Characterization of Solanum tuberosum Multicystatin and Its Structural comparison with Other Cystatins

Potato (Solanum tuberosum) multicystatin (PMC) is a crystalline Cys protease inhibitor present in the subphellogen layer of potato tubers. It consists of eight tandem domains of similar size and sequence. Our in vitro results showed that the pH/PO₄⁻-dependent oligomeric behavior of PMC was due to its multidomain nature and was not a characteristic of the individual domains. Using a single domain of PMC, which still maintains inhibitor activity, we identified a target protein of PMC, a putative Cys protease. In addition, our crystal structure of a representative repeating unit of PMC, PMC-2, showed structural similarity to both type I and type II cystatins. The N-terminal trunk, α-helix, and L2 region of PMC-2 were most similar to those of type I cystatins, while the conformation of L1 more closely resembled that of type II cystatins. The structure of PMC-2 was most similar to the intensely sweet protein monellin from Dioscorephyllum cumminisii (serendipity berry), despite a low level of sequence similarity. We present a model for the possible molecular organization of the eight inhibitory domains in crystalline PMC. The unique molecular properties of the oligomeric PMC crystal are discussed in relation to its potential function in regulating the activity of proteases in potato tubers.     

Resolving UV Bioactinometric Results with Intensity and Time Distributions

A UV reactor with an annular design, a total liquid volume of 460[emsp4 ]ml, and outfitted with a single lamp with 1690[emsp4 ]mW of germicidal power was tested. Coliphage MS2 was used as a bioactinometer to measure the UV dose at a flow rate of 56.7[emsp4 ]ml/sec in water with a very low absorbance. The Beers Law coefficient was A₁₀0.003. The measured dose (MS2 bioactinometry) was 35.2±1.1[emsp4 ]mW-sec/cm².A retention time distribution was generated with a dye tracer study. The reactor was modeled as if flow was confined to ten equal volume paths existing as concentric rings around the lamp. The UV intensity along each path (ith intensity) was calculated to generate a simulated distribution of UV intensity in the reactor. The retention time distribution was subdivided to estimate the retention time associated with each decile jth time) of the total flow.Seven methods of associating the ith intensity with the jth retention time were used to produce simulated dose distributions for the reactor. The average UV dose for each distribution was calculated as the average of the products of I and t (AP protocol) and by the apparent survival (AS protocol), in which the predicted survival along each path was averaged to back-calculate dose from the reference batch inactivation curve. The average dose predicted assuming that time and intensity were independent was 51.5[emsp4 ]mW-sec/cm² based on the arithmetic average (AP protocol). Using the apparent survival method, the predicted dose for the independent distribution (I independent of t) was 36.4[emsp4 ]mW-sec/cm². Three methods of developing dependent structure between time and intensity were tested. In the best possible case for stratified flow (I negatively correlated with t) the calculated (AS) intensity was 46.3[emsp4 ]mW-sec/cm^2. In the worst case for stratified flow (I positively correlated with t) the AS intensity was 32.0[emsp4 ]mW-sec/cm². In a rational case where flows were assumed to be distributed parabolically (low flow at the wall and at the lamp) produced an AS intensity of 37.7[emsp4 ]mW-sec/cm². When either time or intensity was averaged, while the other variable was allowed to keep its distribution, the (AS) dose (time averaged 43.3[emsp4 ]mW-sec/cm², intensity averaged 41.0[emsp4 ]mW-sec/cm²), yielded a poor prediction compared to the measured value.The errors associated with averaging time, intensity, or both, far outweigh the errors associated with choosing a rational distribution or an independent distribution of time and intensity in the prediction. This observation is generally true whenever an organism is exposed to UV light in a flow through reactor such that the range of doses is within the portion of the inactivation curve exhibiting strong exponential decay.