Biochemical and developmental characterization of carbonic anhydrase II from chicken erythrocytes

Background  

Carbonic anhydrase (CA) of the chicken has attracted attention for a long time because it has an important role in the eggshell formation. The developmental profile of CA-II isozyme levels in chicken erythrocytes has not been determined or reported. Furthermore, the relations with CA-II in erythrocyte and egg production are not discussed. In the present study, we isolated CA-II from erythrocytes of chickens and determined age-related changes of CA-II levels in erythrocytes.     

Insulin Receptor Substrate-4 Binds to Slingshot-1 Phosphatase and Promotes Cofilin Dephosphorylation

Cofilin plays an essential role in cell migration and morphogenesis by enhancing actin filament dynamics via its actin filament-severing activity. Slingshot-1 (SSH1) is a protein phosphatase that plays a crucial role in regulating actin dynamics by dephosphorylating and reactivating cofilin. In this study, we identified insulin receptor substrate (IRS)-4 as a novel SSH1-binding protein. Co-precipitation assays revealed the direct endogenous binding of IRS4 to SSH1. IRS4, but not IRS1 or IRS2, was bound to SSH1. IRS4 was bound to SSH1 mainly through the unique region (amino acids 335–400) adjacent to the C terminus of the phosphotyrosine-binding domain of IRS4. The N-terminal A, B, and phosphatase domains of SSH1 were bound to IRS4 independently. Whereas in vitro phosphatase assays revealed that IRS4 does not directly affect the cofilin phosphatase activity of SSH1, knockdown of IRS4 increased cofilin phosphorylation in cultured cells. Knockdown of IRS4 decreased phosphatidylinositol 3-kinase (PI3K) activity, and treatment with an inhibitor of PI3K increased cofilin phosphorylation. Akt preferentially phosphorylated SSH1 at Thr-826, but expression of a non-phosphorylatable T826A mutant of SSH1 did not affect insulin-induced cofilin dephosphorylation, and an inhibitor of Akt did not increase cofilin phosphorylation. These results suggest that IRS4 promotes cofilin dephosphorylation through sequential activation of PI3K and SSH1 but not through Akt. In addition, IRS4 co-localized with SSH1 in F-actin-rich membrane protrusions in insulin-stimulated cells, which suggests that the association of IRS4 with SSH1 contributes to localized activation of cofilin in membrane protrusions.     

Design and synthesis of an orally active matrix metalloproteinase inhibitor

A series of 4-(4-phenoxy)benzoylamino-4-methoxymethyloxymethyl butyric acid hydroxamates, which were derived from l-glutamic acid, were synthesized and evaluated as matrix metalloproteinase inhibitors. Most of the compounds listed in exhibited strong inhibitory activity against MMP-2 and MMP-9, as well as even stronger inhibitory activity against MMP-3, but showed relatively weak inhibition of MMP-1. Structure-activity relationships are discussed.     

Primary structure of myosin heavy chain from fast skeletal muscle of Chum salmon Oncorhynchus keta

The nucleotide sequence of the cDNA encoding myosin heavy chain of chum salmon Oncorhynchus keta fast skeletal muscle was determined. The sequence consists of 5,994 bp, including 5,814 bp of translated region deducing an amino acid sequence of 1,937 residues. The deduced sequence showed 79% homology to that of rabbit fast skeletal myosin and 84-87% homology to those of fast skeletal myosins from walleye pollack, white croaker and carp. The putative binding-sites for ATP, actin and regulatory light-chains in the subfragment-1 region of the salmon myosin showed high homology with the fish myosins (78-100% homology). However, the Loop-1 and Loop-2 showed considerably low homology (31-60%). On the other hand, the deduced sequences of subfragment-2 (533 residues) and light meromyosin (564 residues) showed 88-93% homology to the corresponding regions of the fish myosins. It becomes obvious that several specific residues of the rabbit LMM are substituted to Gly in the salmon LMM as well as the other fish LMMs. This may be involved in the structural instability of the fish myosin tail region.     

Separation of Akazara scallop and rabbit troponin components by a single-step chromatography on CM-Toyopearl

Three components of Akazara scallop (Chlamys nipponensis akazara) troponin were well separated from each other by a single-step chromatography on CM-Toyopearl, although they were hardly separated on DEAE-Sephadex A-25. Moreover, by means of this CM-chromatography, the troponin components of rabbit were also readily separated with high purities and in high yields. The components thus separated were readily reconstituted and the Ca2+ regulatory function was fully recovered.     

Reversible changes in the ATPase activity and in the regulatory light chain content upon heat (30 degrees C)-treatment of "Akazara" striated adductor myosin

Myosin from striated adductor muscle of "Akazara" scallop was incubated at 30 degrees C for 5 min in a medium containing 2 mM MgCl2 and various concentrations of Ca2+ ions. It was observed that the 30 degrees C-treatment resulted in a decrease in the Ca2+-sensitivity of myosin-ATPase as well as in the release of the regulatory light chain (EDTA-LC) of myosin. The 30 degrees C-treated myosin was then subjected to a cooling treatment, being kept for 18 h at 0 degrees C. It was found that EDTA-LC recombined with myosin and that Ca2+-sensitivity of myosin-ATPase was restored. It was also found that Ca2+ alone was about 70 times more effective than Mg2+ alone in preventing the heat-induced release of EDTA-LC from occurring and also in recombination of EDTA-LC with the heat-treated myosin.