scholarly journals Effects of Tissue Protectants on the Kinetics of Lactate Dehydrogenase in Cells

1997 ◽  
Vol 45 (10) ◽  
pp. 1417-1425 ◽  
Author(s):  
Yoshiko Nakae ◽  
Peter J. Stoward
Keyword(s):  
Skeletal Muscle ◽  
Lactate Dehydrogenase ◽  
Regression Line ◽  
Muscle Fibers ◽  
Agarose Gel ◽  
Image Analysis System ◽  
Reaction Products ◽  
Isobestic Point ◽  
Skeletal Muscle Fibers ◽  
Kinetics Of

We studied the effects of two tissue protectants, polyvinyl alcohol (PVA) and agarose gel, on a kinetic parameter of lactate dehydrogenase LDH that is assumed to be related to the extent of diffusion of the enzyme out of tissue sections during its histochemical assay. The kinetics of the enzyme in mouse gastrocnemius (skeletal) muscle fibers and periportal hepatocytes were determined in unfixed sections incubated either on substrate (l-lactate)-containing agarose gel films or in aqueous assay media in the presence or absence of 18% PVA. The absorbances of the formazan final reaction products at their isobestic point were measured continuously in the cytoplasm of individual cells as a function of incubation time, using a real-time image analysis system. Whichever incubation medium was used, the absorbances in the two cell types increased nonlinearly during the first minute of incubation but linearly for incubation times between 1 and 3 min. The nonlinearity of the LDH reaction was analyzed using the equation vi - v = a° A, where vi is the observed initial velocity determined from the absorbance changes during the first 10 sec of incubation and v and ° A are respectively the gradient and intercept on the absorbance axis of the linear regression line of the absorbance on incubation times between 1 and 3 min. The plots of the observed ( vi - v) against ° A were linear. Their gradients a were characteristic for each cell type and tissue protectant. The a values for skeletal muscle fibers were 12–43% lower than those for hepatocytes. The a value for hepatocytes obtained with the PVA method was 32% lower than that determined with the gel film method. For skeletal muscle fibers, the a values determined by the two methods were almost the same. Addition of excess pyruvate to the aqueous assay medium had no effects on a for either muscle fibers or hepatocytes. In contrast, a was zero for sections of polyacrylamide gels containing purified enzyme, whether incubated on agarose films or in PVA media. These data confirmed that the constant a is related to the extent to which the enzyme diffuses out of sections during incubation but not to product inhibition of LDH by pyruvate. PVA was more effective for protecting diffusion of LDH from hepatocytes than from skeletal muscle fibers, possibly because hepatocytes contain a greater proportion of diffusable (unbound) LDH than skeletal muscle fibers.

scholarly journals Kinetic Parameters of Lactate Dehydrogenase in Liver and Gastrocnemius Determined by Three Quantitative Histochemical Methods

1997 ◽  
Vol 45 (10) ◽  
pp. 1427-1431 ◽  
Author(s):  
Yoshiko Nakae ◽  
Peter J. Stoward
Keyword(s):  
Skeletal Muscle ◽  
Lactate Dehydrogenase ◽  
Kinetic Parameters ◽  
Muscle Fibers ◽  
Lactate Concentration ◽  
Agarose Gel ◽  
Image Analysis System ◽  
Maximal Velocity ◽  
Skeletal Muscle Fibers ◽  
Assay Methods

We determined the Michaelis constant ( Km) and maximal velocity ( Vmax) of lactate dehydrogenase (LDH) in periportal hepatocytes and skeletal muscle fibers by three different histochemical assay methods. Unfixed sections of mouse liver and gastrocnemius were incubated at 37C either on substrate (l-lactate)-containing agarose gel films or in aqueous assay media with and without 18% polyvinyl alcohol (PVA) as a tissue protectant. The absorbances of the formazan final reaction products were continuously measured at 584 nm in the cytoplasm of individual cells as a function of incubation time, using an image analysis system. The kinetic parameters of purified rabbit skeletal muscle LDH incorporated into polyacrylamide gel sections were similarly determined. The intrinsic initial velocities ( vi) of LDH, corrected for “nothing dehydrogenase,” were determined as described in the previous article. The Km and Vmax were calculated from Hanes plots of s/ vi on l-lactate concentration ( s). The Km values obtained with three assay methods were similar and in the range of 21.1–21.9 mM for pure LDH, 8.62–13.5 mM for LDH in mouse periportal hepatocytes, and 13.3–17.9 mM for LDH in mouse skeletal muscle fibers. The Vmax values determined on agarose gel substrate films and in aqueous assay media without PVA were in good agreeement but were 53–65% lower when 18% PVA was included in the medium. These results indicate that catalytic center activity kcat of LDH is retarded by the high viscosity of PVA media because PVA hardly inhibited the enzyme. The Km values of LDH determined histochemically in skeletal muscle fibers and periportal hepatocytes were respectively three to five times and two to three times higher than those determined biochemically. These differences may be due to interactions of LDH with intracellular components.

Determinants of relaxation rate in skinned frog skeletal muscle fibers

1998 ◽  
Vol 274 (6) ◽  
pp. C1608-C1615 ◽  
Author(s):  
Philip A. Wahr ◽  
J. David Johnson ◽  
Jack. A. Rall
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Slow Phase ◽  
Frog Skeletal Muscle ◽  
Force Of Contraction ◽  
Thin Filaments ◽  
Fast Phase ◽  
Skeletal Muscle Fibers ◽  
Kinetics Of ◽  
Overall Kinetics

The influences of sarcomere uniformity and Ca2+ concentration on the kinetics of relaxation were examined in skinned frog skeletal muscle fibers induced to relax by rapid sequestration of Ca2+ by the photolysis of the Ca2+ chelator, diazo-2, at 10°C. Compared with an intact fiber, diazo-2-induced relaxation exhibited a faster and shorter initial slow phase and a fast phase with a longer tail. Stabilization of the sarcomeres by repeated releases and restretches during force development increased the duration of the slow phase and slowed its kinetics. When force of contraction was decreased by lowering the Ca2+concentration, the overall kinetics of relaxation was accelerated, with the slow phase being the most sensitive to Ca2+ concentration. Twitchlike contractions were induced by photorelease of Ca2+ from a caged Ca2+ (DM-Nitrophen), with subsequent Ca2+ sequestration by intact sarcoplasmic reticulum or Ca2+ rebinding to caged Ca2+. These twitchlike responses exhibited relaxation kinetics that were about twofold slower than those observed in intact fibers. Results suggest that the slow phase of relaxation is influenced by the degree of sarcomere homogeneity and rate of Ca2+ dissociation from thin filaments. The fast phase of relaxation is in part determined by the level of Ca2+ activation.

scholarly journals Kinetics of contractile activation in voltage clamped frog skeletal muscle fibers

1997 ◽  
Vol 73 (4) ◽  
pp. 1999-2011 ◽  
Author(s):  
P. Szentesi ◽  
Z. Papp ◽  
G. Szücs ◽  
L. Kovács ◽  
L. Csernoch
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Frog Skeletal Muscle ◽  
Skeletal Muscle Fibers ◽  
Contractile Activation ◽  
Kinetics Of

Fall in intracellular Po 2 at the onset of contractions in Xenopus single skeletal muscle fibers

2001 ◽  
Vol 90 (5) ◽  
pp. 1871-1876 ◽  
Author(s):  
Michael C. Hogan
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Ringer Solution ◽  
Work Period ◽  
Force Development ◽  
Delayed Onset ◽  
Maximal Force ◽  
Skeletal Muscle Fibers ◽  
Lumbrical Muscle ◽  
Kinetics Of

It remains uncertain whether the delayed onset of mitochondrial respiration on initiation of muscle contractions is related to O2 availability. The purpose of this research was to measure the kinetics of the fall in intracellular Po 2 at the onset of a contractile work period in rested and previously worked single skeletal muscle fibers. Intact single skeletal muscle fibers ( n = 11) from Xenopus laevis were dissected from the lumbrical muscle, injected with an O2-sensitive probe, mounted in a glass chamber, and perfused with Ringer solution (Po 2 = 32 ± 4 Torr and pH = 7.0) at 20°C. Intracellular Po 2 was measured in each fiber during a protocol consisting sequentially of 1-min rest; 3 min of tetanic contractions (1 contraction/2 s); 5-min rest; and, finally, a second 3-min contractile period identical to the first. Maximal force development and the fall in force (to 83 ± 2 vs. 86 ± 3% of maximal force development) in contractile periods 1 and 2, respectively, were not significantly different. The time delay (time before intracellular Po 2 began to decrease after the onset of contractions) was significantly greater ( P < 0.01) in the first contractile period (13 ± 3 s) compared with the second (5 ± 2 s), as was the time to reach 50% of the contractile steady-state intracellular Po 2(28 ± 5 vs. 18 ± 4 s, respectively). In Xenopus single skeletal muscle fibers, 1) the lengthy response time for the fall in intracellular Po 2 at the onset of contractions suggests that intracellular factors other than O2 availability determine the on-kinetics of oxidative phosphorylation and 2) a prior contractile period results in more rapid on-kinetics.

scholarly journals Structural Kinetics of Troponin during Activation of Skeletal Muscle Fibers by Photolysis of Nitrophenyl-EGTA (NP-EGTA)

2012 ◽  
Vol 102 (3) ◽  
pp. 230a
Author(s):  
Luca Fusi ◽  
Elisabetta Brunello ◽  
Ivanka Sevrieva ◽  
Yin-Biao Sun ◽  
Malcolm Irving
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Skeletal Muscle Fibers ◽  
Kinetics Of

scholarly journals Age affects myosin relaxation states in skeletal muscle fibers of female but not male mice

2018 ◽  
Author(s):  
Lien A. Phung ◽  
Sira M. Karvinen ◽  
Brett A. Colson ◽  
David D. Thomas ◽  
Dawn A. Lowe
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Natural Aging ◽  
Psoas Muscle ◽  
Male Mice ◽  
Skeletal Muscle Fibers ◽  
Significant Difference ◽  
Potential Contributor ◽  
Aging Muscle ◽  
Kinetics Of

AbstractThe recent discovery that myosin has two distinct states in relaxed muscle – disordered relaxed (DRX) and super-relaxed (SRX) – provides another factor to consider in our fundamental understanding of the aging mechanism in skeletal muscle, since myosin is thought to be a potential contributor to dynapenia. The primary goal of this study was to determine the effects of age on DRX and SRX states and to examine their sex specificity. We have used quantitative fluorescence microscopy of the fluorescent nucleotide analog 2′/3′-O-(N-methylanthraniloyl) ATP (mantATP) to measure single-nucleotide turnover kinetics of myosin in skinned skeletal muscle fibers under relaxing conditions. We examined changes in DRX and SRX in response to the natural aging process by measuring the turnover of mantATP in skinned fibers isolated from psoas muscle of adult young (3-4 months old) and aged (26-28 months old) C57BL/6 female and male mice. Fluorescence decays were fitted to a multi-exponential decay function to determine both the time constants and mole fractions of fast and slow turnover populations, and significance was analyzed by a t-test. We found that in females, both the DRX and SRX lifetimes of myosin ATP turnover at steady state were shorter in aged muscle fibers compared to young muscle fibers (p≤0.033). However, there was no significant difference in relaxation lifetime of either DRX (p=0.202) or SRX (p=0.804) between young and aged male mice. No significant effects were measured on the mole fractions (populations) of these states, as a function of sex or age (females, p=0.100; males, p=0.929). The effect of age on the order of myosin heads at rest and their ATPase function is sex specific, affecting only females. These findings provide new insight into the molecular factors and mechanisms that contribute to aging muscle dysfunction in a sex-specific manner.

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