The effectiveness of clove extracts in the inhibition of hydroxyl radical oxidation-induced structural and rheological changes in porcine myofibrillar protein.

Oxidation is a major cause of protein quality deterioration during the storage and processing of food. This study investigated the effects of clove extract (CE) on structural and rheological changes in porcine longissimus myofibrillar proteins (MP) and the effects of oxidizing radicals produced by a Fenton reaction system (FRS). Increased oxidation time was accompanied by increased carbonyl content, reduced Ca-ATPase activity, decreased enthalpy of denaturation, decreased thermal transition temperatures (P<0.05), and increased protein susceptibility to thermal aggregation. The addition of CE significantly inhibited carbonyl formation (P<0.05), enhanced solubility and thermal stability, and improved the gel formation ability (storage modulus, loss modulus) of MP. The protective effect of CE on protein denaturation was demonstrated by its efficacy in maintaining Ca-ATPase activity and decreasing the degree of protein aggregation. Overall, the hydroxyl radical-induced loss of the structural and functional properties of MP was significantly reduced by the presence of CE.

Amino acid and mineral composition of protein and other components and their recovery yields from whole Antarctic krill (Euphausia superba) using isoelectric solubilization/precipitation.

Proteins and insolubles were recovered from whole Antarctic krill via novel isoelectric solubilization/precipitation using different pH treatments. The protein recovery yield was 45% to 50% (dry basis). The recovered proteins had higher (P < 0.05) content of essential amino acids (EAAs) and non-EAAs as well as higher (P < 0.05) ratio of total EAA/total AA than whole krill. The EAAs constituted almost 50% of total AAs. The least extreme pH treatments (pHs 3 and 12) yielded highest (P < 0.05) content of EAAs. The quality of recovered proteins was high based on EAAs meeting FAO/WHO/UNU recommendations for adults and infants. The basic pH yielded proteins with the lowest (P < 0.05) amount of minerals and the highest (P < 0.05) amount of Ca, P, and Mg in the insolubles when compared to the acidic treatments. However, both basic and acidic treatments effectively removed minerals from recovered proteins without the removal of the exoskeleton before processing. Therefore, besides high-quality proteins, the insolubles may provide a mineral supplement in the animal diet.

Purification of novel kinesins from embryonic systems.

Several kinesin holoenzymes, including the heterotrimeric kinesin-II and bipolar KLP61F complexes described here, are being purified in our laboratory using microtubule affinity precipitation and conventional biochemical fractionation procedures. These protocols have been optimized by using pan-kinesin peptide antibodies and subunit-specific antibodies to monitor the enrichment of kinesin-related polypeptides in particular fractions by immunoblotting. Protein purification represents the most direct route available for determining the oligomeric state and subunit composition of a kinesin holoenzyme, for identifying tightly associated accessory subunits such as SpKAP115, and for determining the molecular architecture and functional properties of native kinesin motors. Protein purification methods therefore represent an important complementary approach to molecular genetic approaches that are being pursued in many other laboratories.

Sequence and submolecular localization of the 115-kD accessory subunit of the heterotrimeric kinesin-II (KRP85/95) complex.

The heterotrimeric kinesin-II holoenzyme purified from sea urchin (Strongylocentrotus purpuratus) eggs is assembled from two heterodimerized kinesin-related motor subunits of known sequence, together with a third, previously uncharacterized 115-kD subunit, SpKAP115. Using monospecific anti-SpKAP115 antibodies we have accomplished the molecular cloning and sequencing of the SpKAP115 subunit. The deduced sequence predicts a globular 95-kD non-motor "accessory" polypeptide rich in alpha-helical segments that are generally not predicted to form coiled coils. Electron microscopy of individual rotary shadowed kinesin-II holoenzymes also suggests that SpKAP115 is globular, with a somewhat asymmetric morphology. Moreover, the SpKAP115 subunit lies at one end of the 51-nm-long kinesin-II complex, being separated from the two presumptive motor domains by a approximately 26-nm-long rod, in a manner similar to the light chains (KLCs) of kinesin itself. This indicates that SpKAP115 and the KLCs may have analogous functions, yet SpKAP115 does not display significant sequence similarity with the KLCs. The results show that kinesin and kinesin-II are assembled from highly divergent accessory polypeptides together with kinesin related motor subunits (KRPs) containing conserved motor domains linked to divergent tails. Despite the lack of sequence conservation outside the motor domains, there is striking conservation of the ultrastructure of the kinesin and kinesin-II holoenzymes.

Kinesin-related proteins in the mammalian testes: candidate motors for meiosis and morphogenesis.

The kinesin superfamily of molecular motors comprises proteins that participate in a wide variety of motile events within the cell. Members of this family share a highly homologous head domain responsible for force generation attached to a divergent tail domain thought to couple the motor domain to its target cargo. Many kinesin-related proteins (KRPs) participate in spindle morphogenesis and chromosome movement in cell division. Genetic analysis of mitotic KRPs in yeast and Drosophila, as well as biochemical experiments in other species, have suggested models for the function of KRPs in cell division, including both mitosis and meiosis. Although many mitotic KRPs have been identified, the relationship between mitotic motors and meiotic function is not clearly understood. We have used sequence similarity between mitotic KRPs to identify candidates for meiotic and/or mitotic motors in a vertebrate. We have identified a group of kinesin-related proteins from rat testes (termed here testes KRP1 through KRP6) that includes new members of the bimC and KIF2 subfamilies as well as proteins that may define new kinesin subfamilies. Five of the six testes KRPs identified are expressed primarily in testes. Three of these are expressed in a region of the seminiferous epithelia (SE) rich in meiotically active cells. Further characterization of one of these KRPs, KRP2, showed it to be a promising candidate for a motor in meiosis: it is localized to a meiotically active region of the SE and is homologous to motor proteins associated with the mitotic apparatus. Testes-specific genes provide the necessary probes to investigate whether the motor proteins that function in mammalian meiosis overlap with those of mitosis and whether motor proteins exist with functions unique to meiosis. Our search for meiotic motors in a vertebrate testes has successfully identified proteins with properties consistent with those of meiotic motors in addition to uncovering proteins that may function in other unique motile events of the SE.

Selenoprotein W of rat muscle binds glutathione and an unknown small molecular weight moiety.

When purified from rat muscle, selenoprotein W is fractionated into four forms distinguished by slightly different chromatographic behavior. Precise masses of the four forms were determined by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. The mass distribution of the forms (9549, 9592, 9853, and 9898 d) suggests that they occur through derivatization of the lowest mass form with two moieties of approximate masses 44 and 305 d. The apparent 305 d moiety was demonstrated to be glutathione (307 d) by reductive release from the 9853 d protein form with 1000-fold excess of dithiothreitol at 50 degrees C. Milder conditions failed to remove the glutathione. The reduction produced nearly stoichiometric amounts of free glutathione as determined by HPLC of a fluorometric derivative. HPLC retention of the protein changed to match that of the 9549 d form, and a change of mass to 9550 d was observed by MALDI mass spectrometry. The identity of the 44 d moiety is unknown. The presence of glutathione in isolated selenoprotein W may suggest its involvement in the metabolism of this tripeptide.

Association of sorcin with the cardiac ryanodine receptor.

Sorcin is a 22-kDa calcium-binding protein initially identified in multidrug-resistant cells; however, its patterns of expression and function in normal tissues are unknown. Here we demonstrate that sorcin is widely distributed in rodent tissues, including the heart, where it was localized by immunoelectron microscopy to the sarcoplasmic reticulum. A > 500-kDa protein band immunoprecipitated from cardiac myocytes by sorcin antiserum was indistinguishable in size on gels from the 565-kDa ryanodine receptor/calcium release channel recognized by ryanodine receptor-specific antibody. Association of sorcin with a ryanodine receptor complex was confirmed by complementary co-immunoprecipitations of sorcin with the receptor antibody. Forced expression of sorcin in ryanodine receptor-negative Chinese hamster lung fibroblasts resulted in accumulation of the predicted 22-kDa protein as well as the unexpected appearance of ryanodine receptor protein. In contrast to the parental host fibroblasts, sorcin transfectants displayed a rapid and transient rise in intracellular calcium in response to caffeine, suggesting organization of the accumulated ryanodine receptor protein into functional calcium release channels. These data demonstrate an interaction between sorcin and the ryanodine receptor and suggest a role for sorcin in modulation of calcium release channel activity, perhaps by stabilizing the channel protein.

Electrical currents generated by a partially purified Na/Ca exchanger from lobster muscle reconstituted into liposomes and adsorbed on black lipid membranes: activation by photolysis of Ca2+.

The Na/Ca exchanger from lobster muscle crossreacts specifically with antibodies raised against the dog heart Na/Ca exchanger. Immunoblots of the lobster muscle and mammalian heart exchangers, following SDS-PAGE, indicate that the invertebrate and mammalian exchangers have similar molecular weights: about 120 kDa. The exchanger from lobster muscle was partially purified and functionally reconstituted into asolectin vesicles which were loaded with 160 mM NaCl. 45Ca uptake by these proteoliposomes was promoted by replacing 160 mM NaCl in the external medium with 160 mM KCl to produce an outwardly-directed Na+ concentration gradient. When the proteoliposomes were adsorbed onto black lipid membranes (BLM), and DM-Nitrophen-Ca2+ ("caged Ca2+") was added to the KCl medium, photolytically-evoked Ca2+ concentration jumps elicited transient electric currents. These currents corresponded to positive charge exiting from the proteoliposomes, and were consistent with the Na/Ca exchanger-mediated exit of 3 Na+ in exchange for 1 entering Ca2+. The current was dependent upon the Ca2+ concentration jump, the protein integrity, and the outwardly directed Na+ gradient. KCl-loaded proteoliposomes did not produce any current. Low external Na+ concentrations augmented the current, whereas Na+ concentrations > 25 mM reduced the current. The dependence of the current on free Ca2+ was Michaelis-Menten-like, with half-maximal activation (KM(Ca)) at < 10 microM Ca2+. Caged Sr2+ and Ba2+, but not Mg2+, also supported photolysis-evoked outward current, as did Ni2+, but not Mn2+. However, Mg2+ and Mn2+ augmented the Ca-dependent current, perhaps by facilitating the adsorption of proteoliposomes to the BLM. The Ca-dependent current was irreversibly blocked by La3+ (added as 200 microM DMN-La3+). The results indicate that the properties of the Na/Ca exchanger can be studied with these electro-physiological methods.

Identification and characterization of myophilin, a muscle-specific antigen of Echinococcus granulosus.

A muscle-specific gene of Echinococcus granulosus has been identified and characterized. A lambda gt11 clone (10P1), containing an incomplete copy of the gene, was originally isolated from a larval E. granulosus cDNA library by serum antibodies from dogs infected with the parasite. The full-length cDNA sequence was obtained by PCR amplification of cDNA from an adult E. granulosus lambda gt22A library. Southern blot analysis indicated the presence of the gene as a single copy in the genome of E. granulosus and also detected homologous genes in genomic DNA of E. multilocularis and Taenia saginata. The 21.2-kDa protein deduced from the complete cDNA sequence contains two regions of 12 amino acids with similarity to the EF-hand motif of calcium binding proteins. Antibodies raised against the purified 10P1-GST fusion protein detected a 22-kDa antigen in the E. granulosus developmental stages examined. Immunoelectron microscopy localized the native protein in the muscle of the parasite. The amino-acid sequence of the E. granulosus protein shows significant homology to the muscle proteins mp20 of Drosophila melanogaster, chicken SM22 alpha and mammalian calponin, and also to the neuronal protein NP25 of rats. A conserved carboxy-terminal motif of 17 amino acids is present in all the homologous proteins and is proposed to be the characteristic feature of a novel protein family. The term myophilin is proposed for the E. granulosus protein due to its localization and homology to other muscle proteins.

A novel EF-hand Ca(2+)-binding protein from abdominal muscle of crustaceans with similarity to calcyphosine from dog thyroidea.

The amino acid sequence of a novel EF-hand Ca(2+)-binding protein from the abdominal muscle of the crayfish, Orconectes limosus, has been elucidated by tandem mass spectrometry and automated Edman degradation. The name CCBP-23 (23-kDa crustacean Ca(2+)-binding protein) is proposed. The protein can also exist as a disulfide-linked homodimer. The sequence of the monomeric form spans 200 residues with an acetylated N-terminal Ser and reveals four EF-hand domains. The 174-mass-unit difference between the calculated average molecular mass of 22,669.6 Da deduced from the sequence and the obtained electrospray ionization mass spectroscopy (ESI-MS) mass of 22,844 Da has not yet been explained. Partial sequence analysis (137 residues) of CCBP-23 from the lobster, Homarus americanus, showed a sequence identity of 74% with the crayfish protein. Homology searches revealed a 44% sequence identity of CCBP-23 from crayfish to calcyphosine, a Ca(2+)-binding protein from dog thyroidea (Lefort et al., 1989). Although CCBP-23 also shows a 44% identity to R2D5 antigen (Nemoto et al., 1993), we believe that both proteins represent two distinct subgroups within the family of EF-hand proteins.

Biochemical and molecular characterization of the chicken cysteine-rich protein, a developmentally regulated LIM-domain protein that is associated with the actin cytoskeleton.

LIM domains are present in a number of proteins including transcription factors, a proto-oncogene product, and the adhesion plaque protein zyxin. The LIM domain exhibits a characteristic arrangement of cysteine and histidine residues and represents a novel zinc binding sequence (Michelsen et al., 1993). Previously, we reported the identification of a 23-kD protein that interacts with zyxin in vitro (Sadler et al., 1992). In this report, we describe the purification and characterization of this 23-kD zyxin-binding protein from avian smooth muscle. Isolation of a cDNA encoding the 23-kD protein has revealed that it consists of 192 amino acids and exhibits two copies of the LIM motif. The 23-kD protein is 91% identical to the human cysteine-rich protein (hCRP); therefore we refer to it as the chicken cysteine-rich protein (cCRP). Examination of a number of chick embryonic tissues by Western immunoblot analysis reveals that cCRP exhibits tissue-specific expression. cCRP is most prominent in tissues that are enriched in smooth muscle cells, such as gizzard, stomach, and intestine. In primary cell cultures derived from embryonic gizzard, differentiated smooth muscle cells exhibit the most striking staining with anti-cCRP antibodies. We have performed quantitative Western immunoblot analysis of cCRP, zyxin, and alpha-actinin levels during embryogenesis. By this approach, we have demonstrated that the expression of cCRP is developmentally regulated.

Sodium dodecyl sulfate and heat induce two distinct forms of lobster muscle multicatalytic proteinase: the heat-activated form degrades myofibrillar proteins.

A multicatalytic proteinase (MCP) purified from lobster claw and abdominal muscles degrades a variety of peptide and protein substrates. The enzyme is activated by low concentrations (0.03%) of sodium dodecyl sulfate (SDS) and brief (1 min) heating at 60 degrees C. The lobster MCP can assume three stable and functionally distinct states in vitro; these are classified as the basal, heat-activated, and SDS-activated forms. The basal MCP possessed high trypsin-like peptidase activity and low chymotrypsin-like peptidase, peptidylglutamyl-peptide hydrolase, and caseinolytic activities; incubation of the basal form with SDS stimulated the peptidylglutamyl-hydrolase activity about 30-fold and inhibited the other three activities 80% to 100%. Heating the basal form stimulated caseinolytic activity about 6-fold with little effect on the peptidase activities. The heat-activated enzyme also degraded myosin, tropomyosin, troponin, and actin depolymerizing factor; alpha-actinin was resistant to proteolysis. Incubation of the heat-activated MCP with SDS inhibited the trypsin-like, chymotrypsin-like, and proteinase activities 95 to 100% and stimulated the peptidylglutamyl-hydrolase activity about 16-fold. Incubation of myosin with either the basal or the heat-activated forms in the presence of SDS generated identical proteolytic fragments of the myosin heavy chain, suggesting that SDS induced a third form that can be produced from either the basal or the heat-activated forms. The heat-activated form produced proteolytic fragments of myosin heavy chain different from those generated by either basal or heat-activated enzymes in the presence of SDS. Furthermore, 100 mM KCl stimulated the caseinolytic activity of the heat-activated form 24% and inhibited the trypsin-like and peptidylglutamyl-hydrolase activities 56 and 20%, respectively. These results, though indirect, suggest that heating induced a proteinase activity that was distinct from the three peptidase activities. Activation of the basal form with SDS was reversible, since precipitation of dodecyl sulfate with 100 mM KCl restored trypsin-like activity and inhibited peptidylglutamyl-hydrolase activity. In contrast, removal of dodecyl sulfate from the SDS-activated form that was derived from the heat-activated MCP induced its conversion to the basal form. Thus, although heat-activation was irreversible, the heat-activated form was converted back to the basal form via the SDS-activated form.

Trypsin digestion of junctional sarcoplasmic reticulum vesicles.

A putative constituent of the junctional processes, connecting the terminal cisternae of sarcoplasmic reticulum and the transverse tubules of skeletal muscle fibers, is a greater than or equal to 350,000-dalton (Da) protein that displays ryanodine binding and Ca2+ channel properties. Ryanodine modulation of Ca2+ fluxes suggests that the ryanodine receptor and calcium channel are integral parts of one functional unit corresponding to the greater than or equal to 350,000-Da protein [Inui, M., Saito, E., & Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747; Campbell, K. P., Knudson, C. M., Imagawa, T., Leung, A. L., Sutko, J. L., Kahl, S. D., Raab, C. R., & Madson, L. (1987) J. Biol. Chem. 262, 6460-6463]. We subjected vesicular fragments of junctional-cisternal membrane to stepwise trypsin digestion. The greater than or equal to 350,000-Da protein is selectively cleaved in the early stage of digestion, with consequent disappearance of the corresponding band in electrophoretic gels. The Ca2+-ATPase is cleaved at a later stage, while calsequestrin is not digested under the same experimental conditions. While the Ca2+-ATPase yields two complementary fragments that are relatively resistant to further digestion, the greater than or equal to 350,000-Da protein yields fragments that are rapidly broken down to small peptides. Under conditions producing extensive digestion of the greater than or equal to 350,000-Da protein, the junctional processes are still visualized by electron microscopy, with no discernible alterations of their ultrastructure. The functional properties of the Ca2+ release channel are also maintained following trypsin digestion, including blockage by Mg2+ and ruthenium red and activation by Ca2+ and nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative studies of light meromyosin paracrystals derived from red, white, and cardiac muscle myosins.

Tryptic and chymotryptic light meromyosin paracrystals from red and cardiac muscles of rabbit show a negative and positive staining pattern with uranyl acetate and phosphotungstate that sharply differs from that of white muscle light meromyosin paracrystals. The main periodicity of about 430 A is the same regardless of the source of light meromyosin. The results are discussed in terms of the molecular structure and the functional properties of various myosins.