It is not yet known whether the particulate and cytosolic enzymes are related. A second calcium-stimulated protein I kinase is found only in the cytosol and phosphorylates a region of protein I that is phosphorylated in intact synaptosomes in response to either calcium or cyclic AMP. The calcium stimulation of this latter kinase is probably mediated by calmodulin, judging from its inhibition by low concentrations of trifluoperazine. Both of the calcium-stimulated protein I kinases are more highly concentrated in brain than in other tissues. The two cytosolic kinases are distinguishable from each other and from myosin light chain kinase and phosphorylase b kinase by their substrate specificities and their chromatographic behavior on DEAE-cellulose.     

Molecular approaches to the diagnosis of male infertility

We investigated the effects of aging and/or swimming training on the antioxidant enzyme system in diaphragm of mice. Young (2 months old) and old (26 months old) male mice were swimming-trained for 6 weeks (1 h/day, 5 days/week). Cu,Zn-Superoxide dismutase (Cu,Zn-SOD) activity was significantly upregulated with aging, and swimming training definitely enhanced the activity only in young mice. Neither aging nor swimming training had overt effect on Mn-SOD activity. Glutathione peroxidase activity in young mice was significantly increased after training, but not in old mice. Both of immunoreactive Cu,Zn-SOD and Mn-SOD were significantly increased with aging but were unaffected by swimming training. Consequently, physical training significantly enhanced the specific activity of Cu,Zn-SOD in young mice, but not in old mice. Meanwhile, swimming training significantly increased xanthine oxidase activity in both age groups, the extent of the increase being greater in old mice than in young mice. We concluded that the antioxidant enzyme system in mouse diaphragm trends to be upregulated with aging, but that swimming training improved the system only in young mouse diaphragm.     

Comparative studies of kinetic and optical properties of rabbit muscle, sturgeon muscle, and yeast pyruvate kinase

The kinetic and optical properties of pyruvate kinase isolated from rabbit muscle, sturgeon muscle, and yeast were compared using various activating divalent metal ions as probes for functional features and using ultraviolet circular dichroism (cd) measurements for conformational features, respectively. All three preparations of pyruvate kinase were similar in many aspects, such as activating efficiencies of the four activating metal ions, Mg(II), Co(II), Mn(II), and Ni(II) and pH-rate profiles, suggesting the presence of a similar metal binding locus of these enzymes as well as a common underlying mechanism of action. L-Phe inhibited the rabbit muscle enzyme and turned the hyperbolic kinetics into a sigmoidal kinetic with respect to phosphoenolpyruvate at alkaline pH, while fructose-1,6-biphosphate activated the sturgeon muscle and yeast enzymes and turned the sigmoidal kinetics into hyperbolic kinetics with respect to phosphoenolpyruvate. The ultraviolet cd spectral changes qualitatively correlated well with kinetic observations of all three native enzymes in the presence and absence of allosteric effectors. Our results suggested that there are at least two conformational states of pyruvate kinase which are inducible by the binding of substrate and (or) allosteric effectors. The conformational changes from one form to another in these enzymes are very similar, especially between the rabbit and sturgeon muscle enzymes.