Chicken skeletal muscle has three Ca2+-dependent proteinases.

Chicken breast muscle has three Ca2+-dependent proteinases, two requiring millimolar Ca2+ (m-calpain and high m-calpain) and one requiring micromolar Ca2+ (mu-calpain). High m-calpain co-purifies with mu-calpain through successive DEAE-cellulose (steep gradient), phenyl-Sepharose, octylamine agarose, and Sephacryl S-300 columns, but elutes after mu-calpain when using a shallow KCl gradient to elute a DEAE-cellulose column. The mu- and m-calpains have 80 and 28 kDa polypeptides and are analogous to the mu- and m-calpains that have been purified from bovine, porcine and rabbit skeletal muscle. High m-calpain, which seems to be a new Ca2+-dependent proteinase, is still heterogeneous after the DEAE-cellulose column eluted with a shallow KCl gradient. Additional purification through two successive HPLC-DEAE columns and one HPLC-SW-4000 gel permeation column produces a fraction having six major polypeptides and 6-8 minor polypeptides on SDS-PAGE. A 74-76 kDa polypeptide in this fraction reacts in Western blots with monospecific, polyclonal anti-calpain antibodies that react with both the 80 kDa and the 28 kDa polypeptides of mu- or m-calpain. High m-calpain also is related to mu- and m-calpain in that it causes the same limited digestion of skeletal muscle myofibrils, has a similar pH optimum near pH 7.9-8.4, requires Ca2+ for activity, and reacts with the calpain inhibitor, calpastatin, and a variety of serine and cysteine proteinase inhibitors in a manner identical to mu- and m-calpain. High m-calpain differs from mu- and m-calpain in its elution off DEAE-cellulose columns and its requirement of 3800 microM Ca2+ for one-half maximal activity compared with 5.35 microM Ca2+ for mu-calpain and 420 microM Ca2+ for m-calpain. The physiological significance of high m-calpain in unclear. The presence of mu-calpain in chicken breast muscle suggests that all skeletal muscles contain both mu- and m-calpain, although the relative proportions of these two proteinases may vary in different species.

Failure to find Ca2(+)-dependent proteinase (calpain) activity in a plant species, Elodea densa.

Five and nine-tenth kg of Elodea densa (Anacharis), a common aquarium plant, was extracted, and the extract was subjected to column chromatographic procedures that successfully purify the two Ca2(+)-dependent proteinases (calpains) and their protein inhibitor (calpastatin) from a variety of animal tissues. Although these procedures purified a protein having 55- and 16-kDa polypeptides, neither this protein nor any of the other chromatographic fractions contained detectable proteinase or calpastatin activity. Moreover, the purified 55- and 16-kDa polypeptides did not react on immunoblots with polyclonal antibodies that were monospecific for the calpains or calpastatin. We conclude that Elodea densa contains no calpain nor calpastatin at the level of 4 micrograms per g plant protein (1 part per 250,000), which was the sensitivity of our assay.

Effects of low voltage electrical stimulation on mature cow carcasses: Part 1-Selected quality attributes of beef.

The quality characteristics of semitendinosus (ST) and longissimus dorsi (LD) roasts obtained from mature carcasses subjected to extra low voltage (50-100 V) electrical stimulation (ES) were determined. ES was applied for 2 min with voltage increases to either 50 V (ES1) or 100 V (ESII). Evaluationss were conducted on meat obtained from control sides (no ES) aged for either 48h or 7 days and from ES carcasses aged for 48h. ES caused a reduction (P < 0·1) in pH values 1 and 4h post mortem. At 24h, the pH of each of the muscles from ES and control carcasses was similar. There were no differences in raw rib-eye muscle colour, firmness or texture due to ES. Cooking losses of ST and LD did not appear to be affected by either ES or increased ageing of control roasts. For both ST and LD roasts, trained panellists generally detected no significant effects of ES or ageing time (for no ES roasts) on palatability. Consumer panel judgements of ST roasts were similar to those of the trained judges. Water-holding capacity, tenderness (OTMS) and softness (penetrometer) data for ST and LD roasts generally support findings from sensory evaluation. Cooking and eating quality characteristics of ST and LD roasts from mature cow carcasses subjected to ES were similar to those of the controls.

An assessment of extracellular space measurements in post-mortem muscle.

Extracellular space measurements of early post-mortem muscle were made by incubating muscle strips in inulin [(14)C] carboxylic acid. The extracellular space measurements provide a means to assess the functionality of the muscle membrane. This may lead to an understanding of variations in the water-holding capacity of meat and the effects of stress on the muscle membranes of stress susceptible animals. Secondly, a level of intrafibre water affinity between carcasses may be determined which may relate to the interfilamental spacings between myofibrils in the fibre and thus meat quality.

Evidence for differences in post mortem intramuscular phospholipase activity in several muscle types.

The neutral and polar lipids of chicken adductor longus and pork semitendinosus muscles have been analysed at several periods of post mortem ageing. A complete fatty acid analysis for pork muscle has also been carried out. The results revealed small changes in chicken leg muscle phosphatidyl ethanolamine and free fatty acid fractions, suggesting limited phospholipase A activity in this muscle. The pork muscle showed no changes. The results are considerably different from results obtained for chicken pectoralis major. The differences are discussed in the light of the structural and biochemical differences between the sarcotubular systems of red and white muscle fibres.

Effect of post-mortem storage temperatures on isometric tension, pH, ATP, glycogen and glucose-6-phosphate for selected bovine muscles.

Relationships between isometric tension development and pH, ATP, glucose-6-phosphate and glycogen levels as a function of post-mortem storage temperature were examined for three bovine muscles. Tension responses in the range of 5-37°C were similar for B. femoris, Vastus lateralis and outer M. semitendinosis. At 0°C, the three muscles developed considerably higher tension than at 5°C. Cold shortening developed only in the outer M. semitendinosis strips at 0°C. At low temperatures the drop in pH lagged compared to the decline in ATP and glycogen and maximum tension was attained several hours after ultimate pH and minimum levels of ATP and glycogen were reached. Glucose-6-phosphate was found to accumulate rapidly at 0°C in both outer M. semitendinosis and B. femoris, being more pronounced in the latter muscle. The responses of glucose-6-phosphate levels suggest that the relative activities of glycolytic enzymes in muscle stored at 0°C are altered compared to those in muscle stored at higher temperatures.

Rigor related changes in mechanical properties (tensile and adhesive) and extracellular space in beef muscle.

The tensile and adhesive properties of selected beef muscle strips undergoing rigor mortis are presented at various times post mortem. The changes in these mechanical properties of the muscle correlated well with pH and its rate of fall. Additionally, and perhaps most importantly, the shape of the curves generated over the post-mortem ageing times correlate well with changes in extracellular space. The results are discussed and the conclusion drawn that intrafibre water must be considered as a potentially important third factor, in addition to myofibrillar contraction and connective tissue orientation, in the evaluation of meat tenderness.

Studies of extra low voltage electrical stimulation of mature beef carcasses.

The quality characteristics of biceps femoris (BF) and semimembranosus (SM) roasts obtained from mature cow carcasses treated with a commercial extra low voltage (30 V) electrical stimulation (LVES) system were determined. LVES was applied for either 2 (ESII) or 4 min (ESIII). Evaluations were conducted on meat obtained from control sides (no ES) aged for either 48 h (Ia) or 7 days (Ib) and from ES sides aged 48 h. ES caused a reduction (P<0·001) in pH values at 2 and 6 h post mortem. At 24 h, the pH of muscles from all carcasses was about 5·5. ES duration did not influence muscle pH. Rib-eye muscle colour for ESII and ESIII carcasses was lighter and brighter (P<0·05) than that of control carcasses. Generally stimulated BF roasts had greater cooking losses than control Group Ib roasts; SM roasts from ES carcasses had lower losses than comparable to Group Ib roasts. ES duration had no effect on per cent cooking losses. Trained panelists generally detected few significant effects in BF roasts due to ES. Warner-Bratzler data indicated that ESII and ESIII BF roasts were similar and significantly more tender than comparable control Group Ib samples: OTMS data indicated that all BF roasts were similar in tenderness. However, SM roasts from ES carcasses were judged more soft (Groups II and III) and tender (ESII) than comparable control roasts. Instrumental measurements of tenderness for SM roasts tended to support the taste panel results. Generally, duration of LVES had no effect on the eating quality of either BF or SM roasts. Since LVES effects on the palatability of SM roasts were evident but the effects of stimulation of BF roasts were few, further studies of this LVES system are needed before its use can be recommended. Generally, increasing post-mortem ageing time for mature control carcasses did not influence either BF or SM roast quality.

Effects of dietary alpha-linolenic acid and strain of hen on the fatty acid composition, storage stability, and flavor characteristics of chicken eggs.

A study was conducted to determine the effect of dietary alpha-linolenic acid on the fatty acid compositions of egg yolk lipids, tocopherols, and internal quality of raw eggs during storage and the sensory characteristics of hard-boiled eggs from six different laying hen strains. Laying hens (total 300 birds, 72 wk old) from six strains (Rhode Island Red, Barred Plymouth Rock, New Hampshire, Light Sussex, Brown Leghorn, and White Leghorn) were distributed in 12 floor pens (2 pens per strain, 25 birds per pen) with male roosters. One of the pens for each strain was fed with tallow-based control diet and another was assigned with 3% alpha-linolenic acid (LNA) enriched diet with 120 U of mixed tocopherol/kg diet for 3 mo. Ten eggs from each pen were collected every day after 2 wk with the experimental diets, and stored in a cold room at 4 C up to 4 wk. Total lipids, fatty acid compositions, Haugh units, and tocopherols of egg yolk were determined once a week during the 4-wk storage periods. Sensory studies were also conducted using the eggs stored for 2 wk at 4 C. Dietary LNA increased the amount of n-3 fatty acids (6.5%) in total lipid, and over 70% was C18:3n3, and the rest was C22:6n3 (20 to 25%) and C22:5n3 (5 to 10%). Only minor differences in fatty acids among strains were observed. The differences and the changes in tocopherols during storage periods by strain and diet appeared randomly and lacked consistency.(ABSTRACT TRUNCATED AT 250 WORDS)

Autolysis of the millimolar Ca2+-requiring form of the Ca2+-dependent proteinase from chicken skeletal muscle.

The millimolar Ca2+-requiring form of the Ca2+-dependent proteinase from chicken breast skeletal muscle contains two subunit polypeptides of 80 and 28 kDa, just as the analogous forms of this proteinase from other tissues do. Incubation with Ca2+ at pH 7.5 causes rapid autolysis of the 80-kDa polypeptide to 77 kDa and of the 28-kDa polypeptide to 18 kDa. Autolysis of the 28-kDa polypeptide is slightly faster than autolysis of the 80-kDa polypeptide and is 90-95% complete after 10 s at 0 degrees C. Autolysis for 15 s at 0 degrees C converts the proteinase from a form requiring 250-300 microM Ca2+ to one requiring 9-10 microM Ca2+ for half-maximal activity, without changing its specific activity. The autolyzed proteinase has a slightly lower pH optimum (7.7 vs. 8.1) than the unautolyzed proteinase. The autolyzed proteinase is not detected in tissue extracts made immediately after death; therefore, the millimolar Ca2+-requiring proteinase is largely, if not entirely, in the unautolyzed form in situ.