The ERD14 protein (early response to dehydration) is a member of the dehydrin family of proteins which accumulate in response to dehydration-related environmental stresses. Here we show the Arabidopsis dehydrin, ERD14, possesses ion binding properties. ERD14 is an in vitro substrate of casein kinase II; the phosphorylation resulting both in a shift in apparent molecular mass on SDS-PAGE gels and increased calcium binding activity. The phosphorylated protein bound significantly more calcium than the nonphosphorylated protein, with a dissociation constant of 120 M and 2.86 mol of calcium bound per mol of protein. ERD14 is phosphorylated by extracts of cold-treated tissues, suggesting that the phosphorylation status of this protein might be modulated by cold-regulated kinases or phosphatases. Calcium binding properties of ERD14 purified from Arabidopsis extracts were comparable with phosphorylated Escherichia coli-expressed ERD14. Approximately 2 mol of phosphate were incorporated per mol of ERD14, indicating a minimum of two phosphorylation sites. Western blot analyses confirmed that threonine and serine are possible phosphorylation sites on ERD14. Utilizing matrix assisted laser desorption ionization-time of flight/ mass spectrometry we identified five phosphorylated peptides that were present in both in vivo and in vitro phosphorylated ERD14. Our results suggest that the polyserine (S) domain is most likely the site of phosphorylation in ERD14 responsible for the activation of calcium binding.

A vacuole membrane-associated calcium-binding protein with an apparent mass of 45 kD was purified from celery (Apium graveolens). This protein, VCaB45, is enriched in highly vacuolate tissues and is located within the lumen of vacuoles. Antigenically related proteins are present in many dicotyledonous plants. VCaB45 contains significant amino acid identity with the dehydrin family signature motif, is antigenically related to dehydrins, and has a variety of biochemical properties similar to dehydrins. VCaB45 migrates anomalously in sodium dodecyl sulfate-polyacrylamide gel electrophoresis having an apparent molecular mass of 45 kD. The true mass as determined by matrix-assisted laser-desorption ionization time of flight was 16.45 kD. VCaB45 has two characteristic dissociation constants for calcium of 0.22 Ϯ 0.142 mm and 0.64 Ϯ 0.08 mm, and has an estimated 24.7 Ϯ 11.7 calcium-binding sites per protein. The calcium-binding properties of VCaB45 are modulated by phosphorylation; the phosphorylated protein binds up to 100-fold more calcium than the dephosphorylated protein. VCaB45 is an "in vitro" substrate of casein kinase II (a ubiquitous eukaryotic kinase), the phosphorylation resulting in a partial activation of calcium-binding activity. The vacuole localization, calcium binding, and phosphorylation of VCaB45 suggest potential functions.The vacuole is a reservoir for calcium (Machlon, 1984) and consequently plays an important role in calcium homeostasis (Miller et al., 1990;Allen and Sanders, 1995;Sanders et al., 1999). Regulation of vacuole calcium levels is complex involving a variety of calcium channels and pumps (Sanders et al., 1999;Sze et al., 2000). Sustained elevated levels of cytosolic calcium can be toxic (Hepler and Wayne, 1985), so under normal conditions, cytosolic calcium levels increase only transiently. Proteinaceous calcium buffers may serve as homeostats to attenuate the signal transduction system. Well-characterized protein calcium buffers include calreticulin and calsequestrin (Ostwald and MacLennon, 1974;Campbell et al., 1983b). Homologs of calsequestrin (Krause et al., 1989;Xing et al., 1994), calreticulin (Chen et al., 1994;Napier et al., 1995;Nelson et al., 1997), and calnexin (Li et al., 1998) have been identified in plants. These calcium-binding proteins can bind on the order of 20 to 50 calcium ions with both high-(1-3 sites per protein) and low-(20-50 sites per protein) affinity sites. The levels of calcium binding proteins may have a significant impact on signaling processes and may regulate second messenger transmission (Camacho and Lechleiter, 1995;Mery et al., 1996;Coppolino et al., 1997). In an alternative role, calcium-dependent interactions of calnexin and calreticulin have been characterized with a variety of proteins (Nigam et al., 1994;Peterson et al., 1995) and both are implicated in the promotion of correct protein folding (Hebert et al., 1996). These latter activities clearly suggest a molecular chaperone role. Recently, a high-capacity, low-affinity calcium-binding protei...

The active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), N-methyl-4-phenylpyridinium (MPP(+)), selectively destroys the dopaminergic neurons and induces the symptoms of Parkinson's disease. Inhibition of mitochondrial complex I and/or the perturbation of dopamine metabolism through cellular and granular accumulation have been proposed as some of the major causes of neurotoxicity. In the present study we have synthesized and characterized a number of MPTP and MPP(+) derivatives that are suitable for the comparative neurotoxicity and complex I inhibition versus dopamine metabolism perturbation studies. Structure-activity studies with bovine chromaffin granule ghosts show that 3'-hydroxy-MPP(+) is one of the best known substrates for the vesicular monoamine transporter (VMAT). A series of compounds that combine the structural features of MPP(+) and a previously characterized VMAT inhibitor, 3-amino-2-phenyl-propene, have been identified as the most effective VMAT inhibitors. These derivatives have been used to define the structural requirements of the VMAT substrate and inhibitor activities.

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Assessment instruments designed to measure student conceptual understanding and skills proficiency related to biochemistry are important to transform undergraduate biochemistry education. The purpose of this study was to develop an assessment instrument to measure student understanding of protein structure and enzyme inhibition in a new context, that of saturable binding. A community of biochemistry educators was involved in an iterative process of designing and testing of this assessment, which consists of true/false and open-ended questions that map to low and high levels in Bloom's taxonomy. A total of 188 students' responses were collected from seven different institutions and were graded by two independent raters using a rubric. Results from this administration indicate that most students were able to answer the questions related to lower-levels in Bloom's taxonomy; however for higher-level questions, students had more difficulty. The results from this assessment can give instructors insight into the ways in which persistent incorrect or incomplete ideas related to protein structure and binding events can hinder knowledge application. Also, the use of a community of practice to develop assessment instruments of this type and the format of the instrument itself could be a useful model for development of assessment instruments in the future.

This article describes a biochemistry educator community-driven effort to design and implement a course-based instrument to assess undergraduate students’ understanding of metabolic pathway dynamics and regulation.