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 Mg 2؉ concentration is increased from 1-5 mM. With increasing Zn 2؉ concentration, the reaction rate is stimulated and then depressed. Experimental work on the interaction between Mg 2؉ and Zn 2؉ in the reaction is suggested for more capable students.Keywords: Alkaline phosphatase, enzyme kinetics, enzyme inhibition, divalent cation cofactors.Experience has shown that enzyme assays used to teach introductory enzyme kinetics should ideally meet a number of criteria. (i) The assay should be reliable and show a prolonged linear rate of reaction to maximize the possibility that inexperienced students will obtain satisfactory data; (ii) given the funding situation at most institutions, the reagents should be inexpensive; and (iii) if preparation time is limited because multiple laboratory classes are offered throughout the week, reagents should be stable. For 2 years we have successfully used variants of one such kinetic assay as the basis for teaching enzyme kinetics in a 2nd-year undergraduate laboratory class. The assay presented in this article involves the hydrolysis of p-nitrophenyl phosphate by a commercially available preparation of the enzyme alkaline phosphatase.A number of publications describe class experiments with acid phosphatase [1][2][3][4], which is easily prepared by aqueous extraction of wheat germ and which can be colorimetrically assayed. However, these enzymatic studies all involve a fixed-time assay where a single measurement of product concentration is made after stopping the reaction following a given period of time. Fixed-time assays can be time-consuming because a basic assumption in the Michaelis-Menten approach to enzyme kinetics is that the enzyme is substrate-saturated. Consequently, initial velocities must be determined from reaction data that are linear with respect to the decline in substrate concentration (or increase in product concentration) over time. In fixed-time assays considerable effort is required to establish the duration in which the reaction between enzyme and substrate is linear. Moreover, the validity of the linear relationship must be confirmed each time that the reaction mixture is changed to accommodate the study of, for example, enzyme inhibitors or cofactors. Conversely, if one uses a kinetic assay, in which the reaction is continuously monitored, each data set reveals how long the reaction is linear, and any deviations from linearity are readily apparent. It is not possible to run a kinetic assay with acid phosphatase using p-nitrophenyl phosphate as substrate because the product, p-nitrophenol, is colorless at acidic pH. I consequently suggest that...

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.Keywords: Ammonium chloride, isolated chloroplasts, uncoupling, dichlorophenolindophenol.We teach a section of a second year undergraduate laboratory course entitled "Scientific Methods in Biology." The course encompasses experimental design, instrumentation, the evaluation of experimental data, and how to communicate results to the scientific community. Because students write about some of their work in the form of a scientific paper, we try to find a variety of experimental systems that exemplify similar principles. This allows different groups of students to write on different topics. The measurement of light-dependent rates of electron transport in the thylakoid membranes of isolated chloroplasts has been particularly useful in generating different questions. In this assay, the decline in absorbance of oxidized dichlorophenolindophenol (DCPIP) 1 is measured over time. DCPIP becomes colorless as it is reduced by intercepting electrons from the electron transport chains in the thylakoid membranes. It has been shown previously that rates of electron transport change with irradiance levels [1], in response to pH and in the presence of inhibitory reagents [2]. In this paper we demonstrate that rates of electron transport in the thylakoid membrane of illuminated chloroplasts can be dramatically increased in the presence of millimolar concentrations of ammonium chloride. Reagents that act like ammonium chloride are called uncouplers because, by a variety of mechanisms, they prevent the formation of a proton gradient across the thylakoid membrane and consequently uncouple electron transport from ATP synthesis (photophosphorylation).We believe that the information presented could be useful for teaching students from grade 10 to the senior undergraduate level. For younger students, the assay can be simply used as a demonstration to correct a common misconception [3] that light quantity limits the rate of photosynthetic reactions when the process is light-saturated. The more detailed studies are appropriate for second and third year undergraduates. We have, in addition, included information from studies using oxygen electrodes that we regard as unsuitable for the majority of undergraduates but wh...

A simple, rapid, and novel procedure for purifying ferritin from the postnuclear supernatant of red blood cell lysates is described. This report establishes the resistance of commercially available holo- and apo-ferritins to proteinase-K digestion, and documents how the use of this enzyme, in conjunction with the well-documented resistance of ferritins to heat denaturation (75-80 degrees C for 10 min), makes it possible to obtain high yields (greater than 90%) of pure, undegraded ferritin from the postnuclear supernatant of hypotonically or Triton X-100 lysed red blood cells. The resultant purified ferritin contains the same amount of iron as ferritin not treated with proteinase-K and, as judged by one- and two-dimensional gel electrophoresis and electron microscopy, consists of intact ferritin with a subunit isoform composition identical in molecular mass and isoelectric points to that obtained from ferritin prepared in the absence of this enzyme.

Larvae of Calpodes ethlius (Lepidoptera) reared at 22 degrees C die after 1 h at 45 degrees C, but they acquire the ability to tolerate 1 h at 45 degrees C if they are first exposed to 37 degrees C for 1 or 2 h. Incubation of intact larvae at 37 degrees C for 1 h induces the new and (or) enhanced synthesis of a similar family of polypeptides (relative masses (MrS) approximately 22 000) in all tissues examined (silk gland, fat body, wing discs, central nervous system, and muscle). When these same tissues are cultured at 37 degrees C for 1 h, the new and (or) enhanced synthesis of at least 5 polypeptides (MrS approximately 95 000, 81 000, 74 000, 26 000, and 22 000) is evident. The more dramatic response elicited from cultured tissues by heat shock results, presumably, from the added stress of dissection. The new and (or) enhanced synthesis of polypeptides with MrS of approximately 22 000 by tissues of intact larvae, incubated at 37 degrees C for 1 h, may be related to the acquisition of thermal tolerance by similarly treated larvae. These results suggest that the induced synthesis of these polypeptides is an integral part of a ubiquitous molecular mechanism involved in the development of thermal tolerance.

Subjecting 9-day-old quail embryos to an elevated temperature in ovo causes limb, breast, and brain tissues to shift their patterns of protein synthesis from the production of a broad spectrum of different proteins to the new and (or) enhanced synthesis of a small number of heat-shock proteins (HSPs). The HSPs synthesized by undifferentiated breast tissue in ovo (relative masses (Mrs) 88 000, 82 000, 64 000, and 25 000) are similar to those synthesized by explanted breast tissue or by primary cultures of breast myoblasts heat-shocked in culture. Heat-shocked, 120-hour-old myotube cultures synthesize HSPs similar to those detected in heat-shocked myoblasts except that myotubes also exhibit enhanced synthesis of a 55 000 dalton polypeptide and little or no synthesis of a 25 000 dalton HSP; the failure to thermally induce a 25 000 dalton polypeptide in myotubes is related to the fused nature of these cells rather than to their state of differentiation. Myoblasts, as well as myotubes, cultured in the presence of elevated amounts of arsenite, copper, or zinc also synthesize new and (or) enhanced amounts of polypeptides with isoelectric points and immunochemical properties similar to the 25 000 and 64 000 dalton HSPs. However, elevated levels of these metal ions fail to stimulate new and (or) enhanced synthesis of other HSP-like proteins. These results demonstrate that, although the protein synthetic response of myogenic cells to chemical and thermal stress may be similar in some respects, a number of the synthetic responses are clearly different.