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.

Effect of water temperature on a herpesvirus infection of sea turtles.

The role of water temperature in the induction and maintenance of a dermal herpesvirus infection (gray-patch disease) of young, green sea turtles (Chelonia mydas) was studied under carefully controlled experimental conditions, in which the influence of other recognized stress factors was negligible. A nimals that were subjected to a gradual temperature increase from 25 to 30 degrees C, with subsequent maintenance at 30 degrees C, and those that were abruptly shifted from water at 25 degrees C to water at 30 degrees C showed a significantly shorter period before the onset of clinical signs and an increase in the severity of the lesions when compared with control animals. Animals that were subjected to a gradual increase in water temperature from 25 to 30 degrees C and a subsequent decrease to 25 degrees C, where they were maintained, had a period before onset of clinical signs and severity closer to that of control animals. Our findings indicate that both the induction of clinical gray-patch disease and the severity of the lesions are affected by water temperature and suggest that one possible means of control of this herpesvirus infection under intensive aquaculture conditions might be water temperature manipulation.

Colostral immunoglobulin concentration in two fractions of first milking postpartum and five additional milkings.

Colostrum removed from mammary glands of parturient cows before letdown contained no greater concentration of immunoglobulins than colostrum collected after letdown and complete milking. Greater absorption of colostral immunoglobulins in calves suckling their dams initially postpartum over those hand fed by buckets or bottles is not from higher concentration of immunoglobulins in the gland and teat cistern. Colostral concentrations of three immunoglobulin classes (G, M, and A) obtained from one partial and six consecutive complete milkings at 12-h intervals postpartum decreased at different rates over time or number of milkings. Potential prophylactic value is discussed of continued lacteal secretion of immunoglobulins A and M available to the calf after intestinal closure to systemic absorption of colostral immunoglobulins.

Immunofluorescent localization of the Ca2+-dependent proteinase and its inhibitor in tissues of Crotalus atrox.

The native 108,000 dalton Ca2+-dependent proteinase (CDP) and its 115,000 dalton protein inhibitor (CDPI) were purified from bovine skeletal muscle using native polyacrylamide gel electrophoresis and were used to elicit antibody production in rabbits and BALB/c mice. Polyclonal antibodies were purified as IgG fractions by column chromatography; monoclonal antibodies were produced by the hybridoma technique. Indirect immunofluorescence localization of CDP and CDPI in tissues of Crotalus atrox show both proteins to be ubiquitous. Both occur in the cytoplasm and are absent from the cell membrane and the nucleus; CDPI is also present in the I-band of skeletal muscle.

Localization of the Ca(2+)-dependent proteinases and their inhibitor in normal, fasted, and denervated rat skeletal muscle.

Immunofluorescence and immunogold localization studies show that the two Ca(2+)-dependent proteinases (mu-calpain for the micromolar Ca(2+)-requiring proteinase and m-calpain for the millimolar Ca(2+)-requiring proteinase) and their protein inhibitor (calpastatin) are located exclusively intracellularly in normal rat soleus muscle. Quantitative immunogold studies indicate that binding of antibodies to both calpains and to calpastatin is approximately two times greater at the Z-disk of myofibrils than it is at the I-band or A-band regions. Mitochondria and nuclei in muscle cells contain both calpains and calpastatin at concentrations approximately one-tenth and one-fifth, respectively, of the concentration at the Z-disk, as estimated by antibody binding. Very little calpain or calpastatin was seen in the cytoplasmic intermyofibrillar spaces, and most of the calpain and calpastatin in muscle cells is associated with intracellular structures. Immunofluorescence results suggest that concentration of m-calpain but not mu-calpain or calpastatin is, in some instances, slightly higher near the intracellular surface of the plasma membrane than elsewhere in the muscle cell. Most m-calpain, however, is distributed throughout the interior of mature rat skeletal muscle cells. Denervation, or fasting and refeeding increases the concentration of the calpains and calpastatin in the muscle cell but does not change their distribution. Some mu- and m-calpain and calpastatin is found extracellularly in denervated soleus muscle or soleus muscle from fasting rats, but the extracellular calpains and calpastatin seem to originate from "leakage" of these proteins out of the cell because serum creatine kinase levels are much higher than normal in denervated or fasting rats.