Brain type II calcium and calmodulin-dependent protein kinase: characterization of a brain-region specific isozyme and regulation by autophosphorylation

A variety of molecular approaches have been used to investigate the structural and enzymatic properties of rat brain type ll Ca^(2+) and calmodulin-dependent protein kinase (type ll CaM kinase). This thesis describes the isolation and biochemical characterization of a brain-region specific isozyme of the kinase and also the regulation the kinase activity by autophosphorylation.

The cerebellar isozyme of the type ll CaM kinase was purified and its biochemical properties were compared to the forebrain isozyme. The cerebellar isozyme is a large (500-kDa) multimeric enzyme composed of multiple copies of 50-kDa α subunits and 60/58-kDa β/β’ subunits. The holoenzyme contains approximately 2 α subunits and 8 β subunits. This contrasts to the forebrain isozyme, which is also composed of and β/β'subunits, but they are assembled into a holoenzyme of approximately 9 α subunits and 3 β/β ' subunits. The biochemical and enzymatic properties of the two isozymes are similar. The two isozymes differ in their association with subcellular structures. Approximately 85% of the cerebellar isozyme, but only 50% of the forebrain isozyme, remains associated with the particulate fraction after homogenization under standard conditions. Postsynaptic densities purified from forebrain contain the forebrain isozyme, and the kinase subunits make up about 16% of their total protein. Postsynaptic densities purified from cerebellum contain the cerebellar isozyme, but the kinase subunits make up only 1-2% of their total protein.

The enzymatic activity of both isozymes of the type II CaM kinase is regulated by autophosphorylation in a complex manner. The kinase is initially completely dependent on Ca^(2+)/calmodulin for phosphorylation of exogenous substrates as well as for autophosphorylation. Kinase activity becomes partially Ca^(2+) independent after autophosphorylation in the presence of Ca^(2+)/calmodulin. Phosphorylation of only a few subunits in the dodecameric holoenzyme is sufficient to cause this change, suggesting an allosteric interaction between subunits. At the same time, autophosphorylation itself becomes independent of Ca^(2+) These observations suggest that the kinase may be able to exist in at least two stable states, which differ in their requirements for Ca^(2+)/calmodulin.

The autophosphorylation sites that are involved in the regulation of kinase activity have been identified within the primary structure of the α and β subunits. We used the method of reverse phase-HPLC tryptic phosphopeptide mapping to isolate individual phosphorylation sites. The phosphopeptides were then sequenced by gas phase microsequencing. Phosphorylation of a single homologous threonine residue in the α and β subunits is correlated with the production of the Ca^(2+) -independent activity state of the kinase. In addition we have identified several sites that are phosphorylated only during autophosphorylation in the absence of Ca^(2+)/ calmodulin.

Structure-, stereochemistry-, and metal-regulated DNA binding/cleaving molecules

In the cell, the binding of proteins to specific sequences of double helical DNA is essential for controlling the processes of protein synthesis (at the level of DNA transcription) and cell proliferation (at the level of DNA replication). In the laboratory, the sequence-specific DNA binding/cleaving properties of restriction endonuclease enzymes (secreted by microorganisms to protect them from foreign DNA molecules) have helped to fuel a revolution in molecular biology. The strength and specificity of a protein:DNA interaction depend upon structural features inherent to the protein and DNA sequences, but it is now appreciated that these features (and therefore protein:DNA complexation) may be altered (regulated) by other protein:DNA complexes, or by environmental factors such as temperature or the presence of specific organic molecules or inorganic ions. It is also now appreciated that molecules much smaller than proteins (including antibiotics of molecular weight less than 2000 and oligonucleotides) can bind to double-helical DNA in sequence-specific fashion. Elucidation of structural motifs and microscopic interactions responsible for the specific molecular recognition of DNA leads to greater understanding of natural processes and provides a basis for the design of novel sequence-specific DNA binding molecules. This thesis describes the synthesis and DNA binding/cleaving characteristics of molecules designed to probe structural, stereochemical, and environmental factors that regulate sequence-specific DNA recognition.

Chapter One introduces the DNA minor groove binding antibiotics Netropsin and Distamycin A, which are di- and tri(N-methylpyrrolecarboxamide) peptides, respectively. The method of DNA affinity cleaving, which has been employed to determine DNA binding properties of designed synthetic molecules is described. The design and synthesis of a series of Netropsin dimers linked in tail-to-tail fashion (by oxalic, malonic, succinic, or fumaric acid), or in head-to-tail fashion (by glycine, β-alanine, and γ-aminobutanoic acid (Gaba)) are presented. These Bis(Netropsin)s were appended with the iron-chelating functionality EDTA in order to make use of the technique of DNA affinity cleaving. Bis(Netropsin)-EDTA compounds are analogs of penta(N-methylpyrrolecarboxamide)-EDTA (P5E), which may be considered a head-to-tail Netropsin dimer linked by Nmethylpyrrolecarboxamide. Low- and high-resolution analysis of pBR322 DNA affinity cleaving by the iron complexes of these molecules indicated that small changes in the length and nature of the linker had significant effects on DNA binding/cleaving efficiency (a measure of DNA binding affinity). DNA binding/cleaving efficiency was found to decrease with changes in the linker in the order β-alanine > succinamide > fumaramide > N-methylpyrrolecarboxamide > malonamide >glycine, γ-aminobutanamide > oxalamide. In general, the Bis(Netropsin)-EDTA:Fe compounds retained the specificity for seven contiguous A:T base pairs characteristic of P5E:Fe binding. However, Bis(Netropsin)Oxalamide- EDTA:Fe exhibited decreased specificity for A:T base pairs, and Bis(Netropsin)-Gaba-EDT A:Fe exhibited some DNA binding sites of less than seven base pairs. Bis(Netropsin)s linked with diacids have C₂-symmmetrical DNA binding subunits and exhibited little DNA binding orientation preference. Bis(Netropsin)s linked with amino acids lack C₂-symmetrical DNA binding subunits and exhibited higher orientation preferences. A model for the high DNA binding orientation preferences observed with head-to-tail DNA minor groove binding molecules is presented.

Chapter Two describes the design, synthesis, and DNA binding properties of a series of chiral molecules: Bis(Netropsin)-EDTA compounds with linkers derived from (R,R)-, (S,S)-, and (RS,SR)-tartaric acids, (R,R)-, (S,S)-, and (RS,SR)-tartaric acid acetonides, (R)- and (S)-malic acids, N ,N-dimethylaminoaspartic acid, and (R)- and (S)-alanine, as well as three constitutional isomers in which an N-methylpyrrolecarboxamide (P1) subunit and a tri(N-methylpyrrolecarboxamide)-EDTA (P3-EDTA) subunit were linked by succinic acid, (R ,R)-, and (S ,S)-tartaric acids. DNA binding/cleaving efficiencies among this series of molecules and the Bis(Netropsin)s described in Chapter One were found to decrease with changes in the linker in the order β-alanine > succinamide > P1-succinamide-P3 > fumaramide > (S)-malicamide > N-methylpyrrolecarboxamide > (R)-malicamide > malonamide > N ,N-dimethylaminoaspanamide > glycine = Gaba = (S,S)-tartaramide = P1-(S,S)-tanaramide-P3 > oxalamide > (RS,SR)-tartaramide = P1- (R,R)-tanaramide-P3 > (R,R)-tartaramide (no sequence-specific DNA binding was detected for Bis(Netropsin)s linked by (R)- or (S)-alanine or by tartaric acid acetonides). The chiral molecules retained DNA binding specificity for seven contiguous A:T base pairs. From the DNA affinity cleaving data it could be determined that: 1) Addition of one or two substituents to the linker of Bis(Netropsin)-Succinamide resulted in stepwise decreases in DNA binding affinity; 2) molecules with single hydroxyl substituents bound DNA more strongly than molecules with single dimethylamino substituents; 3) hydroxyl-substituted molecules of (S) configuration bound more strongly to DNA than molecules of (R) configuration. This stereochemical regulation of DNA binding is proposed to arise from the inherent right-handed twist of (S)-enantiomeric Bis(Netropsin)s versus the inherent lefthanded twist of (R)-enantiomeric Bis(Netropsin)s, which makes the (S)-enantiomers more complementary to the right-handed twist of B form DNA.

Chapter Three describes the design and synthesis of molecules for the study of metalloregulated DNA binding phenomena. Among a series of Bis(Netropsin)-EDTA compounds linked by homologous tethers bearing four, five, or six oxygen atoms, the Bis(Netropsin) linked by a pentaether tether exhibited strongly enhanced DNA binding/cleaving in the presence of strontium or barium cations. The observed metallospecificity was consistent with the known affinities of metal cations for the cyclic hexaether 18-crown-6 in water. High-resolution DNA affinity cleaving analysis indicated that DNA binding by this molecule in the presence of strontium or barium was not only stronger but of different sequence-specificity than the (weak) binding observed in the absence of metal cations. The metalloregulated binding sites were consistent with A:T binding by the Netropsin subunits and G:C binding by a strontium or barium:pentaether complex. A model for the observed positive metalloregulation and novel sequence-specificity is presented. The effects of 44 different cations on DNA affinity cleaving by P5E:Fe were examined. A series of Bis(Netropsin)-EDTA compounds linked by tethers bearing two, three, four, or five amino groups was also synthesized. These molecules exhibited strong and specific binding to A:T rich regions of DNA. It was found that the iron complexes of these molecules bound and cleaved DNA most efficiently at pH 6.0-6.5, while P5E:Fe bound and cleaved most efficiently at pH 7.5-8.0. Incubating the Bis(Netropsin) Polyamine-EDTA:Fe molecules with K₂PdCl₄ abolished their DNA binding/cleaving activity. It is proposed that the observed negative metalloregulation arises from kinetically inert Bis(Netropsin) Polyamine:Pd(II) complexes or aggregates, which are sterically unsuitable for DNA complexation. Finally, attempts to produce a synthetic metalloregulated DNA binding protein are described. For this study, five derivatives of a synthetic 52 amino acid residue DNA binding/cleaving protein were produced. The synthetic mutant proteins carried a novel pentaether ionophoric amino acid residue at different positions within the primary sequence. The proteins did not exhibit significant DNA binding/cleaving activity, but they served to illustrate the potential for introducing novel amino acid residues within DNA binding protein sequences, and for the development of the tricyclohexyl ester of EDTA as a superior reagent for the introduction of EDT A into synthetic proteins.

Chapter Four describes the discovery and characterization of a new DNA binding/cleaving agent, [SalenMn(III)]OAc. This metal complex produces single- and double-strand cleavage of DNA, with specificity for A:T rich regions, in the presence of oxygen atom donors such as iodosyl benzene, hydrogen peroxide, or peracids. Maximal cleavage by [SalenMn(III)]OAc was produced at pH 6-7. A comparison of DNA singleand double-strand cleavage by [SalenMn(III)]⁺ and other small molecules (Methidiumpropyl-EDTA:Fe, Distamycin-EDTA:Fe, Neocarzinostatin, Bleomycin:Fe) is presented. It was found that DNA cleavage by [SalenMn(III)]⁺ did not require the presence of dioxygen, and that base treatment of DNA subsequent to cleavage by [SalenMn(III)]⁺ afforded greater cleavage and alterations in the cleavage patterns. Analysis of DNA products formed upon DNA cleavage by [SalenMn(III)] indicated that cleavage was due to oxidation of the sugar-phosphate backbone of DNA. Several mechanisms consistent with the observed products and reaction requirements are discussed.

Chapter Five describes progress on some additional studies. In one study, the DNA binding/cleaving specificities of Distamycin-EDTA derivatives bearing pyrrole N-isopropyl substituents were found to be the same as those of derivatives bearing pyrrole N-methyl substituents. In a second study, the design of and synthetic progress towards a series of nucleopeptide activators of transcription are presented. Five synthetic plasmids designed to test for activation of in vitro run-off transcription by DNA triple helix-forming oligonucleotides or nucleopeptides are described.

Chapter Six contains the experimental documentation of the thesis work.

Experiments on the production of a highly dispersible calcium carbonate. ('Experimental section' only) [Translation from: Szklo i Ceramiki 30, 133-136, 1979]

This short translation summarises experiments on the production of a highly dispersible precipitate of calcium carbonate. The translation covers the 'Experimental section' (of the original larger paper) only.

Enzyme induction in Neurospora crassa

I. Studies on Nicotinamide Adenine Dinucleotide Glycohydrase (NADase)

NADase, like tyrosinase and L-amino acid oxidase, is not present in two day old cultures of wild type Neurospora, but it is coinduced with those two enzymes during starvation in phosphate buffer. The induction of NADase, like tyrosinase, is inhibited by puromycin. The induction of all three enzymes is inhibited by actinomycin D. These results suggest that NADase is synthesized de novo during induction as has been shown directly for tyrosinase. NADase induction differs in being inhibited by certain amino acids.

The tyrosinaseless mutant ty-1 contains a non-dialyzable, heat labile inhibitor of NADase. A new mutant, P110A, synthesizes NADase and L-amino acid oxidase while growing. A second strain, pe, fl;cot, makes NADase while growing. Both strains can be induced to make the other enzymes. These two strains prove that the control of these three enzymes is divisible. The strain P110A makes NADase even when grown in the presence of Tween 80. The synthesis of both NADase and L-amino acid oxidase by P110A is suppressed by complete medium. The theory of control of the synthesis of the enzymes is discussed.

II. Studies with EDTA

Neurospora tyrosinase contains copper but, unlike other phenol oxidases, this copper has never been removed reversibly. It was thought that the apo-enzyme might be made in vivo in the absence of copper. Therefore cultures were treated with EDTA to remove copper before the enzyme was induced. Although no apo-tyrosinase was detected, new information on the induction process was obtained.

A treatment of Neurospora with 0.5% EDTA pH 7, inhibits the subsequent induction during starvation in phosphate buffer of tyrosinase, L-amino acid oxidase and NADase. The inhibition of tyrosinase and L-amino acid oxidase induction is completely reversed by adding 5 x 10^-5M CaCl₂, 5 x 10^-4M CuSO₄, and a mixture of L-amino acids (2 x 10^-3M each) to the buffer. Tyrosinase induction is also fully restored by 5 x 10^-4M CaCl₂ and amino acids. As yet NADase has been only partially restored.

The copper probably acts by sequestering EDTA left in the mycelium and may be replaced by nickel. The EDTA apparently removes some calcium from the mycelium, which the added calcium replaces. Magnesium cannot replace calcium. The amino acids probably replace endogenous amino acids lost to the buffer after the EDTA treatment.

The EDTA treatment also increases permeability, thereby increasing the sensitivity of induction to inhibition by actinomycin D and allowing cell contents to be lost to the induction buffer. EDTA treatment also inhibits the uptake of exogenous amino acids and their incorporation into proteins.

The lag period that precedes the first appearance of tyrosinase is demonstrated to be a separate dynamic phase of induction. It requires oxygen. It is inhibited by EDTA, but can be completed after EDTA treatment in the presence of 5 x 10^-5M CaCl₂ alone, although no tyrosinase is synthesized under these conditions.

The time course of induction has an early exponential phase suggesting an autocatalytic mechanism of induction.

The mode of action of EDTA, the process of induction and the kinetics of induction are discussed.

Experiments using an artificial stream to investigate the seasonal growth of chalk-stream algae

Research into the production ecology of chalk streams using a large artificial recirculating stream is described. Physical chemical processes including calcium and inorganic phosphate levels, and exchange of gaseous carbon dioxide in both a simple closed system and a circulating system with gravel substrate have been monitored in both light and dark conditions. Further experiments were concerned with the seasonal changes in algal growth over the gravel substrate with constant water velocities and replenishment. The algal population, composed mainly of the diatoms Achnanthes minutissima, Meridion circulare, Nitzschia fonticola and Synedra ulna reached a peak in mid May and declined rapidly during June. Concentrations of phosphate phosphorus fell as the diatoms grew but was not thought to limit growth. Silicate concentrations followed the diatom cycle closely but never fell below 0.8 mg/l Si. It is possible that one of the nutrients may have been limiting the rate of growth due to steep diffusion gradients through the algal mat. In the last summer and autumn a hard calcareous crust composed of the green alga Gongrosira incrustans and the blue green alga Homeothrix varians , developed. The channel stream is compared with the natural conditions found in chalk streams.

Subsurface damage of Nd-doped phosphate glasses in optical fabrication

We present a destructive method for detecting and measuring subsurface damage of Nd-doped phosphate glasses. An instrument based on the dimple method - a destructive method - was developed. Subsurface damage depth produced in each fabrication procedure was obtained. We extend the surface roughness-subsurface damage relation to Nd-doped phosphate glasses. The constant ratio of subsurface damage and surface roughness was obtained as well. We also analyse the relation of abrasive size and subsurface damage experimentally. From a measurement of the surface roughness or abrasive size, one can obtain an accurate estimate of the damage layer thickness that must be eliminated by polishing or subsequent grinding operations. (C) 2007 Elsevier GmbH. All rights reserved.

Effect of Fe impurity on the optical loss of Nd-doped phosphate laser glass

The optical loss coefficient at 1053-nm wavelength, influenced by Fe ions in N31-type Nd-doped phosphate laser glass, was determined precisely and analyzed in detail. It is found that the optical loss coefficient per unit of Fe concentration (cm^(-1)/ppmw) increases with Fe concentration in the range of 0---300 ppmw, but it approaches a constant as the Fe concentration is larger than 300 ppmw. Such a concentration effect is due to a shift in the redox equilibrium between Fe3+ and Fe2+ ions in the glass. The effect of oxygen pressure, temperature, and variable valence states of other metal ions in glass samples on the optical loss is also discussed.

Effect of network modifiers on spectroscopic properties of erbium-doped phosphate glasses

The integrated absorption cross section Sigma(abs), I peak emission cross section sigma(cmi), Judd-Ofeld intensity parameters Omega(iota) ( t = 2,4,6), and spontaneous emission probability A(R) of Er3+ ions were determined for Erbium doped alkali and alkaline earth phosphate glasses. It is found the compositional dependence of sigma(emi) 5 almost similar to that of Sigma(abs), which is determined by the sum, of Omega(1) (3 Omega(2) + 10 Omega(4) + 21 Omega(6)). In addition, the compositional dependence of Omega(1) was studied in these glass systems. As a result, compared with. Omega(4) and Omega(6) the Omega(2) has a stronger compositional dependence on the ionic radius and content of modifers. The covalency of Er-O bonds in phosphate glass is weaker than that in silicate glass, germanate glass, aluminate glass, and tellurate glass, since Omega(6) of phosphate glass is relatively large. A(R) is affected by the covalency of the Er3+ ion sites and corresponds to the Omega(6) value.

Er3+ and Yb3+ codoped phosphate laser glass for high power flashlamp pumping

A novel Vb(3+)-Er-(3+) codoped phosphate glass for high power flashlamp pumping and high repetition rate laser at 1.54 mu m, designated EAT5-2, is developed. The weight-loss rate of is 1.3 x 10(-5) gcm(-2) h(-1) in boiling water, which is comparable to Kigre's QX-Er glass. Some spectroscopic parameters are analysed by Judd-Ofelt theory and McCumber theory The emission cross section is calculated to be 0.73 x 10(-20) cm(2). The thermo-mechanical properties of EAT5-2 are modified after an ion-exchange chemical strengthening process in a KNO3/NaNO3 molten salt bath. The thresholds for optical damage from the flashlamp pumping are tested on glass rods. A repetition rate of 15 Hz is achieved for chemically strengthened glass. The laser experimental results at. 1.54 mu m from flashlamp pumping are also reported.