SOME CHARACTERISTICS OF THE NAD(P)H-DEPENDENT LIPID PEROXIDATION SYSTEM IN THE MICROSOMAL FRACTION OF CHICKEN BREAST MUSCLE

Incubation of the microsomal fraction isolated from chicken breast muscle in the presence of NADPH, ADP and Fe⁺⁺⁺ ions was shown to result in lipid peroxidation. NADH was able to replace NADPH as the source of reducing equivalents but was less efficient. The pH optimum for malonaldehyde (MDA) production was found to be around pH 6. 7. Oxygen uptake by the system was shown to have a pH optimum of 5.5. Oxygen uptake was approximately linearly dependent on temperature but malonaldehyde production exhibited a sharp increase between 25°C and 37°C. Inhibitor studies and the virtual absence of cytochromes indicate that this system is not similar to that isolated from rat liver microsomes. The activity of the lipid peroxidation system was maintained after storage for 7 days at pH 7.25 and 4°C. At pH 5.6 a reduction in activity was noted after 7 days.     

MEASURING THE WATER HOLDING CAPACITY OF NATURAL ACTOMYOSIN FROM CHICKEN BREAST MUSCLE IN THE PRESENCE OF PYROPHOSPHATE AND DIVALENT CATIONS

The water holding capacity (WHC) of natural actomyosin (NAM) extracted at pH 9.2 in 0.6M KCl was measured in the presence and absence of various combination of sodium pyrophosphate (PP_i), MgCl₂ and CaCl₂ using a modification of the classical centrifugation technique. Samples, in the presence of 0.15M NaCl and 20 mM sodium phosphate buffer pH 6, were spun at 30,900 X G (as measured at the bottom of the centrifuge tube) for 15 min at 2–4C. The results show that between 17 and 20 g water/g protein were bound over a wide range of NAM concentrations. In each case the amount of water held by the experimental sample was equal to or less than the amount held by a control run at the same time: 5 mM PP_i= 100%; 5 mM PP_i+ 5 mM MgCl₂= 58%; 5 mM MgCl₂= 85%; 5 mM PP_i+ 5 mM CaCl₂= 68% and 5 mM CaCl₂= 92% of the control. Thus polyphosphate addition in the presence of divalent cations lowers the WHC of NAM. The absence of the organized structure of muscle in NAM is postulated to be the reason that polyphosphate plus divalent cation reduced WHC in these samples. A series of preliminary experiments were run in order to determine the effect of experimental parameters on WHC.     

鸡肉盐溶蛋白质热诱导凝胶质构的研究

采用L9（3^4）正交试验法研究磷酸盐和氯化钙复合作用对鸡胸和鸡腿肉热诱导凝胶的硬度和弹性的影响。结果表明，鸡胸肉和鸡腿肉热诱导凝胶的硬度有极显著差异（p〈0．01），二者凝胶的弹性无显著差异（p〉0．05）。就鸡胸内而言。氯化钙、三聚磷酸盐和焦磷酸盐对其硬度均有极显著影响（p〈0．01），三聚磷酸盐和焦磷酸盐对其凝胶的弹性有显著影响（p〈0．05）；而对于腿内来讲。焦磷酸盐对其硬度有极显著影响（p〈0．01），氯化钙和三种磷酸盐对凝胶的弹性均有显著影响（p〈0．05）。     

IDENTIFICATION AND CHARACTERIZATION OF MATRIX METALLOPROTEINASES FROM THE SARCOPLASMIC FRACTION OF COMMON CARP (CYPRINUS CARPIO) DARK MUSCLE

Two gelatin hydrolyzing proteinases in the sarcoplasmic fraction of common carp muscle were detected using gelatin zymography. A comparative study shows that the gelatin hydrolyzing activity in dark muscle is obviously higher than that in white muscle. The enzymes can transform to their active forms after treated by aminophenylmercuric acetate, an activator of matrix metalloproteinases. Optimum pH and temperature of these enzymes were around 8.0 and 40C using gelatin as substrate. Metalloproteinase inhibitors (ethylenediaminetetraacetic acid and ethylene glycol-bis (2-aminoethylother)-N, N, N', N'-tetraacetic acid) completely suppressed the activities and 1,10-phenanthroline also showed great inhibitory effects. However, other proteinase inhibitors, such as soybean trypsin inhibitor, benzamidine, E-64 and pepstatin A, did not show any effect. Metal ions Ca^{2 +} and Zn^{2 +} are essential for these gelatinolytic activities. All these facts indicate that these proteinases are matrix metalloproteinases. Furthermore, these enzymes hydrolyze collagen effectively and maybe thus proposed to be responsible for the tenderization of fish muscle during the postmortem stage.     

EFFECTS OF ANTI-LMM ANTIBODIES ON THE SOLUBILITY OF CHICKEN SKELETAL MUSCLE MYOSIN

Monoclonal antibody Fab fragments which bind epitopes in the rod domain of skeletal muscle myosin had specific effects on the solubility properties of chicken muscle myosin in vitro. Two antibodies (NA4 and 5C3), which bind at the C-terminal portion of the rod domain caused myosin to remain soluble and monomeric in 0.1 M KCl, pH 7.2, conditions in which myosin normally aggregates into filamentous structures. Other antibodies (EB165 and AB8), which bind in the middle of the rod did not alter either myosin solubility or the morphology of the myosin assemblies formed. These results demonstrate the importance of the C-terminus of the myosin rod as a domain involved in regulating myosin interactions and solubility.     

REACTION MECHANISM OF PYROPHOSPHATE-DEPENDENT PHOSPHOFRUCTOKINASE FROM PINEAPPLE LEAVES

Data obtained from initial velocity, product inhibition and dead-end inhibition patterns indicate that the pyrophosphate-dependent phosphofructokinase from pineapple leaves has a rapid equilibrium random mechanism with E:F6P:MgPP_i and E:MgPP_i:FBP dead-end complexes. The pH dependence of enzyme-reactant dissociation constants suggests that phosphates of F6P (fructose 6-phosphate), FBP (fructose 1,6-bisphosphate), P_i and PP_i must be completely ionized and the lysine is present in the vicinity of the 1- and 6-phosphates of the sugar phosphate and bisphosphate. The pH dependence of kinetic parameters suggests that the enzyme catalyzes the reaction via general acid-base catalysis with the use of a proton shuttle. The base is required to be unprotonated in both reaction directions.     

EFFECT OF MgCl2/SODIUM PYROPHOSPHATE ON CHICKEN BREAST MUSCLE MYOSIN SOLUBILIZATION AND GELATION

Sodium chloride (0.29 M) at pH 7 solubilized about 24% of the myosin of washed, minced chicken breast muscle. At a similar pH, 0.2 M sodium chloride in the presence of 10 mM sodiumpyrophosphate and 10 mM magnesium chloride solubilized almost 60% of the myosin. In spite of the greater solubility of myosin under the latter conditions, when gels were prepared with these concentrations of salt at pH 7, the gels without the sodium pyrophosphate and magnesium chloride were slightly superior in both stress (39.3 kPa vs 28.3 kPa) and true strain (2.3 vs 2.0) values. Gels made at a lower pH (6.1–6.5) made much poorer gels. This was true whether the low pH was obtained naturally in the preparation of the sample or re‐adjusted after bringing the mince to a neutral pH. It appears that conditions of pH and salt content that cause solubilization of myosin at more dilute conditions does not contribute to gel quality, but the neutral pH is an important factor for obtaining good gels at ionic strengths <0.3.     

FACTORS AFFECTING THE SODIUM CHLORIDE EXTRACTABILITY OF MUSCLE PROTEINS FROM CHICKEN BREAST, TROUT WHITE AND LOBSTER TAIL MUSCLES

The amount of protein extracted from chicken breast muscle at low salt (0–50 mM NaCl) increased as the salt concentration of the extracting solutions increased. The addition of 10 mM sodium phosphate buffer pH 7 (P_i) caused a marked increase in protein extractability at all salt concentrations. A particular polypeptide chain of about 150,000 daltons appeared to be particularly sensitive to the extraction conditions. At high salt (0.6M NaCl, 50 mM sodium phosphate buffer pH 7.0) a second extraction still contained significant amounts of protein. The amount of protein extracted was maximized at a 1/20 dilution. On the other hand, the protein extract-ability of trout white muscle, showed a smaller P_i effect and very little dependence on low salt concentration. The protein extractability of lobster flexor muscle showed little change with either increased salt or P_i. For all three muscles extraction over time with either high or low salt remained essentially constant after the first day with the most protein being extracted from lobster muscle and the least from chicken muscle.     

EFFECT OF POSTMORTEM AGING ON CHICKEN MUSCLE LIPIDS

Neutral lipids of fresh chicken breast muscles are shown to be triglycerides, sterols and sterol esters with only traces of mono- and diglycerides and free fatty acids. Phospholipids include measurable quantities of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine, sphingomyelin, diphosphatidyl glycerol, lysophosphatidyl choline and lysophosphatidyl ethanolamine. Fatty acid analyses of several of the lipid fractions are also included. Decreases in phosphatidyl choline and phosphatidyl ethanolamine coupled with increases in lysophosphatidyl choline, lysophosphatidyl ethanolamine and free fatty acids after 48 hr postmortem in the cold indicate phospholipase A activity concurrent with other postmortem changes. The significance of the results is discussed.     

SOME PROPERTIES OF MYOFIBRILLAR PROTEINS OBTAINED FROM LOW IONIC STRENGTH EXTRACTS OF WASHED MYOFIBRILS FROM PRE- AND POST-RIGOR CHICKEN PECTORAL MUSCLE

SUMMARY— Changes in extractability of the proteins associated with the fragmentation phenomenon of myofibrils in chicken pectoral muscle were studied. The results indicate that the protein fractions extracted by neutralized water from muscle residue. from which water-soluble proteins have been washed out, increase in post-rigor muscle. The extracts from pre- and post-rigor muscle were fractionated with ammonium sulfate into two fractions: the fraction precipitated by 1.7 M ammonium sulfate (Fr.1) and the supernatant (Fr. 2). Depressing effect on the onset of ATP-induced superprecipitation of trypsin-treated myosin 6 which was initially present in Fr. 2 from pre-rigor muscle decreased to a great extent in that from post-rigor muscle, whereas promotive effect on gelation of F-actin and superprecipitation of the myosin 6 which was little in Fr. 1 from pre-rigor muscle appeared in that from post-rigor muscle. It is proposed that an increasing amount of protein which indicates α-actinin activity is released along with the destruction and final dissolution of the Z-line structure during postmortem storage of chicken pectoral muscle.     

EFFECT OF RADURIZATION ON SHEAR RESISTANCE AND FRAGMENTATION OF CHICKEN MUSCLE

SUMMARY —Excised pectoralis muscles of chicken received radurizing doses of γ-radiation at varying times post-mortem to determine the effect on shear resistance, myofibrillar fragmentation (F-ratio) and pH. Irradiation doses between 0.1 and 0.3 Mrad produced decreases in F-ratio and increases in shear resistance. The magnitude of the changes was directly proportional to the duration of postmortem aging prior to irradiation. Muscle pH subsequent to irradiation between 2 and 12 hr post-mortem was not significantly affected by any of the dose-time treatment combinations. Shear resistance and F-ratio changes were strongly correlated on an average basis (r =+ .857; n = 6) but not on an individual sample basis.