A new, simple classroom technique helps cell biology students understand principles of Michaelis-Menten enzyme kinetics. A student mimics the enzyme and the student's hand represents the enzyme's active site. The catalytic event is the transfer of marbles (substrate molecules) by hand from one plastic container to another. As predicted, increases in marble concentration increase the number of marbles transferred per unit time (initial rate, V 0 ) until the turnover number becomes rate limiting and V 0 approaches the maximum velocity (V max ), as described by the Michaelis-Menten equation. With this demonstration, students visualize an important concept: the turnover number is constant and independent of marble concentration. A student assessment of this exercise showed that it helped students visualize the turnover number and V max but not K m , the marble concentration at which V 0 is one-half V max . To address the concept of K m , we use supplemental laboratory and lecture exercises. This exercise with plastic containers and marbles is equally suited to demonstrate the kinetics of carrier-mediated membrane transport. We conclude that this exercise helps students visualize the turnover number and V max and gives students important insights into the kinetic parameters used to characterize the catalytic activity of enzymes and membrane transporters.

Changes in intracellular ion concentrations have been correlated with the activation of an endogenous endonuclease and thus internucleosomal DNA cleavage during apoptosis in many cell types. We investigated whether intracellular pH could play a significant role in apoptotic initiation and progression in C3H-10T1/2 cells, a cell strain that does not exhibit double-stranded DNA cleavage during apoptosis. Protein kinase C and the Na+/H+ antiporter, known regulators of intracellular pH, also were assessed for their involvement in apoptosis of C3H-10T1/2 cells. When a H+ ionophore was used to clamp intracellular pH to 6.0 or below, a significant level of apoptosis was induced in these cells within 6 h, whereas clamping at pH 6.75 did not induce significant amounts of apoptosis until 36 h after acidification. The acidified cells exhibited classic apoptotic morphology and chromatin condensation, similar to serum withdrawn cells, but failed to show internucleosomal DNA cleavage with electrophoresis of genomic DNA. Our results also suggest that the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated inhibition of apoptosis in serum withdrawn C3H-10T1/2 cells functions through a sequential activation of protein kinase C and the Na+/H+ antiporter; thus, an alkalinization or an inhibition of acidification is involved in this apoptotic block. Serum withdrawal itself does not appear to act through a negative effect on either protein kinase C or the Na+/H+ antiporter. TPA was also capable of inhibiting the apoptosis induced by specific inhibitors of protein kinase C and the Na+/H+ antiporter, but the inhibition was successful only if the TPA was administered at least 20 min prior to the addition of the enzyme inhibitor. These results indicate that apoptosis in C3H-10T1/2 cells follows a pathway that involves intracellular acidification, but is independent of detectable endonuclease activity.

We have developed a simple laboratory exercise that uses an inexpensive dialysis membrane (molecular weight cutoff = 100) to illustrate the generation of membrane potentials (Vm) across plasma membranes of animal cells. A piece of membrane approximately 2.0 cm2 is mounted in an Ussing-like chamber. One chamber half is designated cytosol and the other half external. Chamber sidedness helps students relate their findings to those of real cells. As in real cells, outward directed K+ concentration gradients [high cytosolic K+ concentration ([K+]c) and low extracellular K+ concentration] generate cytosol electrically negative Vm with a slope of approximately -45 mV/decade change in [K+]c. The polarity of Vm reflects the outward flow of potassium ions because flow of the larger counterion, H2PO4-, is restricted to the pores in the membrane. A slope less than Nernstian (<59 mV/decade) suggests that the membrane is slightly permeable to H2PO4-. Importantly, this facilitates teaching the use of the Nernst equation to quantify the relationship between ion concentration ratios across membranes and magnitude of Vm. For example, students use their data and calculate a permeability ratio PK/PH2PO4 that corresponds to a slope of approximately 24% less than Nernstian. This calculation shows that Nernstian slopes are achieved only when permeability to the counterion is zero. Finally, students use the concept of membrane capacitance to calculate the number of ions that cross the membrane. They learn where these ions are located and why the bulk solutions conform to the principle of electroneutrality.