Bifunctional derivatives of the alkaloid galanthamine, designed to interact with both the active site of the enzyme acetylcholinesterase (AChE) and its peripheral cation binding site, have been assayed with Torpedo californica AChE (TcAChE), and the three-dimensional structures of their complexes with the enzyme have been solved by X-ray crystallography. Differences were noted between the IC(50) values obtained for TcAChE and those for Electrophorus electricus AChE. These differences are ascribed to sequence differences in one or two residues lining the active-site gorge of the enzyme. The binding of one of the inhibitors disrupts the native conformation of one wall of the gorge, formed by the loop Trp279-Phe290. It is proposed that flexibility of this loop may permit the binding of inhibitors such as galanthamine, which are too bulky to penetrate the narrow neck of the gorge formed by Tyr121 and Phe330 as seen in the crystal structure.

In accordance with previous results, the activity of extracellular proteases from Pseudomonas fluorescens MFO is maximal at a growth temperature of 17.5°C, well below the optimal growth temperature. In addition, the activities of three periplasmic phosphatases display the same growth temperature optimum. Chemostat experiments have shown that it is the growth temperature itself and not the value of the growth rate that regulates these activities. In contrast, a foreign periplasmic phosphatase, expressed under the control of its own promoter, displays a different sensitivity toward temperature. We conclude that in the psychrotrophic strain P. fluorescens MFO, growth temperature exerts a specific control upon the activity of certain enzymes. The critical temperature (17.5C) is within the range of normal growth, suggesting that this control is probably different from a cold shock or heat shock response.Pseudomonas fluorescens is well known as a major psychrotrophic contaminant of raw milk stored in refrigerated tanks (16). Many studies of this bacterium have been concerned with the production of deleterious extracellular enzymes, such as thermostable proteases (8,15,27). Among the numerous observations concerning these enzymes, it has been repeatedly shown that most strains maximally produce proteases at a temperature (15 to 20°C) well below the optimal growth temperature (25 to 30°C) (13,19,25). However, no studies have yet dealt with the mechanism of regulation of protease production with regard to temperature.At this stage two main questions can be raised regarding the elucidation of this mechanism. The first one relates to the specificity of this temperature effect, i.e., whether it is restricted to the production of proteases or extended to the production of other enzymes. To this end, the activities of extracellular proteases as a function of growth temperature were compared with those of several periplasmic phosphatases. The exported enzymes all showed the same regulation by temperature even though they are clearly differentially regulated by other growth conditions. Thus, it was important to determine whether this temperature effect might involve protein export through the cytoplasmic membrane. If so, any foreign exported protein should be submitted to the same effect. The expression of a gene from the mesophilic species Escherichia coli, under the control of its own promoter, was studied in P. fluorescens at different growth temperatures. In this case, a temperature effect similar to that observed with the native enzymes was not demonstrated.The second question is whether the temperature itself is the direct cause of the regulation or an indirect effector acting through the growth rate variation; such an indirect effect has indeed been demonstrated for several activities or proteins in mesophilic bacteria (5). To answer this question, the activities of the two acidic phosphatases were assayed in cells grown in a chemostat at two different temperatures and * Corresponding author. several dilution rates. The ...

The variations in the maximal specific growth rate of the psychrotrophic bacterium Pseudomonas fluorescens MF0 with respect to temperature were studied between 0 and 30؇C (optimal for growth). The Arrhenius plot showed a drastic change in slope at the intermediate temperature of 17؇C. Over the cold domain from 0 to 17؇C, the temperature characteristic was twofold higher than over the suboptimal domain from 17 to 30؇C. The macromolecular composition of exponentially growing cells was invariant over the entire range from 0 to 30؇C. Variations of temperature and growth rate were independently investigated through chemostat experiments in order to characterize their respective effects on cell macromolecular composition and size. The effect of growth rate in this psychrotrophic strain is identical to that of all other bacteria assayed so far. In contrast, an original biphasic variation of total protein concentration was demonstrated in strain MF0 with respect to temperature, with a maximum at 17 to 20؇C. Indeed, increasing the temperature in the chemostat resulted in a biphasic decrease in the net protein production rate: a very slight decrease below 17؇C and a much larger decrease from 17 to 28؇C. These results could signify an increase in the cellular protein degradation rate with increasing temperature, especially above 17؇C.

At chill temperatures, Psez&monas ji'uorescens produces inducible extracellular protease and lipase. Their regulation by growth temperature and conditions was studied in chemostat experiments and by the use of a transcriptional fusion in the structural lipase gene. The findings suggest two levels of temperature regulation of lipase production, resulting in maximal activity at about 17°C well below the optimum for growth.