scholarly journals Impact of Sampling and Storage Techniques on Beef Muscle Measurements During Aging

2019 ◽  
Vol 3 (2) ◽  
Author(s):  
M. Diss ◽  
W. Keller ◽  
K. Carlin
Keyword(s):  
Phosphate Buffer ◽  
Sarcomere Length ◽  
Troponin T ◽  
Colorimetric Assay ◽  
Thiobarbituric Acid ◽  
Band 3 ◽  
Posterior Portion ◽  
Aging Period ◽  
Storage Type ◽  
Beef Muscle

ObjectivesThis objective was to determine if variations in beef sampling techniques utilized by meat researchers have a significant impact on beef muscle measurements during aging.Materials and MethodsTen beef short loins (IMPS 180) were purchased from a commercial packing plant within 48 h of slaughter. Loins were transported to the NDSU Meat Science laboratory where they were mapped into four sections from most anterior (1) to most posterior (4). Within sections, two, 40-g samples were removed; one sample was vacuum packaged (SMALL-VAC) and the other sample was stored in a wire-closure sealed bag (SMALL-BAG). The remaining whole short loin was vacuum packaged. All samples and whole short loins were stored at 4°C for 10 d. At 10 d, the short loins were sampled again where one, 40-g sample was removed from each mapped section (WHOLE-VAC). Purge loss was measured by weighing each sample prior to packaging treatment and at the end of the 10-d aging period; percentage change in weight was calculated. Troponin-T degradation was determined by western blot. Briefly, protein was extracted in an SDS-phosphate buffer, separated by SDS-PAGE under reducing conditions, and transferred to PVDF membranes. Western analysis was done using an anti-troponin-T antibody (clone JLT 12), and immunoreactive bands (Band 1 = doublet ∼42 to 45 kDa; Band 2 = doublet ∼ 36 to 38 kDa, Band 3 = 30 kDa) were analyzed for differences in density. Sarcomere length was determined using HeNe laser diffraction. Thinly sliced samples (∼50 to 100 mg) were placed in a sucrose-phosphate buffer and subjected to beadmill homogenization. A drop of the homogenate was placed on a glass slide, diffraction patterns were measured, and sarcomere length was calculated. Thiobarbituric acid reactive substances (TBARS) were assessed using a colorimetric assay. Analysis was conducted using Proc Mixed procedure of SAS where storage type, section location, and their interaction were used as fixed effects.ResultsThere was a storage type by section interaction (P = 0.017) that occurred with purge loss. SMALL-VAC samples released more purge than SMALL-BAG from the more posterior samples. Troponin-T Band 1 tended to be less (P = 0.07) in WHOLE-VAC samples compared with SMALL-VAC and SMALL-BAG. There was a storage type by section interaction (P = 0.02) where the most posterior SMALL-BAG samples had greater Band 2. There were no differences (P ≥ 0.25) in Band 3 between treatments. There was no difference (P = 0.29) in sarcomere length due storage type. However, there was a difference (P = 0.01) in sarcomere length between sections, where the shortest sarcomeres were in the center of the strip loin and longest sarcomeres on either end. There was a storage type by section interaction (P = 0.02) for TBARS where concentration was greatest in the most posterior portion of SMALL-BAG compared with WHOLE-VAC.ConclusionCollection of smaller samples for aging studies may not be representative of samples aged in a whole primal cut and may influence research outcomes.

scholarly journals PSIII-1 A preliminary study to investigate the contribution of different tenderness factors to beef loin, tri-tip and heel tenderness

2020 ◽  
Vol 98 (Supplement_3) ◽  
pp. 233-234
Author(s):  
Colin Chun ◽  
Wanjun Wu ◽  
Amelia Welter ◽  
Travis O'Quinn ◽  
Geraldine Magnin ◽  
...  
Keyword(s):  
Muscle Tissue ◽  
Lipid Content ◽  
Crosslink Density ◽  
Troponin T ◽  
Collagen Content ◽  
Aging Period ◽  
Relative Contribution ◽  
Beef Carcasses ◽  
Trained Panel ◽  
Longissimus Lumborum

Abstract The objective of this study was to identify the relative contribution of tenderness factors for 3 beef muscles with similar tenderness ratings. Longissimus lumborum (loin), tensor fascia latae (tri-tip) and gastrocnemius (heel) were collected from 10 USDA choice beef carcasses, fabricated into steaks and assigned to a 5 or 21 d aging period (n=60). Heel had the greatest relative troponin-T degradation %, followed by tri-tip and loin (68.10, 53.42 and 35.01 % respectively; P< 0.01). Tri-tip had the longest sarcomere, followed by heel and loin (3.01, 2.59 and 1.71 µm, respectively; P < 0.01), and heel increased in sarcomere length from 5 to 21 d of postmortem storage (2.49 vs 2.70 µm; P< 0.05). As expected, heel had the greatest collagen content, followed by tri-tip and loin (6.06, 3.98 and 2.76 mg/g of muscle tissue, respectively; P< 0.01), and collagen content decreased for all cuts from 5 to 21 d of postmortem storage (4.64 vs 3.90 mg/g muscle tissue; P< 0.05). Out of the 3 cuts, heel had the highest collagen crosslink density (0.20 mol/mol collagen; P< 0.05), while loin and tri-tip did not differ (0.13 and 0.15 mol/mol collagen, respectively; P >0.05). Collagen crosslink density increased for all cuts from 5 to 21 d of postmortem storage (0.14 vs 0.20; P< 0.01). Heel had lower lipid content than the others (2.68 %; P< 0.01), while tri-tip and loin did not differ in lipid content (8.24 vs. 6.99 %; P >0.05). Loin was ranked by the trained panel to have the highest overall tenderness, while tri-tip and heel did not differ in overall tenderness (P >0.05). The overall tenderness value for heel was positively correlated with troponin-T degradation (P< 0.01). On the other hand, overall tenderness value for loin was negatively correlated with lipid content (P< 0.05), and overall tenderness value for tri-tip was negatively correlated with collagen content (P< 0.05). These results indicated that each beef cut had a unique profile of tenderness contributors.

scholarly journals A Proposed Mechanism for Texture Property of Woody Breast in Broilers

2019 ◽  
Vol 3 (2) ◽  
Author(s):  
A. Welter ◽  
W. J. Wu ◽  
T. O’Quinn ◽  
T. Houser ◽  
E. Boyle ◽  
...  
Keyword(s):  
Sarcomere Length ◽  
Troponin T ◽  
Chicken Breast ◽  
Free Calcium ◽  
Processing Plant ◽  
Breast Meat ◽  
Collagen Crosslinks ◽  
White Striping ◽  
Chicken Breast Meat ◽  
Early Postmortem

ObjectivesWoody breast is a myopathy observed in chicken breast meat (Pectoralis major) characterized by its tough and rubbery texture. However, the exact causation of woody breast texture is still unknown. We hypothesize that sarcoplasmic reticulum (SR) dysfunctionality early postmortem results in rapid leakage of intracellular calcium may partially contribute to the abnormal meat texture observed in woody breast meat. The objective of this preliminary study was to investigate this hypothesis.Materials and MethodsFourteen Ross line broiler breast fillets (7 severe woody breast and 7 normal) were collected at 3 h postmortem from a commercial processing plant located in the southeast United States. The 7 woody breast samples also exhibited moderate to severe white striping. The 7 normal samples did not exhibit any signs of white striping or woody breast. Each sample was trimmed, weighed, vacuum packaged and frozen at –20°C at approximately 8 h postmortem. One 1.9 cm strip across the cranial end of each fillet was fabricated and pulverized in liquid nitrogen to measure sarcomere length (Laser Scan Confocal Microscope with a 100x/NA 1.4 objective), calpain activity (immunoblotting for µ-calpain autolysis), proteolysis (immunoblotting for troponin-T degradation) and collagen content (hydroxyproline content). Purge was also collected from each sample to evaluate protein (bicinchoninic acid assay) and free calcium concentration (atomic absorption).ResultsWoody breast fillets were heavier than normal chicken breast fillets (522.9 vs. 446.9g; P < 0.05). Woody breast samples tended to have shorter sarcomeres (1.70 vs. 2.02 µm; P = 0.0543) and less intact troponin-T compared to normal breast samples (relative intact troponin-T band density: 49.98 vs. 56.97%; P = 0.0515) at 8 h postmortem. It was interesting to note that no µ-calpain band was detected through immunoblotting for both the woody breast and normal samples at 8 h postmortem. Other studies have found similar results as poultry µ-calpain autolyzed at a much rapid rate than µ-calpain in mammalian species. In addition, the purge from woody breast samples also had higher levels of free calcium compared to normal samples (6.2 vs. 4.2 nmol calcium/mg protein; P < 0.05). Lastly, there was more collagen present in the woody breast samples compared to normal chicken breast samples (3.89 vs. 2.08 mg collagen/g muscle tissue; P < 0.05).ConclusionThe results indicated that the cause of texture abnormality of woody breast may be the combined effects of more calcium being released from the SR early postmortem resulting in shorter sarcomere length and more collagen being deposited in the chicken breast meat. Additional research with the focus on SR integrity and functionality as well as collagen crosslinks are needed to further elucidate the basic mechanism of woody breast texture formation.

scholarly journals The Impacts of Feeding Natursafe (an Immune Support Product) on Beef Quality

2019 ◽  
Vol 3 (2) ◽  
Author(s):  
M. Henriott ◽  
K. Hart ◽  
N. Herrera ◽  
F. Ribeiro ◽  
N. Bland ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Beef Cattle ◽  
Meat Quality ◽  
Shear Force ◽  
Sarcomere Length ◽  
Troponin T ◽  
Quality Characteristics ◽  
Insoluble Collagen ◽  
Plate Counts ◽  
Retail Display

ObjectivesNaturSafe® (Diamond V, USA) is a Saccharomyces cerevisiae fermentation product developed as an animal feed supplement for the further manufacture of nutritionally balanced feeds for beef cattle. This immune support product (Association of American Feed Control Officials number 96.8, 73.046 and International Feed Name number 7–05–520, 8–08–034) has been specifically formulated to optimize beef cattle health and performance, antibiotic stewardship, and food safety. Research has shown that NaturSafe supports optimal rumen and liver health, overall health and immune function, consistency of feed intake, daily gain, feed conversion, and antibiotic effectiveness. As no research previously has assessed the impacts of this feed ingredient on the quality of meat, the objective of this research was to characterize the effects of feeding NaturSafe on meat quality characteristics in beef.Materials and MethodsCrossbred steers (N = 60, n = 12 per treatment; mean hot carcass weight = 421 kg), through an antibiotic free production system, were individually fed diets containing 12, 15, or 18 g/d of NaturSafe or a control diet without (–AB) antibiotics or a control with antibiotics (+AB; 330 mg monensin + 110 mg tylosin·steer–1·d–1) for 112 d. Strip loins were collected and aged for 13 or 29 d postmortem prior to fabrication. Steaks (m. Longissimus) were then evaluated for Warner-Bratzler shear force, pH, sarcoplasmic calcium concentration, troponin-T degradation, fatty acid profile, proximate composition, sarcomere length, total collagen and insoluble collagen. After each aging period, steaks were evaluated for lipid oxidation, and color characteristics (L*, a*, b*, discoloration percentage, and percentage surface oxymyoglobin, metmyoglobin and deoxymyoglobin), during and/or after a 7 d simulated retail display period. A subset of samples at various aging and retail display periods were analyzed for lactic acid bacteria (LAB), psychotrophic plate counts (PPC), and aerobic plate counts (APC). Animal was considered the experimental unit and hot carcass weight and marbling score were used as covariates in the analysis.ResultsTreatment had no effect on pH, sarcomere length, troponin-T degradation, fatty acid profile, proximate composition, total collagen, insoluble collagen, LAB, PPC, APC, lipid oxidation, oxymyoglobin percentage, or metmyoglobin percentage. Meat from cattle fed 18 g/d of NaturSafe was (1) equal to–AB controls and had higher shear force values compared to all other treatments (P < 0.01), (2) had higher (P < 0.05) sarcoplasmic calcium levels than +AB controls and cattle fed 12 g of NaturSafe/d, (3) was redder (higher a* values, P < 0.05) than all other treatments, and (4) was yellower (higher b* values, P < 0.01) than the 12 or 15 g dose and the–AB control. There were no differences among treatments fed NaturSafe for lightness (L*) at either aging time. There were no differences for meat from animals fed 12 or 15 g NatureSafe/d, except deoxymyoglobin percent and discoloration, which were both minimal. Discoloration values were low for all treatments (< 10%).ConclusionThese data indicate that feeding NaturSafe had few discernible effects on meat quality characteristics.

scholarly journals Hypertrophic cardiomyopathy mutation in cardiac troponin T (R95H) attenuates length-dependent activation in guinea pig cardiac muscle fibers

2017 ◽  
Vol 313 (6) ◽  
pp. H1180-H1189 ◽  
Author(s):  
Alexis V. Mickelson ◽  
Murali Chandra
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Guinea Pig ◽  
Central Region ◽  
Cardiac Troponin ◽  
Sarcomere Length ◽  
Troponin T ◽  
Cardiac Troponin T ◽  
Cross Bridge ◽  
Cardiac Muscle Fibers ◽  
Length Dependent Activation

The central region of cardiac troponin T (TnT) is important for modulating the dynamics of muscle length-mediated cross-bridge recruitment. Therefore, hypertrophic cardiomyopathy mutations in the central region may affect cross-bridge recruitment dynamics to alter myofilament Ca2+ sensitivity and length-dependent activation of cardiac myofilaments. Given the importance of the central region of TnT for cardiac contractile dynamics, we studied if hypertrophic cardiomyopathy-linked mutation (TnTR94H)-induced effects on contractile function would be differently modulated by sarcomere length (SL). Recombinant wild-type TnT (TnTWT) and the guinea pig analog of the human R94H mutation (TnTR95H) were reconstituted into detergent-skinned cardiac muscle fibers from guinea pigs. Steady-state and dynamic contractile measurements were made at short and long SLs (1.9 and 2.3 µm, respectively). Our results demonstrated that TnTR95H increased pCa50 (−log of free Ca2+ concentration) to a greater extent at short SL; TnTR95H increased pCa50 by 0.11 pCa units at short SL and 0.07 pCa units at long SL. The increase in pCa50 associated with an increase in SL from 1.9 to 2.3 µm (ΔpCa50) was attenuated nearly twofold in TnTR95H fibers; ΔpCa50 was 0.09 pCa units for TnTWT fibers but only 0.05 pCa units for TnTR95H fibers. The SL dependency of rate constants of cross-bridge distortion dynamics and tension redevelopment was also blunted by TnTR95H. Collectively, our observations on the SL dependency of pCa50 and rate constants of cross-bridge distortion dynamics and tension redevelopment suggest that mechanisms underlying the length-dependent activation cardiac myofilaments are attenuated by TnTR95H. NEW & NOTEWORTHY Mutant cardiac troponin T (TnTR95H) differently affects myofilament Ca2+ sensitivity at short and long sarcomere length, indicating that mechanisms underlying length-dependent activation are altered by TnTR95H. TnTR95H enhances myofilament Ca2+ sensitivity to a greater extent at short sarcomere length, thus attenuating the length-dependent increase in myofilament Ca2+ sensitivity.

Free and bound malondialdehyde measured as thiobarbituric acid adduct by HPLC in serum and plasma

1991 ◽  
Vol 37 (8) ◽  
pp. 1423-1429 ◽  
Author(s):  
M A Carbonneau ◽  
E Peuchant ◽  
D Sess ◽  
P Canioni ◽  
M Clerc
Keyword(s):  
High Risk ◽  
Human Serum ◽  
Cancer Patients ◽  
Flow Rate ◽  
Phosphate Buffer ◽  
Healthy Subjects ◽  
Thiobarbituric Acid ◽  
Acid Precipitation ◽  
Hplc Separation ◽  
Acid Adduct

Abstract Assay of free and total malondialdehyde (MDA) in human serum and plasma from healthy subjects and from patients with high risk of lipoperoxidation was performed as follows: (a) acidic (HClO4, pH 1, at 20 degrees C) or basic (NaOH, pH 13, at 60 degrees C) treatments for 30 min; (b) reaction of the protein-free extract (obtained by acid precipitation) with thiobarbituric acid (TBA); (c) HPLC separation on C18 columns with an eluting solution of methanol/phosphate buffer, 10 mmol/L, pH 5.8 (40/60, by vol), at a flow rate of 1.5 mL/min. Free MDA averaged 0.042 (SEM 0.008) and 0.043 (SEM 0.007) mumol/L, respectively, in serum and plasma from healthy subjects. Free (+/- SEM) MDA increased significantly in the plasma from cancer patients (0.270 +/- 0.047 mumol/L) and from hemodialyzed patients (0.214 +/- 0.035 mumol/L). In serum of hemodialyzed patients, analyses for total MDA were unsuitable because of interfering peaks. MDA bound to NH2 groups constituted 83.2% and 83.5% of total MDA in serum and plasma of healthy subjects, respectively, and only 58% in plasma of hemodialyzed patients.

scholarly journals Regional Phenotypic Differences of the Opener Muscle in Procambarus clarkii: Sarcomere Length, Fiber Diameter, and Force Development

Biology ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 118 ◽  
Author(s):  
Rachel C. Holsinger ◽  
Robin L. Cooper
Keyword(s):  
Sarcomere Length ◽  
Procambarus Clarkii ◽  
Fiber Diameter ◽  
Troponin T ◽  
Input Resistance ◽  
The Other ◽  
Phenotypic Differences ◽  
Synaptic Structure ◽  
Frequency Of Stimulation ◽  
40 Hz

The opener muscle in the walking legs of the crayfish (Procambarus clarkii) has three distinct phenotypic regions although innervated by only one excitatory motor neuron. These regions (distal, central, and proximal) have varied biochemistry and physiology, including synaptic structure, troponin-T levels, fiber diameter, input resistance, sarcomere length, and force generation. The force generated by the central fibers when the excitatory neuron was stimulated at 40 Hz was more than the force generated by the other regions. This increase in force was correlated with the central fibers having longer sarcomeres when measured in a relaxed claw. These data support the idea that the central fibers are tonic-like and that the proximal fibers are phasic-like. The addition of serotonin directly on the fibers was hypothesized to increase the force generated by the central fibers more than in the other regions, but this did not occur at 40-Hz stimulation. We hypothesized that the central distal fibers would generate the most force due to the arrangement on the apodeme. This study demonstrates how malleable the motor unit is with modulation and frequency of stimulation.

Beef muscle troponin: evidence for multiple forms of troponin-t

1976 ◽  
Vol 427 (2) ◽  
pp. 617-626 ◽  
Author(s):  
F.M. Clarke ◽  
S.J. Lovell ◽  
C.J. Masters ◽  
D.J. Winzor
Keyword(s):  
Troponin T ◽  
Multiple Forms ◽  
Beef Muscle

Effects of aging and dietary supplementation with polyphenols from Pinus taeda hydrolysed lignin on quality parameters, fatty acid profile and oxidative stability of beef

2020 ◽  
Vol 60 (5) ◽  
pp. 713 ◽  
Author(s):  
A. Maggiolino ◽  
J. M. Lorenzo ◽  
A. Salzano ◽  
M. Faccia ◽  
F. Blando ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Oxidative Stability ◽  
Pinus Taeda ◽  
Meat Quality ◽  
Shear Force ◽  
Control Diet ◽  
Thiobarbituric Acid ◽  
Control Group ◽  
Thiobarbituric Acid Reactive Substances ◽  
Aging Period

Context The inclusion of Pinus taeda hydrolysed lignin (PTHL) in beef diets could improve quality and stability of meat, but effects could vary through the aging period (AP). Aim The aim was to evaluate the effects of the PTHL inclusion in the diet of finishing beef cattle on meat quality, fatty acid composition and oxidative stability at Days 1, 8, 11 and 15 of aging. Methods Forty Limousin bulls (340 ± 42 kg) were fed ad libitum on a total mixed ration (TMR). The control group received exclusively TMR for 120 days, while the experimental group received the same TMR as the control group but supplemented with PTHL (Oxifenol, I-Green, Padua, Italy; 35 g/day per head at 1–90 days and 70 g/day per head at 91–120 days). Key results Diet did not influence the chemical composition, pH, cooking loss, Warner–Bratzler shear force and hydroperoxide content. The Warner–Bratzler shear force (P &lt; 0.001) decreased, while lightness (P &lt; 0.01) and hydroperoxides (P &lt; 0.001) increased through the aging period. Thiobarbituric acid reactive substances were similar for both diets at 1 and 15 days. However, thiobarbituric acid reactive substances at 8 and 11 days were higher for control than for the PTHL diet (P = 0.023 for interaction). Protein carbonyls were higher for control than for the PTHL diet at 8 days (P = 0.003 for interaction), but similar for both diets for the other dates. Saturated, monounsaturated and polyunsaturated fatty acids varied through the AP with PTHL diet, while no changes were observed with control diet (P &lt; 0.01 for interactions). At 11 days, the n-6:n-3 ratio passed from being the minimum value with the PTHL diet to be the maximum with control diet (P &lt; 0.01 for interaction). Conclusions The effects of PTHL inclusion in bull finishing diets depends on the AP but, generally, may result in beef with meat with beneficial effects on human health. Implications Including PTHL in the diet of finishing bulls can be useful to improve meat quality, favouring the use of natural waste substances deriving from vegetal production.

scholarly journals Concomitant Administration of Rosuvastatin and Lefleunamide in Low doses Synergize Against Complete Freunds Adjuvant (CFA)-Induced Rheumatoid Arthritis in Experimental Model

2021 ◽  
pp. 370-382
Author(s):  
Abdel-Aziz Saeed ◽  
Mohamed El-Shafey ◽  
Gouda K. Helal ◽  
El-Sayed Akool
Keyword(s):  
Rheumatoid Arthritis ◽  
Experimental Model ◽  
Colorimetric Assay ◽  
Mrna Level ◽  
Thiobarbituric Acid ◽  
Concomitant Administration ◽  
Thiobarbituric Acid Reactive Substances ◽  
Sod Activity ◽  
Mda Content ◽  
Inflammatory Effect

Aim: The present work was designed to examine of the potential anti-inflammatory effect of rosuvastatin (ROSV) and/or Lefleunamide (LFLU) against Complete Freunds Adjuvant (CFA)-induced arthritis in rats. Methods: The mRNA level of perxisome proliferator-activated receptor-alpha (PPAR-a) was determined using Real-time PCR. The levels of NF-B, iNOS, IL-6, TNF-a and SOD activity were measured using ELISA. The swollen paws were measured using caliper. The GSH level was measured using colorimetric assay. The level of malondialdehyde (MDA) was determined using thiobarbituric acid reactive substances assay kit. Results: ROSV induced the expression of PPAR-a that suppresses NF-kB as demonstrated by a strong reduction in NF-kB level in animals treated with ROSV. Also, ROSV administration reduced the levels of the inflammatory mediators IL-6 and TNF-a. In addition, iNOS and MDA content as well as expression of MMP-9 and MMP-2 induced by CFA is abrogated in animals treated with ROSV. Also GSH content and SOD activity were highly increased in ROSV-treated animals. Furthermore, the size of right paw induced by CFA was reduced in ROSV-treated rats. Moreover, the histopathological alterations induced by CFA were highly improved in animals treated with ROSV. Similar results were also found in animals treated with LFLU. Importantly, similar effects were obtained in rats treated with both ROSV and LFLU in half doses. Conclusion: This study demonstrates that ROSV as well as LFLU has the ability to inhibit rheumatoid arthritis in experimental model induced by CFA. Importantly, concomitant administration of ROSV and LFLU in half doses synergize against rheumatoid arthritis.

Mapping of the Glycolytic Enzyme Binding Sites on Human Erythrocyte Membrane Band 3.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1668-1668
Author(s):  
Haiyan Chu ◽  
Philip S. Low
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Binding Sites ◽  
Troponin T ◽  
Band 3 ◽  
Glycolytic Enzyme ◽  
Human Erythrocyte Membrane ◽  
Binding Studies ◽  
Red Cell Membrane ◽  
N Terminus

Abstract Glycolytic enzymes (GEs) including aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) are known to associate with the inner surface of the human erythrocyte membrane. Previous studies have suggested that the N-terminus of the cytoplasmic domain of band 3 (cdb3) constitutes the likely binding site for aldolase, GAPDH and PFK, however, the membrane docking sites for PK and LDH have not been elucidated. In this study, we demonstrate that PK and LDH exhibit no affinity for band 3, regardless of whether the association is measured by co-immunoprecipitation assay, binding competition studies, or catalytic inhibition analyses. We further find that the binding sites for GAPDH, aldolase and PFK on band 3 are distinct but partially overlapping, as evidenced by the fact that: 1) deletion of residues 1–11 of cdb3 eliminates the binding of aldolase, but not PFK or GAPDH, 2) fusion of thioredoxin (Trx) to the N-terminus of residues cdb3 blocks aldolase binding, but not the association of GAPDH or PFK, 3) deletion of sequences 1–50, 1–40, 1–31, or 1–23 of cdb3 blocks cdb3 association with all three GEs, whereas deletion of residues 12-23 only abrogates aldolase binding (while reducing the affinity of PFK and GAPDH), 4) the presence of both sequences, 6–DDYED-10 and 19-EEYED-23, are necessary for cdb3 association with aldolase, whereas the presence of either sequence alone (especially 19-EEYED-23) is sufficient to maintain association with GAPDH and PFK, 5) mutation of all of the acidic residues in the above two sequences to their corresponding amides (E→Q and D→N) results in loss of affinity for all GEs. Because i) kidney cdb3 (which lacks residues 1-65 of cdb3) shows no affinity for any of the GEs, ii) residues 1–55 of cdb3 show near normal affinity for aldolase, GAPDH and PFK, and iii) GAPDH, aldolase, and PFK all compete with each other for cdb3, we conclude that the binding sites for aldolase, GAPDH and PFK are all located within the first 23 residues of cdb3, with the docking site for aldolase likely residing somewhat more N-terminal than the binding sites for GAPDH and PFK. Finally, because each band 3 monomer contains three homologous sequences (6-DDYED-10, 19-EEYED-23 and 902-DEYDE-906) that are found in other proteins that bind GEs (e.g. actin, β-tubulin, troponin T), and since the first two of these sequences were shown to be required for cdb3 binding, the question naturally arose whether different GEs might also associate with residues 902-DEYDE-906 at the extreme C-terminus of band 3. Similar binding studies demonstrate that such an interaction does not occur. Thus, our data show that GAPDH, aldolase, and PFK all bind near the N-terminus of band 3 and that PK and LDH must dock somewhere else on the red cell membrane.

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