Meat flavor: contribution of proteins and peptides to the flavor of beef.

While the majority of meat flavor is lipid in origin, the contribution of peptides and amino acids to overall meat flavor should not be overlooked. Amino acids and peptide levels have been shown to change with postmortem aging in muscle and with dry-curing, a process similar to PMA. Variation in protein, peptide, and amino acid composition have also been shown to occur with heating and with post-heating storage of meat. This makes a large pool of reactive components that may directly affect flavor or indirectly affect flavor by reacting with reducing sugars to form Maillard reaction products and Strecker degradation products that impact meat flavor. Further research in this area should continue with particular emphasis on natural peptide flavor enhancers, modulators, and potentiators.

A potential index for assessing the tenderness of hydrodynamic pressure (HDP)-treated beef strip loins.

The objective of this research was to obtain physicochemical data that might prove useful in understanding the effect of hydrodynamic pressure (HDP), shock-wave treatment on the structural and functional properties of the HDP-treated beef strip loins. A homogenization protocol that was sufficiently harsh to break-up the tissue yet sufficiently gentle to maintain the integrity of the remaining subcellular structures was therefore developed for HDP-treated beef strip loins. Physicochemical changes resulting from HDP-treatment revealed a relationship between shear resistance value (tenderness level) and protein distribution. The 10,800×g pellet of homogenized HDP-treated strip loins showed a decrease in the protein content associated with a decrease in shear resistance values (increased tenderness). Conversely, the soluble-fraction (post 48,200×g) showed an increase in protein content as the shear resistance value decreased (tenderness increased) from ∼11 to 6 kg. The data presented herein indicate that there is a relationship between the tenderness of hydrodynamic pressure (HDP) treated meat and the protein distribution in the HDP-treated samples.

Isolation, purification, and alteration of some functional groups of major milk proteins: a review.

This review covers selected methods of isolation and purification of mainly alpha s-casein, beta-casein, kappa-casein, beta-lactoglobulin, and alpha-lactalbumin. Selected methods of alteration of some functional groups of these proteins also were reviewed. Isolation and purification of milk proteins per se are methods of modifying the individual milk proteins. Gram quantities of these proteins can now be purified in a relatively short time using ion-exchange resins. Due to the prominent use of non-food-grade reagents in the procedures for preparation of these milk proteins, individual proteins are not maximally utilized for the manufacture of food/feed and pharmaceutical products. Therefore, intensive research efforts are needed to obviate the problems associated with underutilization of milk proteins.

A study of anti-group A streptococcal monoclonal antibodies cross-reactive with myosin.

Anti-group A streptococcal monoclonal antibodies were obtained from BALB c/BYJ mice immunized with purified membranes from M type 5 Streptococcus pyogenes. Two of the anti-streptococcal monoclonal antibodies were previously shown to cross-react with muscle myosin. In this study the monoclonal antibodies were reacted with tissue sections of normal human heart and skeletal muscle. Antibody binding was estimated by indirect immunofluorescence and immunoperoxidase techniques. Both of the monoclonal antibodies (36.2.2 and 54.2.8) investigated in this report reacted with heart and/or skeletal muscle sections. When evaluated by immunofluorescence, monoclonal antibody 54.2.8 demarcated the periphery of cardiac striated muscle cells and reacted to a lesser degree with subsarcolemmal components. Monoclonal antibody 36.2.2 failed to react with heart sections, but both of the monoclonal antibodies reacted strongly with skeletal muscle sections. Results similar to those observed with indirect immunofluorescence were obtained with the immunoperoxidase technique. By Western immunoblotting and competitive inhibition assays, monoclonal antibodies 36.2.2 and 54.2.8 both were found to react with the heavy chain of skeletal muscle myosin. However, only 54.2.8 reacted with the heavy chain of cardiac myosin. The specificity of the monoclonal antibodies for subfragments of skeletal muscle myosin indicated that monoclonal antibody 36.2.2 was specific for light meromyosin fragments, whereas 54.2.8 reacted with both heavy and light meromyosin. The data demonstrated that two monoclonal antibodies against streptococci were specific for skeletal muscle and/or cardiac myosin and for subfragments of the myosin molecule. The reactions of the monoclonal antibodies with human tissue sections were consistent with the immunochemical reactions of the monoclonal antibodies with both denatured and native myosin.

Superoxide dismutase: tissue, cellular, and subcellular distribution in adult canine heart.

Cell-free extracts of canine myocardial tissue were found to contain two biochemically and electrophoretically distinct superoxide dismutases (SOD), an enzyme that provides defense against the deleterious effect of superoxide radicals (O2.-). Polyacrylamide gel (7.5%) electrophoresis revealed two distinct bands of SOD activity: a slower moving band [retardation factor (Rf) = 0.4] resembling the manganese SOD found in bacteria and mitochondria (which is not inhibited by 2.5 mM cyanide) and a faster moving band (Rf = 0.75) that is sensitive to cyanide. In contrast, extracts from isolated adult canine cardiac myocytes were found to contain only the cyanide-insensitive SOD. Extracts of whole myocardium and isolated cardiac myocytes contain 22.3 +/- 1.2 and 27.0 +/- 1.5 U cyanide-insensitive SOD/mg protein, respectively. However, the activity of cyanide-sensitive SOD in these fractions is 7.9 +/- 2.0 (tissue) and 1.5 +/- 1.4 (cells) U/mg protein. Cardiac myocyte SOD activity was particulate in nature, and the major part of the SOD activity was associated with heavy mitochondrial fractions. The biologic significance of this higher activity of SOD in the heavier mitochondrial fraction remains to be elucidated.

Response of canine cardiocyte lysosomes to ATP.

The present study was designed to evaluate the relationship of adenosine triphosphate (ATP) to maintenance of cardiac lysosome latency. Highly latent lysosomes were isolated from adult canine ventricular myocytes or cardiocytes and exhibited latency values (based on % free N-acetyl-beta-glucosaminidase, NAGA) of 29.3 +/- 4.7% (SE), minimal cross contamination by mitochondria (less than 2% cardiolipin by weight) and only 1.68% of the total cytochrome c oxidase activity; enzymatic enrichments ranged from 10-fold for NAGA to almost 50-fold for cathepsin B. Incubations of cardiocyte lysosomes at pH 7.0 and 25 degrees C for up to 1 h resulted in changes in the rate of loss in lysosomal latency when ATP levels were adjusted from 0.0 to 5.0 mM; under these conditions as ED50 of 0.62 mM ATP was determined. The protection afforded by ATP was reversed by addition of the H+ ionophore m-chlorocarbonylcyanide phenylhydrazone (CCCP) at 10 microM to the lysosomal suspensions. A significant increase in lysosomal membrane fluidity, measured by fluorescent polarization of diphenylhexatriene, was also seen in the absence of ATP. Adenosine diphosphate (ADP) afforded significant protection only at the very highest concentration (5.0 mM); inorganic pyrophosphate (PPi) did not protect against loss of latency at any concentration. Thus ATP exerts a significant stabilizing effect on cardio(myo)cyte lysosomes in vitro and may be one of the metabolites regulating lysosomal integrity in vivo.

Characterization of sarcolemma from calcium-tolerant canine cardiocytes.

Large numbers of calcium-tolerant canine cardiocytes can be isolated from the collagenase-perfused canine myocardium. An average yield of 500 million cells provides abundant tissue for the preparation of subcellular fractions. Using nitrogen cavitation, along with extraction by 0.6 M potassium chloride/sucrose buffer, we have been able to prepare, after differential and sucrose gradient centrifugation, membrane fractions that are more than 100-fold enriched in sarcolemmal marker enzymes. This preparation of sarcolemma has the advantage of being essentially free of plasma membranes from endothelial, smooth muscle, and other cell types residing in the myocardium.

Structural and functional characteristics of cardiac myocytes.

Canine and rat cardiac myocytes, prepared by proteolytic disaggregation, retain morphological and topographical features of intact tissue. Cells are rod-shaped and are cleanly separated at their sarcolemmal and intercalated disc borders. Cells demonstrate glucose and fatty acid uptake and metabolic features comparable to intact organ, and are responsive to insulin and epinephrine, but nor carnitine. Although rat cardiac cells contain immunodetectable lipoprotein lipase, they are incapable of metabolizing extracellular lipoprotein triglyceride, nor the glycerol or monoglyceride moieties of the triglyceride. These and other metabolic characteristics suggest that cardiac cells can provide a useful cellular model for studies on cardiac pathophysiology.

Rapid agonist-induced decrease of 125I-pindolol binding to beta-adrenergic receptors. Relationship to desensitization of cyclic AMP accumulation in intact heart cells.

Cyclic AMP accumulation in embryonic chick heart cells and binding of the beta-adrenergic antagonist 125I-pindolol to intact cells has been examined during the first 30 min of (-)-isoproterenol-induced desensitization. Myocardial beta-adrenergic receptors exist in two states which bind agonists with high (KD congruent to 10 nM) and low (KD congruent to 10 microM) affinities. Both activation and desensitization of cyclic AMP accumulation were mediated by (-)-isoproterenol binding to high affinity receptors. (-)-Isoproterenol-induced desensitization of cyclic AMP accumulation occurred with a t1/2 of 3.8 min. Desensitization was accompanied by a decrease in the number of 125I-pindolol binding sites assessed by equilibrium radioligand binding assays conducted at 4 degrees C or short (80 s) binding assays conducted at 37 degrees C. There was an excellent temporal correlation between loss of binding and loss of (-)-isoproterenol-stimulated cyclic AMP accumulation. After (-)-isoproterenol-induced desensitization, most of the remaining receptors assayed at 4 degrees C bound (-)-isoproterenol with high affinity. A rapid (-)-isoproterenol-induced decrease in the number of 125I-pindolol binding sites also occurred in adult canine heart cells and rat adipocytes. The data suggest that agonists do not cause uncoupling of surface receptors. Receptors may be uncoupled as a consequence of rapid sequestration into a hydrophobic compartment.

Replacement perfusion of cultured eucaryotic cells: a method for the accurate measurement of the rates of growth, protein synthesis, and protein turnover.

The fractional rates of protein synthesis (ks) and degradation (kp) were studied in the myeloma cell line SP2/0-AG14 grown at different rates (kg). Cells in spinner flask suspension cultures were maintained at constant cellular density for prolonged periods by replacement perfusion of labeling medium at a rate equivalent to the rate of growth. Total protein synthesis was calculated from the specific radioactivity of labeled L-leucine in the precursor (medium) and cellular protein. Fractional synthesis rates determined by approach to equilibrium labeling were the same as those determined by equilibrium-pulse labeling kinetics and pulse-chase kinetics. The rate of protein degradation was determined from the established relationship Kg = ks - kp. Protein synthesis rates remained constant over a threefold range in the rate of cell growth. At relatively slow growth rates (kg = 0.017/hr) turnover represented a major fraction of total synthesis (kp = 0.032/hr = 0.65ks). At rapid growth rates (kg = 0.058/hr) the value of kp was less than 0.005/hr. No major difference was observed between the ks determined for individual cellular proteins (separated by SDS-polyacrylamide (7.5%) gel electrophoresis) from rapid- and slow-growing cultures. Thus, with an invariable ks, any change in growth rate is due to an inverse change in the rate of turnover. Since turnover is the balance between synthesis and degradation and since synthesis is unchanging, then changes in the growth rate of SP2/0-AG14 should be due to changes in the rate of protein degradation. Experiments were therefore performed to determine the origin of the degradative machinery, ie, cytosolic or lysosomal; autolysis of prelabeled cellular protein (in vitro) was observed only at acidic pH (4.2) and was totally inhibited by addition of leupeptin (10 microM) and pepstatin (2 microM), the specific inhibitors of lysosomal cathepsins B (&L) and D, respectively. Since growth rate appears to be regulated by the alterations in the rate of protein degradation and degradation (in vitro) in SP2/0-AG14 appears to be lysosomal, then one should be able to alter the rate of cellular growth by interfering with rate of lysosomal proteolysis. Indeed, when the lysosomotropic amine NH4Cl (10 mM) is added to cells growing with a kg of 0.018/hr +/- 0.001 (ks = 0.050/hr +/- 0.002) the growth rate increased to 0.051/hr +/- 0.002 without change in the rate of protein synthesis (ks = 0.049/hr +/- 0.003).(ABSTRACT TRUNCATED AT 400 WORDS)

Ca2+-tolerant adult canine myocytes: preparation and response to anoxia/acidosis.

A procedure is described for the production of large numbers of Ca2+-tolerant adult canine ventricular myocytes. Gentle tissue dissociation is achieved in Spinner flasks using 320 mosM enzyme buffer containing collagenase hyaluronidase, and trypsin in the presence of millimolar levels of Ca2+. The technique allows for the complete removal of erythrocytes, the gentle removal of closely adhering nonmuscle cells (less than 3% contamination), and the selective removal of damaged from nondamaged myocytes. Total myocyte yields averaged 4-6 x 10(6)/g wet wt; 61.0 +/- 6.5% of the cells have rod-shaped morphology and are "viable" based on trypan blue exclusion. Only 3.9 +/- 1.1% of the rod-shaped cells beat spontaneously when challenged with 2 mM Ca2+. Exposure to 2 mM Ca2+ at 37 degrees C results in minimal loss of viability (2.1 +/- 1.3%/h) based on both trypan blue uptake and creatine kinase release. Simulation of cellular (i.e., membrane) injury in vitro is performed by the separate application of the perturbations of anoxia, acidosis, and low glucose to the canine myocyte suspensions; the data suggest that these myocytes are affected independently by these perturbations and are in good agreement with the results obtained by others using the isolated rat myocyte system.

Endogenous cathepsin B inhibitor activity in normal and myopathic red and white skeletal muscle.

Despite extensive biochemical and morphological studied on the degenerative muscle diseases, the primary chemical lesions are still obscure, both in humans and animals. In this report we examine the activities of the lysosomal endoproteinase cathepsin B and its endogenous inhibitor(s) in the red and white skeletal muscles of guinea pigs with nutritional muscular myopathy induced by vitamin E deficiency. We observed a twofold increase (P less than 0.005) in the activity of cathepsin B in the white skeletal muscles of the vitamin E-deficient (E-) animals over that of the normal (N) and control (E+) groups. Assessment of the activity of endogenous cathepsin B inhibitor revealed a one and a half times greater amount of inhibitor in N when compared to E-; this difference in inhibitor activity applied to both red (masseter) and white (medial head, gastrocnemius) muscle. When the specific activity of cathepsin B in the E-tissue was corrected for inhibitor activity, the corrected value was not significantly different from either the E+ or the N tissue.

Proteinases in cardiac and skeletal muscle.

Although changes in proteolysis in muscle tissue are now well documented for a variety of physiological and pathological conditions, the mechanism of degradation of cellular protein during normal protein turnover remains to be elucidated. Data from several laboratories have suggested the involvement of alkaline serine proteinases. Recent studies have questioned these results, and demonstrated that the serine proteinases are of mast cell origin and are not present in muscle cells. The only proteinases to date that have been shown to be present in muscle cells and capable of degrading myofibrillar proteins are Ca2+-activated proteinase, cathepsin B, and cathepsin D. Recent interest and developing awareness of endogenous enzyme inhibitors in cells may unmask many new enzymes.

Degradation of myofibrillar proteins by cathepsins B and D.

Cathepsin B and cathepsin D were purified from rat liver and skeletal muscle. Electrophoretic analyses revealed that the enzymes were highly purified, and isoelectric focusing demonstrated multiple forms of both enzymes. Purified actin and myosin, as well as actin and myosin in myofilaments and myofibrils, were degraded by the purified cathepsins B and D. Degradation of myosin was completely blocked by the cathepsin B and D inhibitors, leupeptin and pepstatin, respectively. Cathepsins B and D were visualized by electron microscopy, using CBZ-Ala- Arg-Arg-4-methoxy-beta-naphthylamine and BZ-Arg-Gly-Phe-Leu-4-methoxy-beta-naphthylamine as substrates.