Interaction of aqueous inorganic orthophosphate and phosphate rock

An experimental study was made of the interaction of phosphate rock and aqueous inorganic orthophosphate, calcium, and hydroxyl ions. A model of the reaction was developed by observing electron diffraction patterns in conjunction with concentration changes of chemical components. The model was applied in explaining the performance of batch suspensions of powdered phosphate rock and packed columns of granular phosphate rock. In both cases the reaction consisted initially of a rapid nucleation phase that occurred in a time period of minutes. In the batch system the calcium phosphate nuclei then ripened into larger micro-crystals of hydroxyapatite, which eventually became indistinguishable from the original phosphate rock surface. During column operation the high supersaturation ratio that existed after the rapid nucleation phase resulted in a layer of small nuclei that covered a slowly growing hydroxyapatite crystal.

The column steady-state rate constant was found to increase with increasing temperature, pH, and fluoride concentration, and to decrease with increasing concentrations of magnesium sulfate, ammonium chloride, and bicarbonate ion.

An engineering feasibility study indicated that, based on economic considerations, nucleation of apatite on phosphate rock ore has a potential use as a wastewater phosphate removal treatment process.

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.

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.