Robert C. Bateman

Evidence for tissue-specific forms of glutaminyl cyclase

Glutaminyl cyclase (QC) is responsible for the presence of pyroglutamyl residues in many neuroendocrine peptides. An examination of the bovine tissue distribution of QC immunoreactivity, enzyme activity, and mRNA confirmed that QC was abundant in brain and pituitary by all three measures. However, enzymatic activity was considerably more widespread than either immunoreactivity or mRNA, suggesting multiple enzyme forms. Partially purified QC from bovine spleen differed significantly from the known bovine pituitary QC in physical and catalytic properties. We propose that this form of glutaminyl cyclase plays a role in the posttranslational processing of constitutively secreted pGlu‐containing proteins.

Inhibition of Cysteine Proteinases by Carica-Papaya Cystatin Produced in Escherichia coli

A papaya cystatin (Cst)-encoding cDNA clone was isolated from a papaya leaf cDNA library and the active protein produced in Escherichia coli. The amino-acid sequence reveals a protein of 11,262 Da with over 40% identity to other published plant Cst. Unique features of the papaya Cst include a single Cys residue, variation in the papain-binding region, and the first reported inhibition of papaya proteinase IV by a Cst.

Teaching and Learning with Three-dimensional Representations

Computer-based visualizations play a profoundly important role in chemistry instruction. In this chapter, we review the role of visualization tools and possible ways in which they may influence thinking about chemistry. There are now several visualization systems available that allow students to manipulate important variables in obtain a solution to a scientific problem. We discuss the fundamental differences between these tools, and we emphasize the use of each within the context of constructivist curricula and pedagogies. We also consider the impact such tools may have on visuo-spatial thinking. We suggest that although visuo-spatial ability may be important in visualization use, its role has at times been overemphasized. We argue for a more nuanced, richer understanding of the many ways in which visuo-spatial reasoning is used in solving chemistry problems. This discussion leads to a set of design principles for the use of visualization tools in teaching chemistry. Finally, we present our work on the Kinemage Authorship Project, a program designed to assist students in understanding spatial structures in complex, biochemical molecules. The Kinemage Authorship Project allows students to construct their own molecular visualizations, and we discuss how this may lead to greater understanding of the spatial properties of molecules. This constructivist program embodies many of the design principles that we present earlier in the chapter.

A rapid dipstick test for histamine in tuna

Abstract This study describes the production of a solid phase assay (dipstick test) for histamine in tuna based on the coupling of diamine oxidase to a peroxidase/dye system. The optimized dipstick preparation consisted of a diamine oxidase/peroxidase/Stabilcoat mixture layered onto a tetramethylbenzidine color strip. The assay was linear to 1.0 mM histamine (RSQ = 0.994) and the minimum detectable concentration was 0.07 mM corresponding to 2.3 mg% in tuna extracts. Quality control samples gave intra and interassay precisions of 9.14–9.84 %CV and 7.08–13.86 %CV respectively. Similarly, the assay was accurate (p> 0.05) for both low and high controls. Histidine and cadaverine did not interfere with the assay whereas putrescine reacted slightly with the dipstick. Histamine determinations of fresh and spoiled tuna gave good agreement between the dipstick method and both the official reference and modified AOAC flurometric methods.

Human pituitary glutaminyl cyclase: expression in insect cells and dye affinity purification.

Human pituitary glutaminyl cyclase (hQC) was expressed in Drosophila S2 cells under the control of an inducible metallothionene promoter and fused to the Drosophila immunoglobulin-binding protein signal sequence to enable secretion into the culture media. Expression levels reached 50 microg/mL culture media after 7 days of induction. The enzyme was purified to homogeneity directly from culture media by affinity chromatography on Reactive Blue 4-agarose using a step pH elution. The identity of the expressed protein was confirmed by peptide mass mapping and Western blotting. Glutaminyl cyclase was expressed as a fully active 37 kDa enzyme with kcat/Km values of 14.3, 9.3, and 2.4 mM(-1)s(-1) for the substrates Gln-Gln, Gln-NH(2), and Gln-t-butyl ester, respectively. The two cysteines were disulfide bonded, and the lone predicted glycosylation site, asparagine 49, was shown by both enzymatic deglycosylation of the expressed enzyme and site-directed mutagenesis to be glycosylated.