Analysis of phosphofructokinase-1 activity as affected by pH and ATP concentration.

A key player in energy metabolism is phosphofructokinase-1 (PFK1) whose activity and behavior strongly influence glycolysis and thus have implications in many areas. In this research, PFK1 assays were performed to convert F6P and ATP into F-1,6-P and ADP for varied pH and ATP concentrations. PFK1 activity was assessed by evaluating F-1,6-P generation velocity in two ways: (1) directly calculating the time slope from the first two or more datapoints of measured product concentration (the initial-velocity method), and (2) by fitting all the datapoints with a differential equation explicitly representing the effects of ATP and pH (the modeling method). Similar general trends of inhibition were shown by both methods, but the former gives only a qualitative picture while the modeling method yields the degree of inhibition because the model can separate the two simultaneous roles of ATP as both a substrate of reaction and an inhibitor of PFK1. Analysis based on the model suggests that the ATP affinity is much greater to the PFK1 catalytic site than to the inhibitory site, but the inhibited ATP-PFK1-ATP complex is much slower than the uninhibited PFK1-ATP complex in product generation, leading to reduced overall reaction velocity when ATP concentration increases. The initial-velocity method is simple and useful for general observation of enzyme activity while the modeling method has advantages in quantifying the inhibition effects and providing insights into the process.

Inhibition of pyruvate dehydrogenase accelerates anaerobic glycolysis under postmortem simulating conditions.

This research aimed to explore the potential influence of mitochondria on the rate of anaerobic glycolysis. We hypothesized that mitochondria could reduce the rate of anaerobic glycolysis and pH decline by metabolizing a portion of glycolytic pyruvate. We utilized an in vitro model and incorporated CPI-613 and Avidin to inhibit pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), respectively. Four treatments were tested: 400 µM CPI-613, 1.5 U/ml Avidin, 400 µM CPI-613 + 1.5 U/ml Avidin, or control. Glycolytic metabolites and pH of the in vitro model were evaluated throughout a 1440-min incubation period. CPI-613-containing treatments, with or without Avidin, decreased pH levels and increased glycogen degradation and lactate accumulation compared to the control and Avidin treatments (P < 0.05), indicating increased glycolytic flux. In a different experiment, two treatments, 400 µM CPI-613 or control, were employed to track the fates of pyruvate using [13C6]glucose. CPI-613 reduced the contribution of glucose carbon to tricarboxylic acid cycle intermediates compared to control (P < 0.05). To test whether the acceleration of acidification in reactions containing CPI-613 was due to an increase in the activity of key enzymes of glycogenolysis and glycolysis, we evaluated the activities of glycogen phosphorylase, phosphofructokinase, and pyruvate kinase in the presence or absence of 400 µM CPI-613. The CPI-613 treatment did not elicit an alteration in the activity of these three enzymes. These findings indicate that inhibiting PDH increases the rate of anaerobic glycolysis and pH decline, suggesting that mitochondria are potential regulators of postmortem metabolism.

Cattle breed type and anabolic implants impact calpastatin expression and abundance of mRNA associated with protein turnover in the longissimus thoracis of feedlot steers.

Two methods that the beef cattle industry can use to improve efficiency, sustainability, and economic viability are growth promotants and crossbreeding cattle of different breed types. In the United States, over 90% of cattle receive an anabolic implant at some point during production resulting in an overall increase in skeletal muscle growth. Recent research suggests that the two main cattle breed types, Bos indicus and Bos taurus, respond differently to anabolic implants. The objective of this study was to characterize changes that occur in skeletal muscle following implanting in Bos indicus influenced steers or Bos taurus steers. Twenty steers were stratified by initial weight in a 2 × 2 factorial design examining two different breeds: Angus (AN; n = 10) or Santa Gertrudis influenced (SG; n = 10), and two implant strategies: no implant (CON; n = 10) or a combined implant containing 120 mg TBA and 24 mg E2 (IMP; n = 10; Revalor-S, Merck Animal Health). Skeletal muscle biopsies were taken from the longissimus thoracis (LT) 2 and 10 d post-implantation. The mRNA abundance of 24 genes associated with skeletal muscle growth were examined, as well as the protein expression of µ-calpain and calpastatin. Succinate dehydrogenase mRNA abundance was impacted (P = 0.05) by a breed × treatment interaction 2 d post-implanting, with SG-CON having a greater increased abundance than all other steers. A tendency for a breed × treatment interaction was observed for calpain-6 mRNA (P = 0.07), with SG-CON having greater abundance than AN-CON and SG-IMP. Additionally, calpastatin protein expression was altered (P = 0.01) by a breed × treatment interaction, with SG-CON and SG-IMP steers having increased expression (P = 0.01) compared with AN-CON steers. At 2 d post-implanting, a breed × treatment interaction was observed with SG-CON steers having greater (P = 0.05) mRNA abundance of mitogen-activated protein kinase compared with AN-CON steers. Furthermore, breed affected (P = 0.05) calpastatin abundance with AN steers having increased (P = 0.05) abundance 2 d post-implanting compared with SG steers. Meanwhile, implants tended to affect (P = 0.09) muscle RING finger protein-1 mRNA abundance, with CON steers having increased (P = 0.09) abundance compared with that of IMP steers. These findings suggest that cattle breed type and anabolic implants impact calpastatin expression and mRNA abundance associated with protein turnover in the LT of feedlot steers 2 and 10 d post-implantation.

Two methods that the beef cattle industry can use to potentially improve efficiency, sustainability, and economic viability are growth promotants and crossbreeding cattle of different breed types. In the United States, over 90% of cattle receive at least one anabolic implant during the production cycle resulting in improvements in production and overall economic and environmental sustainability. Research suggests that the two main cattle breed types, Bos indicus and Bos taurus, respond differently to different anabolic implant strategies. The objective of this study was to characterize changes that occur in the skeletal muscle following implanting in Bos indicus influenced animals and Bos taurus animals. This research measured mRNA abundance of 24 genes associated with skeletal muscle growth, and protein expression of calpain-1 and calpastatin. The findings of this research suggest that anabolic implants and cattle breed type interact to cause changes in mRNA abundance in the longissimus thoracis that are related to protein turnover of skeletal muscle. Furthermore, calpastatin protein abundance was also altered by this breed × treatment interaction. This research demonstrates that anabolic implants cause molecular changes in skeletal muscle of feedlot steers, with some of these changes being breed dependent.

Spectro 1-A Potential Spectrophotometer for Measuring Color and Myoglobin Forms in Beef.

The objective of this study was to compare the Color Muse Spectro 1 sensor to the HunterLab MiniScan XE Plus spectrophotometer for evaluating beef color. Color coordinates (lightness (L*), redness (a*), yellowness (b*), chroma (C*), and hue (h*)), myoglobin redox forms (metmyoglobin (MMb), deoxymyoglobin (DMb), and oxymyoglobin (OMb)), and metmyoglobin reducing ability (MRA) were measured on beef steaks over a 5-days storage period. The results indicated that L*, b*, C*, MMb%, OMb%, and MRA% values obtained with Spectro 1 were comparable to those of MiniScan. However, Spectro 1 values for a* were overestimated compared to MiniScan (p < 0.05), whereas those for h* and DMb% were underestimated (p < 0.05). Regardless, Spectro 1 had the capability to detect changes in color coordinates, myoglobin forms, and MRA throughout the storage period. Bland−Altman plots demonstrated that L*, b*, and C* are interchangeable between the two instruments, but it was not the case for a*, h*, myoglobin forms, and MRA. Color coordinates measured by Spectro 1 exhibited excellent stability over time, evidenced by the low total color difference (ΔE*ab) values. Collectively, these findings indicate that Spectro 1 is a potential alternative spectrophotometer for studying meat color and myoglobin redox forms.

Ultrasonication of beef improves calpain-1 autolysis and caspase-3 activity by elevating cytosolic calcium and inducing mitochondrial dysfunction.

The objective of this study was to investigate if ultrasonication of bovine longissimus thoracis et lumborum (LTL) steaks increases calpain-1 and caspase-3 activities, and if so, to explore the underlying mechanisms that trigger their activation. Post-rigor bovine LTL steaks were subjected to ultrasonication at 40 kHz and 12 W/cm2 for 40 min and subsequently aged for 14 d at 4 °C. Ultrasonication improved beef tenderness (P < 0.05) without negatively impacting pH, color, or cook loss (P > 0.05). Improved tenderness in the ultrasonicated steaks was associated with greater degradation of titin, desmin, troponin-T, and calpastatin and increased calpain-1 autolysis and caspase-3 activity (P < 0.05). In addition, ultrasonicated steaks had greater levels of cytosolic calcium and reactive oxygen species and lower mitochondrial oxygen consumption rate (P < 0.05). These data indicate that improved beef tenderness following ultrasonication is, in part, a function of increased calpain-1 and caspase-3 activities, potentially by elevating cytosolic calcium and inducing mitochondrial dysfunction, respectively.