Demonstration of a high affinity Ca2+ ATPase in rat liver plasma membranes

Rat liver plasma membranes contained a high affinity Ca²⁺-ATPase which had an apparent half saturation constant of 0.2 μM for calcium. The Ca²⁺-ATPase was not stimulated by adding magnesium and/or calmodulin. Conversely, the addition of these substances diminished the calcium-stimulation of the ATPase. Orthovanadate (7 nM-2 mM), mitochondrial ATPase blockers (NaN₃, KCN, dicyclohexylcarbodiimide), Na⁺, K⁺ and ouabain had no effect on the ATPase activity. The ATPase was separated from nonspecific divalent cation stimulatable ATPase (Mg²⁺-ATPase) by solubilization with Triton X-100 followed by a Sephadex G-200 column chromatography and showed an apparent molecular weight of 200,000.     

β-structure from the microfibillar proteins of Merino wool

The β-structure of S-caboxymethyl derivatives of microfibrillar proteins isolated from Merino wool was investigated by X-ray diffraction for comparison with the structur of β-keratin. The S-carboxymethylated microfibrillar proteins(SCMKA) w well-oriented β-films of SCMKA weer obtained by stretching the SCMKA cast films in steam up to about 300% extesnsion. It was found that the reflections in β-pattern of SCMKA may be indexed on a pseudo-orthorhombic unit cell with a =0.94 nm, b = 0.66 nm and c = nm, where the ab, and c axes are in the direction of the interchain hydrogen bonding, the main chain(fibre axis) and the side chain, respectively. The unit cell dimesnions evaluated for SCMKA were almost the same as those for β-keratin, suggeting that few peptide sequences containing S-carboxymethyl cystine may be involved in the formation of β-structure from SCMKA.     

Isolation and characterization of visible light-sensitive mutants of Escherichia coli K12

Six mutants of Escherichia coli K12 that are sensitive to visible light have been isolated. Five of them, including an amber mutant, are defective in a gene that maps near 11 minutes on the linkage map of the chromosome, and this gene has been designated visA. The sixth mutant, which was isolated from bacteria that carried the visA+/visA+ diploid allele, is defective in a gene that maps near 63 minutes on the linkage map, which has been designated visB. These mutant strains of bacteria are killed by illumination with visible light. The effective wavelength of the light is around 460 nm. The nucleotide sequence of the visA gene was determined. As a result of a search for homologous products, we found that visA may be identical to hemH, the structural gene for ferrochelatase which catalyzes a final step in the biosynthesis of heme. A possible mechanism for the killing of the visA mutant bacteria is discussed.     

Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.

Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.     

A DBL-homologous region of the yeast CLS4/CDC24 gene product is important for Ca(2+)-modulated bud assembly.

The CLS4/CDC24 is essential for the budding process of the yeast Saccharomyces cerevisiae. Disruption of the CLS4/CDC24 gene is lethal, and expression of the CLS4 product under the control of the GAL1 promoter is sufficient for cellular growth. The CLS4 product is detected in yeast cell lysate with an apparent molecular mass of 93 kD (854 amino acid residues) and shows homology with the human DBL oncogene product. Temperature-sensitive cdc24-1 mutation is located in the N-terminal portion of the protein whereas Ca(2+)-sensitive cls4-1 mutation is present after the DBL-homologous region (amino acid residues 281-518) near the putative Ca(2+)-binding site. Mutations within the DBL-homologous region are responsible for the Ca(2+)-sensitive phenotype. Thus the CLS4 gene product seems to have several functional domains within the molecule essential for bud assembly.     

Primate's p53 inhibits SV40 DNA replication in vitro

Previous reports indicated that rodent p53 inhibits simian virus 40 (SV40) DNA replication in vitro as well as in vivo while that from primate cells does not (1-4). Here we report the evidence that p53 of primate origin also inhibits SV40 DNA replication in vitro. p53-SV40 large tumor antigen (T antigen) complex purified from SV40 infected COS-1 cells had little replication activity and inhibited SV40 DNA replication in vitro. These results suggest that inhibition of SV40 DNA replication by p53 should be regarded as general property of the protein and does not determine the mode of species specific replication of SV40 DNA.     

Elastic behavior of zymogen granule membranes in response to changes in pH and pCa.

In the process of secretion, the membrane of secretory granules is expected to change its elastic behavior. Elastic modulus of the membrane of zymogen granules, prepared from the rat pancreas acinar cell, was measured by an osmotic swelling method. The elastic modulus of the granule membrane at pCa 8 reduced from the maximal value of 230 dyn/cm at pH 6.0 to almost zero at pH 7.5. In a cytosol of an acinar cell, calcium ions play an important role as a second messenger in secretion. The elastic modulus of the granule membrane reduced in a sigmoidal fashion at pCa between 7.0 and 6.0. This range of pCa corresponds to a physiological rise of free Ca2+ concentrations in the cell cytosol when stimulated by external secretagogues. Reduction of the elastic modulus indicates that the state of the granule membrane switches to a more flexible one in which the granule is easy to appose to the cell plasma membrane and then swell as a final step of exocytosis.     

Hydrolysis and transport of proline-containing peptides in renal brush-border membrane vesicles from dipeptidyl peptidase IV-positive and dipeptidyl peptidase IV-negative rat strains

In this investigation, we have demonstrated that the renal brush-border membrane of Fischer 344 rats from the Japanese Charles River Inc. specifically lacks dipeptidyl peptidase IV (DPP IV) activity, whereas the renal brush-border membrane of Fischer 344 rats from three different sources within the United States possesses normal levels of DPP IV activity. Comparison of the brush-border proteins between Charles River (U.S.A.) Fischer 344 rats (DPP IV positive) and Japanese Charles River Fischer 344 rats (DPP IV negative) revealed that a protein band (Mr = 100,000), apparently identical with DPP IV, was absent in the membranes from Japanese Charles River Fischer 344 rats. We examined the handling of radiolabeled beta-casomorphin fragment 1-5 (Tyr-Pro-[3H]Phe-Pro-Gly), a specific substrate for DPP IV, in renal brush-border membrane vesicles isolated from DPP IV-positive and DPP IV-negative rats. Although the membrane vesicles from DPP IV-positive rats were able to hydrolyze the pentapeptide to di- and tripeptides with the subsequent active transport of these products via the H+ gradient-dependent peptide transport system, the membrane vesicles from DPP IV-negative rats failed to hydrolyze the pentapeptide and hence lacked the ability to transport the radiolabel actively from the parent peptide. The H+ gradient-dependent glycyl-sarcosine uptake and the Na+ gradient-dependent proline uptake, however, were normal in DPP IV-negative rats. Urine analysis revealed that the DPP IV-negative rats excreted proline- and hydroxyproline-containing peptides in significantly increased amounts in their urine compared with control rats. Furthermore, following intravenous administration of Tyr-Pro-Phe-Pro-NH2, a peptide that is exclusively hydrolyzed by DPP IV, urinary excretion of the peptide in the intact form was many-fold greater in DPP IV-negative rats than in control rats. These data provide conclusive evidence for the obligatory role of DPP IV in the renal handling of proline (and hydroxyproline)-containing peptides.     

A new electrophoretic method for the separation of disaccharides obtained by digestion of chondroitin sulfates and dermatan sulfate with chondroitinases

A new rapid and simple method has been developed for the separation of disaccharides obtained by chondroitinase digestion of chondroitin sulfates and dermatan sulfate using electrophoresis on cellulose acetate plates (Titan III cellulose acetate plates). Three disaccharides are completely separated by electrophoresis in barium acetate or calcium acetate in a short time, and less than 50 μg of glycosaminoglycan samples can be analyzed within 2 h.     

Occurrence of Two Types of Ca2+-Dependent Protein Kinases in the Cytosol Fraction of the Brain

Abstract— Two types of Ca²⁺-dependent protein kinases were demonstrated and partially purified from the cytosol fraction of rat brain by DEAE-cellulose, Sephadex G-200, and calmodulin-affinity column chromatography, using endogenous proteins and chicken gizzard myosin light chains as substrates. The molecular weights of the enzymes were 88,000 (peak I) and 120,000 (peak II) on gel filtration. Peak I had no affinity for calmodulin, whereas peak II had a high affinity for it, with a K _a value of 16.7 n m . The K _a values of peaks I and II for Ca²⁺ were 2.4 and 1.6 μ m , respectively.