Studies on the interactions of glycerol dehydrogenase from Bacillus stearothermophilus with Zn2+ ions and NADH

The interactions of the essential divalent cation, Zn2+, with the binary complex formed between glycerol dehydrogenase (glycerol:NAD+ 2-oxidoreductase, EC 1.1.1.6) and its coenzyme NADH have been examined by fluorescence spectroscopy. Both the metallo and non-metallo form of the enzyme bind the coenzyme NADH. The addition of Zn2+ ions to a solution of the binary complex formed between metal-depleted enzyme and NADH results in a rapid increase in fluorescence emission at 430 nm. This has been used to determine the on rate for Zn2+ to the enzyme/binary complex. A dissociation constant of 3.02 +/- 0.25.10(-9) M for the equilibrium between Zn2+ ions and the enzyme has been determined.     

The rapid purification of 3-hydroxybutyrate dehydrogenase and malate dehydrogenase on triazine dye affinity matrices.

3-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) and malate dehydrogenase (EC 1.1.1.37) were purified to homogeneity on a large scale involving only two sequential affinity-chromatography steps on two triazine dye-Sepharose matrices. Recoveries of both enzymes were in excess of 60%. Malate dehydrogenase could also be purified by a combination of triazine dye affinity chromatography and gel filtration on Ultrogel AcA-44, but this offered no significant advantage over the purely affinity procedure.     

Effect of diffusion limitations and floc size distributions on fermentor performance and the interpretation of experimental data

The biological rate equation that describes the overall rate of substrate uptake by microbial films has been extended to microbial flocs with the aid of a shape parameter. The “solid”- and liquid-phase diffusion limitations are explored and found to depend largely on a dimensionles characteristic size k₂¹V_p/A_p. Procedures are discussed by which k₂¹V_p/A_p can be determined from experimental data on the conversion efficiency in a completely mixed fermentor and measurements carried out on flocs recovered from the fermentor are assessed. Floc size distributions are shown to affect the performance characteristics of a fermentor when some of the flocs are sufficiently large to exhibit a diffusional limitation, and it is concluded that a single mean floc size (k₂¹V_p/A_p)* is sufficient to characterize a given distribution, at least when all the flocs are geometrically similar. The mean floc size closely corresponds to the “surface” mean floc size of the floc size distributions.     

Activation of Phycomyces adenosine 3', 5'-monophosphate phosphodiesterase by blue light.

Cyclic AMP phosphodiesterase can be extracted from sporangiophores of $\text{Phycomyces}\text{blakesleeanus}$. Activity is enhanced by 1–10 μM FAD and FMN but not by riboflavin. Moderate intensity of blue light also activates the enzyme, especially in the presence of 1mM GTP. The enzyme must be extracted and stored in the absence of blue light for this result. Forty times the intensity of red light has no effect. This finding is consistent with the very sudden transient drop in cyclic AMP level upon light stimulation in the intact sporangiophore.     

GUS expression patterns from a tobacco yellow dwarf virus-based episomal vector

We have constructed a binary vector containing elements of the monopartite geminivirus, tobacco yellow dwarf virus (TYDV). The vector is designed to be stably integrated into the plant genome, via Agrobacterium-mediated transfer. Upon expression of the viral replication-associated protein the TYDV elements are released from the T-DNA and then replicate episomally. We refer to these released forms as multicopy plant episomes (MPE). Tobacco plants (Nicotiana tabacum cv `Samsun') transformed with the vector showed MPE release and subsequent episomal replication in 6 of 11 of these plants. An MPE vector containing the uidA gene faithfully replicated and maintained the reporter gene, even though the construct was considerably larger than the wild-type TYDV genome. Histochemical staining revealed a speckled GUS expression phenotype which could be correlated with MPE replication. Received: 11 July 1997 / Revision received: 4 November 1997 / Accepted: 8 December 1997     

Heparan sulfate proteoglycans regulate autophagy in Drosophila

Heparan sulfate-modified proteoglycans (HSPGs) are important regulators of signaling and molecular recognition at the cell surface and in the extracellular space. Disruption of HSPG core proteins, HS-synthesis, or HS-degradation can have profound effects on growth, patterning, and cell survival. The Drosophila neuromuscular junction provides a tractable model for understanding the activities of HSPGs at a synapse that displays developmental and activity-dependent plasticity. Muscle cell-specific knockdown of HS biosynthesis disrupted the organization of a specialized postsynaptic membrane, the subsynaptic reticulum (SSR), and affected the number and morphology of mitochondria. We provide evidence that these changes result from a dysregulation of macroautophagy (hereafter referred to as autophagy). Cellular and molecular markers of autophagy are all consistent with an increase in the levels of autophagy in the absence of normal HS-chain biosynthesis and modification. HS production is also required for normal levels of autophagy in the fat body, the central energy storage and nutritional sensing organ in Drosophila. Genetic mosaic analysis indicates that HS-dependent regulation of autophagy occurs non-cell autonomously, consistent with HSPGs influencing this cellular process via signaling in the extracellular space. These findings demonstrate that HS biosynthesis has important regulatory effects on autophagy and that autophagy is critical for normal assembly of postsynaptic membrane specializations.