Rob L. Dean

Kinetic studies with alkaline phosphatase in the presence and absence of inhibitors and divalent cations

A very robust and inexpensive kinetic assay for determining rates of hydrolysis of p‐nitrophenyl phosphate by the enzyme alkaline phosphatase is presented. The reaction increases in rate with increase in pH. The enzyme is competitively inhibited by the reaction products, uncompetitively inhibited by L‐phenylalanine, and responds to the presence of two cofactors, magnesium and zinc ions. The reaction rate increases as Mg2+ concentration is increased from 1–5 mM. With increasing Zn2+ concentration, the reaction rate is stimulated and then depressed. Experimental work on the interaction between Mg2+ and Zn2+ in the reaction is suggested for more capable students.

Rates of electron transport in the thylakoid membranes of isolated, illuminated chloroplasts are enhanced in the presence of ammonium chloride

The rate of flow of electrons from water to an artificial electron acceptor, dichlorophenolindophenol (DCPIP), through photosystem II in the thylakoid membranes of isolated chloroplasts is greatly enhanced in the presence of 10 mM ammonium chloride. Rate enhancement depends on irradiance levels. Uncoupling reagents like ammonium chloride prevent the formation of a proton gradient across the thylakoid membrane and consequently remove a constraint on the rate of electron transport. The mode of action of ammonium chloride is explained. Evidence obtained using an oxygen electrode that DCPIP itself also partially uncouples the system is presented as background information for instructors. Suggestions on how this reaction may be used in laboratory classes for students from high school to the senior undergraduate level are included.

Synthesis of heat shock proteins in quail red blood cells following brief, physiologically relevant increases in whole body temperature

Cultured RBCs from quail respond to thermal stress (heat shock) by a rapid and dramatic change in gene expression. This change in gene expression includes the new and/or enhanced non-coordinate synthesis of a small group of heat shock polypeptides (HSPs) having molecular masses of 90,000, 70,000 and 26,000. RBCs obtained from hyperthermic quail exhibit a change in gene expression similar to that observed in RBCs heat-shocked in vitro. Since in vitro studies have linked the synthesis of HSPs in heat-stressed cells with thermotolerance, the similar change in gene expression in RBCs from hyperthermic quail suggests that, here too, this cellular response may be an important homeostatic mechanism by which avian RBCs cope with and/or survive hyperthermic conditions.

Diphenyl carbazide restores electron transport in isolated, illuminated chloroplasts after electron transport from water has been eliminated by mild heat treatment

Freshly isolated, illuminated chloroplasts oxidize water and transfer the resulting electrons through the photosynthetic electron transport chains in their thylakoid membranes to the artificial electron acceptor, dichlorophenol indophenol (DCPIP). As a consequence, DCPIP is reduced and the decline in absorbance over time can be used to measure the rate of electron transfer. When gently heated, chloroplasts lose the capacity to oxidize water and the transfer of electrons to DCPIP is eliminated. Electron transport through chloroplasts to DCPIP is restored in the presence of the artificial electron donor diphenylcarbazide (DPC). If students gain experience with the DCPIP photoreduction assay and are given information on normal chloroplast function, they should be able to predict the behavior of heat‐treated chloroplasts in a variety of experimental conditions. A number of such predictions are outlined and tested. The experiments can all be conducted with a limited repertoire of equipment and easily prepared solutions. Consequently, this work is well suited to an investigative study in which each student group, in consultation with instructors, can make and test its own prediction. The ways in which changing different variables can affect the quality of the experimental results is emphasized. Additional studies, on measurements of rates of oxygen evolution and emitted chlorophyll fluorescence, are briefly described to support the inferences that heat‐treated chloroplasts do not oxidize water and that the vectorial transfer of electrons through them to DCPIP is identical to that in untreated chloroplasts.

Using order of magnitude calculations to extend student comprehension of laboratory data.

i previously published an Illuminations article concerning “challenge” questions that encourage students to think imaginatively with approximate quantities, reasonable assumptions, and uncertain information ([2][1]). This article has promoted some interesting discussion, which has prompted me to