Determination of products of acetohydroxy acid synthase by the colorimetric method, revisited

The enzyme acetohydroxy acid synthase (AHAS, EC 4.1.3.18) catalyzes two competing reactions of physiological importance: condensation of two molecules of pyruvate to form acetolactate (AL) or condensation of pyruvate and 2-ketobutyrate to form acetohydroxybutyrate (AHB). The activity of AHAS is most frequently analyzed using the Westerfeld method, in which the acetoin formed upon decarboxylation of AL is determined by colorimetric reaction with creatine and alpha-naphthol. However, there has been confusion as to the interpretation of the results of this assay in the presence of both substrates, conditions which lead to formation of both AL and AHB. By applying this assay to enzymatically prepared samples of AL and AHB which have also been analyzed by two other independent methods, we show here that the color yield for AHB in the commonly used assay is 35-40% that for equivalent amounts of acetoin or AL. The relative color yield is not significantly affected by varying the time or temperature of various steps in the color-forming reaction. This information could in principle be used, together with an independent specific assay for AHB, to determine the composition of an AHAS product mixture; it would, however, be less accurate than a simultaneous chromatographic method.     

Kinetics and mechanism of acetohydroxy acid synthase isozyme III from Escherichia coli

Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) isozyme III from Escherichia coli has been studied in steady-state kinetic experiments in which the rates of formation of acetolactate (AL) and acetohydroxybutyrate (AHB) have been determined simultaneously. The ratio between the rates of production of the two alternative products and the concentrations of the substrates pyruvate and 2-ketobutyrate (2KB) leading to them, R, VAHB/VAL = R[( 2KB]/[pyruvate]), was found to be 40 +/- 3 under a wide variety of conditions. Because pyruvate is a common substrate in the reactions leading to both products and competes with 2-ketobutyrate to determine whether AL or AHB is formed, steady-state kinetic studies are unusually informative for this enzyme. At a given pyruvate concentration, the sum of the rates of formation of AL and AHB was nearly independent of the 2-ketobutyrate concentration. On the basis of these results, a mechanism is proposed for the enzyme that involves irreversible and rate-determining reaction of pyruvate, at a site which accepts 2-ketobutyrate poorly, if at all, to form an intermediate common to all the reactions. In the second phase of the reaction, various 2-keto acids can compete for this intermediate to form the respective acetohydroxy acids. 2-Keto acids other than the natural substrates pyruvate and 2-ketobutyrate may also compete, to a greater or lesser extent, in the second phase of the reaction to yield alternative products, e.g., 2-ketovalerate is preferred by about 2.5-fold over pyruvate. However, the presence of an additional keto acid does not affect the relative specificity of the enzyme for pyruvate and 2-ketobutyrate; this further supports the proposed mechanism. The substrate specificity in the second phase is an intrinsic property of the enzyme, unaffected by pH or feedback inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The specificity of fumarate as a switching factor of the bacterial flagellar motor

Fumarate restores to flagella of cytoplasm-free, CheY- containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another. To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues. Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate. These observations were made both with wild-type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching-promoting activity of the analogues was not caused by their conversion to fumarate. Aspartate and lactate did not promote switching. Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemo-taxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate). All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.     

Conditional Maximum Likelihood Estimation Following a Group Sequential Test

We consider estimation after a group sequential test. An estimator that is unbiased or has small bias may have substantial conditional bias (Troendle and Yu, 1999, Coburger and Wassmer, 2001). In this paper we derive the conditional maximum likelihood estimators of both the primary parameter and a secondary parameter, and investigate their properties within a conditional inference framework. The method applies to both the usual and adaptive group sequential test designs. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

POPULATION STRUCTURE IN ZAMIA DEBILIS (ZAMIACEAE) I. SIZE CLASSES,LEAF PHENOLOGY,AND LEAF TURNOVER

Population structure, leaf phenology and leaf turnover were followed over a 29-month period in Zamia debilis L.f. ex Aiton (Zamiaceae), an understory species in the Cambalache Forest in northern Puerto Rico. It was not possible to determine plant age or to measure the subterranean stems; size classes based on leaf number and leaf × leaflet number indices were used to determine population structure. Despite seasonal and year to year fluctuations in leaf number at the individual and population level, population profiles remained relatively constant. At any one time, over 50% of the population was composed of unbranched individuals with one or two leaves. Only 7% of the plants were branched. Plants with seven or more leaves comprised at a maximum 8% of the population, but accounted for 28% of the total foliage. Size classes based on leaf number and on a leaf × leaflet index gave approximately reverse J-shaped curves typical of trees with shade tolerant seedlings and saplings. New leaves emerged throughout the year, with a peak at the beginning of the rainy season in May or June and lowest production during the dry months of February through April. Average leaf life expectancy was approximately 2.3 years. Leaf death occurred over an extended period of time by the loss of individual leaflets. Patterns in leaf production and loss differed between few- and many-leaved plants. On the average, as the number of mature leaves on a plant increased, time between emergence of new leaves decreased. In many-leaved plants more than one event of new leaf emergence per year was common. Individuals with one to three mature leaves and individuals with four or more mature leaves differed in their response to water stress: few-leaved plants generally reduced the rate of new leaf production and retained old leaves longer. Plants with more than three leaves continued to produce new leaves, but the rate of leaf mortality increased so that most had a net leaf loss. There was no evidence that leaf emergence or retention were affected by cone production or seed maturation.     

Enhanced binding of a 95 kDa protein to p53 in cells undergoing p53-mediated growth arrest.

To explore the biochemical functions of p53, we have initiated a search for cellular p53-binding proteins. Coprecipitation of three polypeptides was observed when cell lines overexpressing a temperature-sensitive (ts) p53 mutant were maintained at 32.5 degrees C (wild-type p53 activity, leading to growth arrest) but not at 37.5 degrees C (mutant p53 activity). One of these three proteins, designated p95 on the basis of its apparent molecular mass, was highly abundant in p53 immune complexes. We demonstrate herein that p95 is a p53-binding protein, which exhibits poor p53-binding in cells overproducing several distinct mutant p53 proteins. Yet, p95 associates equally well with both the wild-type (wt) and the mutant conformations of the ts p53 in transformed cells growth-arrested at 32.5 degrees C. On the basis of our findings we suggest that wt p53 activity increases p53-p95 complex formation and that such interaction may play a central role in p53 mediated tumour suppression.     

Parameters affecting the in vitro maturation of human monocytes to macrophages

In vitro maturation of human monocytes to macrophages was characterized by morphological criteria, cell size and lysosomal enzymes activity. Purified populations of monocytes were maintained in culture at either adherent or nonadherent conditions and their maturation to macrophages was observed in both cases. The addition of external factors such as hydrocortisone and vitamin D3 inhibited monocyte maturation. In the absence of external factors, nonadherent monocytes were inhibited in their maturation for up to 10 days when plated at crowded cell concentrations. In addition, the presence of human serum in the culture media had a higher inhibitory activity than similar concentrations of fetal calf serum. Supernates from crowded macrophages were also inhibitory for monocyte maturation. We suggest the possibility that cell crowding, as well as soluble factors found in the serum and probably secreted by macrophages, participate in the regulation of monocyte development by inhibiting their maturation. Once released from this inhibitory signal or environment, the monocytes mature to macrophages.     

A line-balancing strategy for designing flexible assembly systems

We present a rough-cut analysis tool that quickly determines a few potential cost-effective designs at the initial design stage of flexible assembly systems (FASs) prior to a detailed analysis such as simulation. It uses quantitative methods for selecting and configuring the components of an FAS suitable for medium to high volumes of several similar products. The system is organized as a series of assembly stations linked with an automated material-handling system moving parts in a unidirectional flow. Each station consists of a single machine or of identical parallel machines. The methods exploit the ability of flexible hardware to switch almost instantaneously from product to product. Our approach is particularly suitable where the product mix is expected to be stable, since we combine the hardware-configuration phase with the task-allocation phase. For the required volume of products, we use integer programming to select the number of stations and the number of machines at each station and to allocate tasks to stations. We use queueing network analysis, which takes into account the mean and variance of processing times among different products to determine the necessary capacity of the material-handling system. We iterate between the two analyses to find the combined solution with the lowest costs. Work-in-process costs are also included in the analysis. Computational results are presented.