The effects of N-substitution of chitosan and the physical form of the products on the rate of hydrolysis by chitinase from Streptomyces griseus

N-Formyl, N-chloroacetyl, N-glycyl, N-isobutyryl, and N-pentanoyl derivatives of chitosan have been prepared. N-Acetylchitosan was the derivative most susceptible to chitinase from Streptomyces griseus and lysozyme from chicken egg-white, but the susceptibility was not restrictive. The relative rates of hydrolysis by chitinase with respect to R in the RCONH group were CH₃ > CH₃CH₂ > H > CH₃CH₂CH₂ > (CH₃)₂CH > NH₂CH₂ > ClCH₂. Neither enzyme hydrolysed chitosan or its N-methylene, N-benzylidene, N-benzoyl, N-nicotinyl, and N-fatty acyl (C₅C₁₈) derivatives, and lysozyme did not hydrolyse N-butyrylchitosan. N-Acetylhexanoyl-chitosans, which had d.s. ratios of ~0.7: ~0.3 and ~0.3; ~0.7, were hydrolysed at ~0.75 and ~0.04 of the rate of N-acetylchitosan (powder) by chitinase. O-Acylation of N-acylchitosans caused a decrease in the rates of hydrolysis by chitinase. N-Acetylchitosan gels were hydrolysed at 8–13 times the rate for crab-shell chitin. These results indicate that not only N- and O-substituents but also the physical form of the substrates influence the rates of hydrolysis by these enzymes.     

Structural changes in thin filaments of crab striated muscle.

Low angle X-ray diffraction patterns were recorded from crab leg muscle in living resting state and in rigor (glycerol-extracted). Both resting and rigor patterns showed a series of layer-lines arising from a helical arrangement of actin subunits in the thin filaments. In the resting state, the crossover repeat of the long-pitch actin helices was 36.6 nm, and the symmetry of the genetic actin helix was an intermediate between $\text{26}\text{12}$ and $\text{28}\text{13}$. When the muscle went into rigor, the crossover repeat changed to 38.3 nm and the helical symmetry to $\text{28}\text{13}$.In the living resting pattern, six other reflections were observed on the meridian and in the near-meridional region. These were indexed as orders of 2 × 38.2 nm and could be assigned to troponin molecules; the spacings and the intensity distributions of these reflections could be explained by the model proposed by Ohtsuki (1974) for the arrangement of troponin molecules in the thin filaments.The muscle in rigor gave meridional and near-meridional reflections at orders of 2 × 38.3 nm. These were identified as the same series of reflections as was assigned to troponin in the living resting pattern, but were more intense and could be seen up to higher orders. We consider that the myosin heads attached to the thin filament at regular intervals along its axis also contribute to these reflections in the rigor pattern.     

Effects of purine nucleotides on photosynthetic electron transport in isolated chloroplasts

The effects of guanylates and inosinates (and adenylates) on phosphorylation, ferricyanide reduction, and light-induced H⁺ uptake in spinach chloroplasts were studied. GDP, GTP, IDP, and ITP (but not GMP and IMP) stimulated the light-induced H⁺ uptake and partially inhibited ferricyanide reduction. Phosphate, arsenate, and phlorizin increased the extent of inhibition by these nucleotides and decreased the values of their apparent dissociation constants for the inhibition process. In the presence of phosphate (or arsenate), restoration of ferricyanide reduction from the level inhibited by guanylates and inosinates was observed as phosphorylation (or arsenylation) proceeded. These results suggest that phosphorylation of GDP and IDP as well as ADP takes place after two steps of nucleotide binding to the chloroplast coupling factor 1. The apparent dissociation constants of GDP and IDP for these two binding steps were estimated to be about 34 and 38 µM for the first and 110 and 160 µM for the second step, respectively (at pH 8.3, 15°C). Above pH 9, the ratio (P/e) of the extent of phosphorylation to the increment of electron transport from the basal level measured in the presence of [ATP + Pi] or [ADP + Pi + phlorizin], became increasingly large. When the electron transport level inhibited by dicyclohexylcarbodiimide was taken to be the basal activity, the P/e ratio remained almost constant ( 1) from pH 7.0 up to 10.     

Purification of modulator-deficient myosin light-chain kinase by modulator protein-Sepharose affinity chromatography.

Modulator-deficient myosin light-chain kinase from rabbit skeletal muscle was purified by modulator protein-Sepharose 4B affinity chromatography. The purified protein showed a single band (MW 80,000) on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and it exists as a monomer in the native state as determined by gel filtration. The modulator-deficient myosin light-chain kinase (MW 80,000), modulator protein (MW 16,500) and Ca2+ were essential for the kinase activity. The half-maximal activity of the kinase in the presence of excess modulator protein with 10 mM MgCl2 was at pCa 5.1, where full activity of actomyosin-ATPase is observed in the presence of the troponin--tropomyosin system. Assuming a rapid equilibrium between myosin light-chain kinase and two substrates, ATP and g2 light-chain, Km values for ATP and g2 light chain were evaluated as 0.28 mM and 0.024 mM, respectively. Vm/e was 5.7 s-1.     

Analysis of dansyl amino acids by reverse-phase high-performance liquid chromatography

All 24 dansyl amino acids were separated by reverse-phase high-performance liquid chromatography on Develosil C8-5, using a linear gradient made from Tris-HCl buffer (pH 7.75) and methanol. A linear relationship between the amount of sample and peak area was found over the range of 6 to 300 ng (0.02–1 nmol) of dansyl derivatives. An application of this method to the NH₂-terminal analysis of lysozyme is described.     

Change in the intrinsic fluorescence of acetylcholine receptor purified from Narke japonica upon binding with cholinergic ligands

Effects of various cholinergic ligands on the intrinsic fluorescence of acetylcholine receptor purified from the electric organ of Narke japonica were investigated. Binding with acetylcholine decreased the fluorescence by 7–8%, and that with carbamylcholine by 4–5% at 20 °C. Decamethonium and d-tubocurarine did not affect significantly the fluorescence intensity, while hexamethonium enhanced it. These changes were completely inhibited by preincubation of the receptor with α-bungarotoxin, which indicated that the observed intrinsic fluorescence change was due to the specific binding of each ligand. Data of the quenching experiment using iodide ion as an extrinsic quencher suggested the occurrence of the conformational change in the receptor upon binding with various cholinergic ligands. Considering these results together with those on intrinsic fluorescence change, conformational change provoked by binding with acetylcholine or carbamylcholine seems to differ from that provoked by binding with other cholinergic ligands examined.     

Monoclonal antibodies toward scallop (Patinopecten yessoensis) testis and wheat germ calmodulins.

A monoclonal antibody (IM7) toward scallop testis calmodulin and another one (PBE2) toward wheat germ calmodulin were produced. Ca2+ was required for IM7 to react with scallop calmodulin. IM7 reacted with the C-terminal region (Asp78-Lys148) of the calmodulin. As observed on competitive ELISA, IM7 reacted with chicken calmodulin, but not with Euglena gracilis or wheat calmodulin, troponin C, myosin light chains, or parvalbumin. It is assumed that the cluster of Thr143, Thr146, and Ser147 in the C-terminal region acts as the antigenic site. IM7 (and Fab of IM7) inhibited the activities of myosin light chain kinase and cAMP-phosphodiesterase. PBE2 reacted with wheat germ calmodulin irrespective of the presence or absence of Ca2+, the antigenic site being in the N-terminal region (Ala1-Met37). It reacted with wheat and spinach calmodulins, but not with scallop, chicken, or Euglena calmodulin, troponin C, myosin light chains, or parvalbumin. PBE2 had no effect on the activities of myosin light chain kinase and cAMP-phosphodiesterase.     

Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.     

Amino acid sequence of calmodulin from Euglena gracilis.

The complete amino acid sequence of calmodulin from Euglena gracilis was determined by isolation and sequence analyses of peptides derived from calmodulin by digestion with trypsin and Staphylococcus aureus V8 protease. Euglena calmodulin consists of 148 amino acid residues; it lacks tryptophan and cysteine and contains one tyrosine, three histidine and two NE-trimethyllysine residues/molecule of the protein. Its N-terminus was blocked with an acetyl group and C-terminal lysine was trimethylated. Euglena calmodulin is the first calmodulin so far examined in which the C-terminal lysine is trimethylated. The comparison of amino acid sequences between Euglena and human brain calmodulins indicated 17 amino acid substitutions in Euglena calmodulin.     

Immunoelectron microscopy of carbonic anhydrase isozyme VI in rat submandibular gland: comparison with isozymes I and II.

Carbonic anhydrase (CA) was purified from the saliva of pilocarpine-treated rats by inhibitor-affinity chromatography, and its localization in the rat submandibular gland was studied by the indirect immunoperoxidase technique using a monoclonal antibody (MAb) raised against the enzyme. SDS-polyacrylamide gel electrophoresis of the CA VI gave three bands of 33, 39, and 42 KD. Enzyme digestion experiment showed that the 42 KD molecule was degraded into the 39 KD molecule and the 39 KD molecule into the 33 KD molecule. The cleavage of the 42 KD molecule was independent and that of the 39 KD molecule was dependent on endo-beta-N-acetylglucosaminidase F. The 42 KD molecule was detected in the CA purified from the pilocarpine-treated but not the untreated salivary gland. The MAb recognized all the three components of the enzyme. Immunostaining for CA VI was seen in the cytosol and secretory granules of serous acinar cells and in the duct luminal contents. Staining specific for erythrocyte CA (CA I and CA II) was observed in the cytosol of the epithelial cells of granular, striated, and excretory ducts. Among these duct cells, the agranular varieties in the granular and excretory ducts were essentially devoid of the immunoreactivity.