Isolation and characterization of des(Ala-Lys)calmodulin in porcine brain

A calmodulin-like protein -des(Ala-Lys)calmodulin- was isolated from porcine brain extract, and was characterized in comparison to porcine brain calmodulin. Des(Ala-Lys)calmodulin was distinguishable from calmodulin by its slightly faster mobility in 10% polyacrylamide gels without sodium dodecyl sulfate. The protein gave an amino acid composition very similar to calmodulin, and contained one ?-N-trimethyllysyl residue. Comparative peptide mapping of calmodulin and des(Ala-Lys)calmodulin by high performance anion-exchange liquid chromatography, and the subsequent analyses of the isolated peptides, have indicated that des(Ala-Lys)calmodulin lacks the Ala(147)-Lys (148) sequence at the C-terminus of calmodulin. The content of des(Ala-Lys)-calmodulin was about one-tenth of calmodulin.     

Primary structure of chicken erythrocyte histone H2A

The complete amino acid sequence (128 residues) of the chicken erythrocyte histone H2A was deduced from the data provided by structural studies on the tryptic peptides from the maleylated histone and of the peptides obtained by thermolysin digestion of the native protein. The sequence of chicken histone H2A differs from the calf homologous histone by the deletion of one residue of histidine at position 123 or 124 and three conservative substitutions: a residue of serine replaces a residue of threonine at position 16, a residue of aspartic acid replaces a residue of glutamic acid at position 121 and a residue of alanine replaces a residue of glycine at position 128.     

Affinity labeling of phosphoenolpyruvate carboxykinase with 1, 5-I-AEDANS.

The concentrations of zinc thionein and cytosolic zinc in rat liver were examined in male rats five days after bilateral adrenalectomy. Zinc in metallothionein increased 10 fold, as compared with control animals. Cytosolic zinc increased 79% as compared with controls. 65% of this increase could be accounted for bound to metallothionein. Sham operated animals after five days showed a 4 fold increase in hepatic zinc thionein and a 23% increase in cytosolic zinc, 71% of this increase being bound to metallothionein. Adrenalectomized rats, maintained on daily injections of corticosterone (4mg/100g b.w.), exhibited the same levels of zinc thionein and cytosolic zinc as adrenalectomized rats receiving no treatment. Adrenalectomized rats, maintained on daily injections of aldosterone (5μg/100g b.w.), exhibited the same levels of zinc thionein as the sham operated rats, but the cytosolic zinc remained elevated at the level found in adrenalectomized rats receiving no treatment. These results indicate that there is adrenal involvement in the control of hepatic zinc and zinc thionein levels in the rat.     

Release and activation of phosphorylase phosphatase upon rupture of organelles from rat liver

Liver extracts (8000 × $\text{g}$ for 10 min) from fasted rats contain about 4 times more phosphorylase phosphatase activity when the liver was homogenized in a hypotonic medium or frozen before homogenization. This increase is caused by: (i) release of partially latent phosphatases ($\text{M}$_r=60 000 and 45 000 in sucrose gradient centrifugation) from ruptured organelles; (ii) rapid activation of phosphatase in the ruptured pellet by endogenous protease(s) which can be blocked by p-tosyl-L-lysine chloromethyl ketone. Only the $\text{M}$_r=60 000 enzyme, associated with the nuclei, can be activated proteolytically, with conversion to an $\text{M}$_r=45 000.     

Isolation of micro glutamic acid-rich protein from bovine brain

An acidic protein, designated as micro glutamic acid-rich protein, was purified to homogeneity from bovine brain extract, and was characterized in its physicochemical properties. The protein had an isoelectric point of 3.9, a molecular weight of 10,000, and was composed of very limited amino acid constituents; $\text{Asp}\text{Asn}$, Thr, Ser, $\text{Glu}\text{Gln}$, Pro, Gly, Ala and Lys, with a relative abundance of glutamic acid/glutamine which accounted for 51 % of the total amino acid composition. The yield of the protein was 750 μg/kg of wet brain tissue. The amino-terminal sequence analysis suggested that the protein arose through proteolysis of the 58,000-dalton precursor protein, that had been reported in a previous paper [Ishioka $\text{et al}$, (1980) Biochim. Biophys. Acta, 625, 281–290].     

Purification of a completely inactive renin from hog kidney and identification as renin zymogen

Hog renal inactive renin was separated from active renin and completely purified to an electrophoretically homogeneous state by using a new procedure which consisted of affinity chromatography on pepstatin-Sepharose, octyl-Sepharose, Affil-Gel blue and Con A-Sepharose columns, ion exchange chromatography and gel filtration. By this method a 3,000,000-fold purification was obtained with a 6% recovery from a crude kidney extract. This pure preparation was totally inactive and underwent marked activation by trypsin. It is a glycoprotein as judged by affinity to concanavalin A and has an apparent molecular weight of 50,000 as determined by gel filtration on Sephadex G-100. Treatment of the inactive renin with guanidine, urea and Triton X-100 did not cause activation indicating that the inactive renin isolated in the present study is not a product of renin-inhibitor complex.     

Catalytic activity and arrangement of subunit polypeptides in rat liver cytochrome c oxidase as studied by proteolysis

Cytochrome c oxidase from rat liver was incubated with various proteinases of different specificities and the enzymic activity was measured after various incubation times. A loss of catalytic activity was found after digestion with proteinase K, aminopeptidase M and a mitochondrial proteinase from rat liver. In each case the decrease in enzymic activity was compared with the changes in intensities of the polypeptide pattern obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibilities of the subunit polypeptides of the soluble cytochrome c oxidase to proteinases were very different. Whereas subunit I was most susceptible, subunits V–VII were rather resistant to degradation. From the relative inaccessibility of subunits V–VII to proteinases it is likely that these polypeptides are buried in the interior of the enzyme complex.     

Letter: Effect of an inhibitor of proteolysis on the size of newly-synthesized protein in HeLa cells

The size of newly-synthesized protein molecules in uninfeeted HeLa cells is altered by the addition of TPCK (l-chloro-4-phenyl-3-tosylamido-2-butanone), a specific inhibitor of certain proteolytic enzymes. The data suggests that the drug is inhibiting a polypeptide cleavage mechanism which affects the maturation of at least 5% of the polypeptide molecules of the cell near the time of their synthesis.     

Large-scale purification and characterization of calmodulin from ram testis its metal-ion-dependent conformers

Calmodulin was isolated in large quantities from ram testis by a simple procedure involving sequentially ammonium sulfate fractionation, heat treatment, anion exchange chromatography on DEAE-cellulose and gel filtration on Sephacryl S-200. Divalent cations (Mg²⁺ and/or Ca²⁺) were present throughout the purification which was entirely performed in the absence of chelators. The final yield was approx. 90 mg per kg testis. Ram testis calmodulin appears to be essentially identical to the brain homologous protein by the following criteria: ultraviolet absorption spectrum, amino acid composition showing a single residue of ?-N-trimethyl lysine, and tryptic peptide maps obtained by high performance liquid chromatography. Turkey gizzard myosin light-chain kinase, the activation of which is extremely specific for calmodulin (Walsh, M.P., Vallet, B., Cavadore, J.C. and Demaille, J.G. (1980) J. Biol. Chem. 255, 335–337), was indeed activated by ram testis calmodulin in the presence of calcium. The isolated protein migrated at different rates upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, depending on the absence or presence of divalent metals which probably induce different conformations. The relative migration rates were Ca²⁺ > Mn²⁺ > Mg²⁺ > EDTA. In the presenceof divalent metals, the observed doublet may be ascribed to the equilibrium between ion-free and ion-saturated forms, which exhibited different Stokes radii, as already suggested (Grab, D.J., Berzins, K., Cohen, R.S. and Siekevitz, P. (1979) J. Biol. Chem. 254, 8690–8696).     

Limited proteolysis patterns of the B chain of insulin.

The oxidized B chain of insulin was used as a simple model for further consideration of limited proteolysis with low substrate:enzyme ratios. With low B chain:trypsin ratios, the ordinarily slower cleavage rate of the -Lys₂₉-Ala₃₀ bond essentially equaled the cleavage saturation rate of the -Arg₂₂-Gly₂₃ bond. This led to the disappearance of octapeptide which ordinarily forms most rapidly. Heptapeptide and alanine, formed mainly by cleavage of the octapeptide, decreased somewhat at high enzyme relative levels. Trypsin added to B chain formed a single chromatographic peak.     

Use of specific trifluoroacetylation of lysine residues in cytochrome c to study the reaction with cytochrome b5, cytochrome c1, and cytochrome oxidase

The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b₅, cytochrome c₁ and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, and 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c₁ and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.     

Chromatin structure of histone genes in sea urchin sperms and embryos.

The nucleosomal organization of active and repressed alpha subtype histone genes has been investigated by micrococcal nuclease digestion of P. lividus sperm, 32-64 cell embryo and mesenchyme blastula nuclei, followed by hybridization with 32P-labeled specific DNA probes. In sperms, fully repressed histone genes are regularly folded in nucleosomes, and exhibit a greater resistance to micrococcal nuclease cleavage than bulk chromatin. In contrast, both coding and spacer alpha subtype histone DNA sequences acquire an altered conformation in nuclei from early cleavage stage embryos, i.e., when these genes are maximally expressed. Switching off of the alpha subtype histone genes, in mesenchyme blastulae, restores the typical nucleosomal organization on this chromatin region. As probed by hybridization to D.melanogaster actin cDNA, actin genes retain a regular nucleosomal structure in all the investigated stages.     

Pseudo-inhibitors of neutrophil superoxide production: evidence that soybean-derived polypeptides are superoxide dismutases

We have previously reported the purification of polypeptides from soybean which are potent inhibitors of superoxide production by human neutrophils. We now report that neither oxygen uptake nor hydrogen peroxide production by stimulated neutrophils is affected by these inhibitors. Furthermore, the E-1 and E-3 polypeptides inhibit ferricytochrome c reduction by a xanthine oxidase superoxide generation system. The inhibitory activity of E-3 in the model system is blocked by 1 mM KCN while E-1 is only slightly cyanide sensitive. Atomic absorption analysis of E-1 and E-3 polypeptides reveal copper in the latter and manganese in the former. Thus, E-3 is a copper-containing superoxide dismutase while E-1 appears to be a manganese-containing superoxide dismutase.     

Evolving sea urchin histone genes-nucleotide polymorphisms in the H4 gene and spacers ofStrongylocentrotus purpuratus

Summary We present a comparison of spacer and coding sequences of histone gene repeats from fourStronglycocentrotus purpuratus individuals. Sequences of two previously cloned units (pCO2 and pSp2) were compared with three new histone gene clones, two of them from a single individual. Within a 1.7-kb region, 59 polymorphic sites were found in spacers, in mRNA nontranslated stretches, and at silent sites in codons of the H4 gene. The permitted silent-site changes were as frequent as in any other region studied. The most abundant polymorphisms were single-base substitutions. The ratio of transitions: tranversions: single-base-pair insertions/deletions was 322. A number of larger insertions/deletions were found, as well as differences in the length of (CTA)_n and (CT)_n runs. Two of the five cloned repeats contained an insertion of a 195-bp element that is also present at many other sites in the genomes of everyS. purpuratus individual studied. Pairwise comparisons of the different clones indicate that the variation is not uniformly divergent, but ranges from a difference of 0.34% to 3.0% of all nucleotide sites. A parsimonious tree of ancestry constructed from the pariwise comparisons indicates that recombination between the most distantly related repeats has not occurred in the 1–2 million years necessary for accumulation of the variation. The level of sequence variation found within theS. purpuratus population, for both tandemly repeated and single-copy genes, is 25%–50% of that found betweenS. purpuratus andS. drobachiensis.