Heterogeneity of insulin-like growth factor binding proteins in swine.

Proteins in porcine amniotic fluid and sera (both fetal and adult) were separated electrophoretically in sodium dodecyl sulfate-containing polyacrylamide gels and transferred to nitrocellulose sheets. Western blots were analysed for proteins that would bind (a) radioiodinated insulin-like growth factor-I (IGF-I) and (b) antibodies to a rat insulin-like growth factor binding protein. Multiple insulin-like growth factor binding proteins were identified in sera. The binding proteins ranged in size from Mr 192,000 to 26,000. One immunologically cross-reactive protein (Mr 36,000) was detected. No binding proteins were detected routinely in amniotic fluids. Sera from adult swine were fractionated by preparative isoelectric focusing. Two binding proteins (Mr 192,000, 46,000) were located in acidic fractions which also contained IGF-I and IGF-II. Two binding proteins (Mr 36,000, 26,000) were located in neutral to basic fractions which contained primarily IGF-II. Immunoglobulin-sized material from adult sera fractionated over Sephadex G-200 contained two binding proteins (Mr 46,000, 42,000) whereas albumin-sized material contained one (Mr 36,000). Porcine insulin-like growth factor binding proteins are as heterogeneous as those from humans.

Calpain II involvement in mitosis.

Mitotic spindle disassembly requires major structural alterations in the associated cytoskeletal proteins and mitosis is known to be associated with Ca2+-sequestering phenomena and calcium transients. To examine the possible involvement of a ubiquitous Ca2+-activated protease, calpain II, in the mitotic process, synchronized PtK1 cells were monitored by immunofluorescence for the relocation of calpain II. The plasma membrane was the predominant location of calpain II in interphase. However, as mitosis progressed, calpain II relocated to (i) an association with mitotic chromosomes, (ii) a perinuclear location in anaphase, and (iii) a mid-body location in telophase. Microinjection of calpain II near the nucleus of a PtK1 cell promoted the onset of metaphase. Injection of calpain II at late metaphase promoted a precocious disassembly of the mitotic spindle and the onset of anaphase. These data suggest that calpain II is involved in mitosis.

Effect of post-mortem storage on Ca(++)-dependent proteases, their inhibitor and myofibril fragmentation.

Post-mortem changes in two calcium-dependent proteases, their inhibitor, myofibril fragmentation index (MFI) and collagen (amount and solubility) were studied. Whereas the activity of high Ca(++)-requiring calcium-dependent protease (CDP-II) remained nearly constant throughout post-mortem storage, there was a progressive decrease in the activities of low Ca(++)-requiring calcium-dependent protease (CDP-I) and their specific inhibitor, with the inhibitor being the most susceptible to post-mortem storage. Results indicated that the greatest changes in MFI occur within the first 24 h of post-mortem storage. There were no detectable changes in either total or soluble collagen content with post-mortem storage. Hence, it was concluded that improvement in tenderness resulting from post-mortem storage must be derived from changes in the myofibrils and since CDP-I activities paralleled the myofibrillar changes, it seems reasonable to suggest that CDP-I, not CDP-II, plays an important role in the fragmentation of myofibrils and consequently in improvement of meat tenderness resulting from post-mortem storage.

The reversible inhibition of myoblast proliferation by gamma-hexachlorocyclohexane.

The effects of treating non-fusing myoblast variants, fu-1 and M3A, with two levels (1 X 10(-4) M and 2 X 10(-4) M) of gamma-hexachlorocyclohexane, an inhibitor of phosphatidylinositol synthesis, on myoblast proliferation were evaluated by measuring myoblast proliferation (counting cells) and visual inspection via phase microscopy. In the presence of gamma-hexachlorocyclohexane, these cells were arrested, presumably in G1. The inability of these cells to replicate did not appear to be due to a toxic effect of gamma-hexachlorocyclohexane, because these cells were capable of resuming proliferation once they were transferred to media lacking gamma-hexachlorocyclohexane. Cells were grown in media containing myo-[2-3H]inositol and the radioactive content of water-soluble metabolites, the end product of phosphatidylinositides hydrolysis, was quantitated. Cells were grown in the presence of gamma-hexachlorocyclohexane, in addition to the loss of proliferative ability, also contained significantly less water-soluble metabolites. Therefore, it appears that there is a direct relationship between phosphatidylinositol metabolism and cell proliferation in the cell lines studied.

Identification of calpain II in porcine sperm.

The role that proteolytic enzymes may play in membrane-associated phenomena of sperm has been the subject of extensive investigation. In the present study, we have examined the possibility that a Ca2+-activated, neutral protease, calpain II, may be associated with sperm membranes. Using indirect immunofluorescence with primary antibodies, which are polyclonal and monoclonal antibodies directed against the 80 kDa subunit of calpain II, we have established the presence of this antigen in porcine sperm. Staining by anticalpain II (80 kDa subunit) of the apical segment of the acrosomal cap and basal body (centriolar) region was seen consistently. Variable staining of the sperm tail was also observed. These observations, combined with our positive identification of a 80 kDa protein in acrosomal membranes (via immunoblot), document the association of this protease with sperm membranes. The proximity of calpain II to the acrosome suggests a potential role for the protease in the Ca2+-mediation of the acrosome reaction.

Possible role of calpain I and calpain II in differentiating muscle.

The variable distribution of the 80-kD subunit of two calcium-activated proteases, calpain I and calpain II, has been examined in L8 and L6 myoblasts, and their non-fusing variants, fu-1 and M3A using non-cross-reacting monoclonal antibodies to both subunits. Immunofluorescence results have shown that while the 80-kD subunit of calpain I is localized in the cytoplasm of all the myoblasts, the 80-kD subunit of calpain II appears to be predominantly associated with the plasma membranes of L8 and L6 myoblasts. The distribution of the 80-kD subunit of calpain II in non-fusing myoblasts, fu-1 and M3A, is generally cytoplasmic and diffuse. Immunoblot analysis of membrane and cytosol fractions of all the myoblasts using the monoclonal antibodies described above essentially confirms the immunofluorescence findings. Because calpain II exhibits a peripheral distribution in cells which are fusion-competent, L6 and L8 myoblasts, but not in fu-1 and M3A myoblasts, we suggest that calpain II may play a role in the Ca2+-mediated fusion events of differentiating (prefusion) myoblasts.

Role of Ca2+ and Ca2+-activated protease in myoblast fusion.

In this report, we have examined the effects of a calcium chelator, EGTA, and a calcium ionophore, A23187, on fusion of a cloned muscle cell line, L6. Our results confirm that EGTA essentially blocks all myoblast fusion because the lateral alignment of presumptive myoblasts cannot occur in the absence of extracellular calcium. A23187, however, promotes the precocious fusion of myoblasts, apparently by facilitating Ca2+ transport into myoblasts. We have also demonstrated that a Ca2+-activated protease, CAF (mM), appears to relocate in response to the Ca2+ flux, changing from a random, dispersed distribution in proliferative myoblasts to a predominantly peripheral distribution in prefusion myoblasts. Coincident with the mM CAF relocation is an altered distribution of a surface glycoprotein, fibronectin. Extracellular fibronectin is seen in abundance in proliferating myoblasts, but is essentially absent from the surface of fusing myoblasts. We suggest that mM CAF when activated by Ca2+ influx may act to promote the release of fibronectin from the myoblast cell surface, thus providing a mechanism by which the membrane of the fusing myoblast may be rearranged to accommodate fusion.