No abstract

A one-semester, project-based biochemistry laboratory that combines the best features of a cookbook approach (high success rate, achievement of defined goals) with those of an investigative, discovery-based approach (student involvement in the experimental design, excitement of real research). Individual modules may be selected and combined to meet the needs of different courses and different institutions. The central theme of this lab is protein purification and design.

A molydate-stabilized, 'non-activated' form of the progesterone receptor from the cytosol of oestrogenstimulated chick oviduct has been purified to homogeneity by a three-step procedure. The first step, affinity chromatography using a N-(l2-amino-dodecyl)-3-oxo-4-androsten-17~-carboxamide-substituted Sepharose gel, purified the receptor 1500 -2700-fold with z 50 % recovery. In the second step, ion-exchange chromatography through a DEAE-cellulose column, progesterone receptor was eluted as a single peak at 0.1 M KCI. Purification after this step was >6700-fold. The third step was filtration through Ultrogel AcA 34, resulting in overall purification z 7400-fold with overall recovery z 25 of pure receptor on the basis of 1 binding site/molecule of M, 85000. The purified molybdate-stabilized receptor had a sedimentation coefficient = 7.9 S & 0.1 ( n = 4) in 0.15 M or 0.4 M KCI containing sucrose 5-20% gradient and z 8.9 S k 0.2 (n = 6) in 0.15 M KCI containing glycerol 10-35 "/, gradient, and its Stokes radius was 7.05 k 0.10 nm (n = 3) (calculated M , between 240000 and 280000). Binding specificity of the purified receptor was the same as that found in crude cytosol. SDS-PAGE revealed a single band migrating as a polypeptide of MI z 85000 -+ 2300 (n = 9). PAGE under nondenaturing conditions at total acrylamide concentrations 5 "/,, 7 % and 9 "/, showed a single [3H]ORG 2058-protein band (ORG 2058 is a high-affinity analogue more suitable than progesterone for electrophoretic studies). The data suggest that the high molecular weight molybdate-stabilized progesterone receptor purified from oestrogen-primed chick oviduct is composed of only = 85000-M, polypeptide chains. [5] have recently described a 'non-activated' or 'native' form of the progesterone receptor from chick oviduct cytosol, stabilized and prepared in the presence of sodium molybdate. This form of the receptor sedimented at 8-9 S on sucrose gradients containing 0.15 M KCI, and was eluted from DEAE-cellulose as a single peak at 0.15 M KCI 151. Its Stokes radius R, was z 7.9 nm and data permitted calculation of M , z 290000. These studies prompted us to purify the molybdate-stabilized receptor using an appropriate biospecific affinity material. The chick oviduct progesterone receptor has been extensively studied by Schrader, O'Malley and coworkers during the past decade (for a review, see [6]), and two non-identical binding components have been characterized and separately purified from the cytosol of hen or of oestrogen-primed chick oviduct. These components were reported to be polypeptides of M, z 79000 ('subunit A') and "N I10000 ('subunit B'), respectively. They are readily separated by ion-exchange chromatography. In contrast, using crude cytosol, Murayama et al. [7] found MI = 84000 for a 'native' unit and Miller et al.[8] M , = 87000 for a 'fast' component of progesterone receptor from chick oviduct.We have developed a method to purify the molybdatestabilized form of the progesterone receptor. This involves a three-step procedure including a new aff...

Students commencing studies in biochemistry must transfer and build on concepts they learned in chemistry and biology classes. It is well established, however, that students have difficulties in transferring critical concepts from general chemistry courses; one key concept is “energy.” Most previous work on students' conception of energy has focused on their understanding of energy in the context of physics (including the idea of “work”) and/or their understanding of energy in classical physical and inorganic chemistry contexts (particularly Gibbs Free Energy changes, the second law of thermodynamics, and equilibrium under standard conditions within a closed system). For biochemistry, students must go beyond those basic thermodynamics concepts of work, standard energy changes, and closed systems, and instead they must consider what energy flow, use, and transformation mean in living, open, and dynamic systems. In this study we explored students' concepts about free energy and flow in biological chemical reactions and metabolic pathways by surveys and in-depth interviews. We worked with students in general chemistry classes and biochemistry courses in both an Australian and a US tertiary institution. We address three primary questions (i) What are the most common alternative conceptions held by students when they explain energy-related phenomena in biochemistry?, (ii) What information do students transfer from introductory chemistry and biology when they are asked to consider energy in a biological reaction or reaction pathway?, and (iii) How do students at varying levels of competence articulate their understandings of energy in pathways and biological reactions? The answers to these questions are used to build a preliminary learning progression for understanding “energy” in biochemistry. We also propose crucial elements of content knowledge that instructors could apply to help students better grasp this threshold concept in biochemistry.