Biosynthesis of 2-aceto-2-hydroxy acids: acetolactate synthases and acetohydroxyacid synthases

Two groups of enzymes are classified as acetolactate synthase (EC 4. 1.3.18). This review deals chiefly with the FAD-dependent, biosynthetic enzymes which readily catalyze the formation of acetohydroxybutyrate from pyruvate and 2-oxobutyrate, as well as of acetolactate from two molecules of pyruvate (the ALS/AHAS group). These enzymes are generally susceptible to inhibition by one or more of the branched-chain amino acids which are ultimate products of the acetohydroxyacids, as well as by several classes of herbicides (sulfonylureas, imidazolinones and others). Some ALS/AHASs also catalyze the (non-physiological) oxidative decarboxylation of pyruvate, leading to peracetic acid; the possible relationship of this process to oxygen toxicity is considered. The bacterial ALS/AHAS which have been well characterized consist of catalytic subunits (around 60 kDa) and smaller regulatory subunits in an alpha2beta2 structure. In the case of Escherichia coli isozyme III, assembly and dissociation of the holoenzyme has been studied. The quaternary structure of the eukaryotic enzymes is less clear and in plants and yeast only catalytic polypeptides (homologous to those of bacteria) have been clearly identified. The presence of regulatory polypeptides in these organisms cannot be ruled out, however, and genes which encode putative ALS/AHAS regulatory subunits have been identified in some cases. A consensus sequence can be constructed from the 21 sequences which have been shown experimentally to represent ALS/AHAS catalytic polypeptides. Many other sequences fit this consensus, but some genes identified as putative 'acetolactate synthase genes' are almost certainly not ALS/AHAS. The solution of the crystal structures of several thiamin diphosphate (ThDP)-dependent enzymes which are homologous to ALS/AHAS, together with the availability of many amino acid sequences for the latter enzymes, has made it possible for two laboratories to propose similar, reasonable models for a dimer of catalytic subunits of an ALS/AHAS. A number of characteristics of these enzymes can now be better understood on the basis of such models: the nature of the herbicide binding site, the structural role of FAD and the binding of ThDP-Mg2+. The models are also guides for experimental testing of ideas concerning structure-function relationships in these enzymes, e.g. the nature of the substrate recognition site. Among the important remaining questions is how the enzyme suppresses alternative reactions of the intrinsically reactive hydroxyethylThDP enamine formed by the decarboxylation of the first substrate molecule and specifically promotes its condensation with 2-oxobutyrate or pyruvate.     

A diagnostic formula for alcoholism

Clinical pathologies with unusually high morbidities in alcoholic populations were analyzed to determine their capacity to diagnose alcoholism. On the basis of five systemic variables it was possible to diagnose correctly nearly 75% of alcoholic and matched control subjects.     

The influence of cardiac frequency on the hemodynamic consequences of mitral stenosis (author's transl)

In eleven patients with isolated mitral stenosis and regular sinus rhythm a right cardiac catheterization was performed and the wedged pulmonary capillary pressure recorded at rest and during electrical pacing of the right atrium at successive frequencies of 100, 120, 140, and, occasionally, 160 and 180 beats/min, while cardiac output was estimated by the Fick's principle. In all cases a significant elevation of pulmonary capillary pressure with a simultaneous reduction in cardiac output was obtained. The rise of wedged pulmonary pressure was proportional to the increment in cardiac frequency and related also to the calculated area of the mitral valve. The influence of active atrial contraction upon pulmonary pressure and cardiac output is discussed and comparisons with other studies are made. Emphasis is made on the value of atrial pacing as a diagnostic method in mitral stenosis, especially in cases in whom classical effort manoeuvres can not be applied or are insufficient to rise cardiac frequency.     

Standardization of milk using cold ultrafiltration retentates for the manufacture of parmesan cheese

The effects of using cold ultrafiltered (UF) retentates (both whole and skim milk) on the coagulation, yield, composition, and ripening of Parmesan cheese were investigated. Milks for cheese making were made by blending cold UF retentates with partially skimmed milk to obtain blends with 14.2% solids and a casein:fat ratio of 1.1. Cutting times, as selected by the cheese-maker, were approximately 15 and approximately 20 min for experimental and control milks, respectively. Storage modulus values at cutting were similar, but yield stress values were significantly higher in UF retentate standardized milks. Cheese yields were significantly higher in UF retentate standardized milks (approximately 12%) compared with control milk (cream removed) (approximately 7 to 8%). Significantly higher protein recoveries were obtained in cheeses manufactured using cold UF retentates. There were no differences in the pH and moisture contents of the cheeses prior to brining, and there was no residual lactose or galactose left in the cheeses. Using UF retentates resulted in a significant reduction in whey volume as well as a higher proportion of protein in the solids of the whey. Proteolysis, free fatty acids, and sensory properties of the cheeses were similar. The use of milk concentrated by cold UF is a promising way of improving the yield of Parmesan cheese without compromising cheese quality. The question remaining to be answered by the cheesemaker is whether it is economical to do so.     

Use of multi-angle laser light scattering and size-exclusion chromatography to characterize the molecular weight and types of aggregates present in commercial whey protein products

In this study, a range of commercial whey protein products were characterized by the use of size-exclusion chromatography coupled with a multi-angle laser light scattering (MALLS) detector. The MALLS system detected some very large-sized material that eluted close to void volume in all samples; this material was hardly detected by concentration detector. It was demonstrated by chitosan treatment that this peak was very small lipid globules or phospholipids, which gave the residual "cloudy" appearance in upper layers after ultracentrifugation of whey products. Composition, molecular weight, and the photo diode array (PDA) spectrum (200 to 400 nm) of the major protein peaks, including: beta-lactoglobulin (BLG), alpha-lactalbumin (ALA), bovine serum albumin (BSA), immunoglobulin G (IgG) and some minor components were analyzed. The molecular weight of BLG, ALA, BSA, and IgG peaks in whey protein isolates (WPI) were 2.3 to 3.7 x 10(4), 1.4 to 1.6 x 10(4), 4.8 to 6.7 x 10(4), and 1.2 to 2.5 x 10(5) Da, respectively. Compared with WPI, WPC has similar major proteins, but more large-sized residual lipid material and different minor constituents such as lactose and nonprotein nitrogen, depending on various commercial samples and protein content. An improved TCA-precipitation method was applied to quantify glycomacropeptide (GMP) in whey proteins, which demonstrated that there was a very low concentration of GMP in WPI manufactured using an ion-exchange process. The molecular weight of GMP was found to be approximately 8600 Da. Size-exclusion chromatography MALLS was demonstrated to be a powerful technique for detailed analysis of the molecular weight of various proteins, aggregates and minor components, such as GMP, in whey protein products.     

trans-Dominant and non-trans-dominant mutant simian virus 40 large T antigens show distinct responses to ATP

Simian virus 40 (SV40) DNA replication requires the coordinated action of multiple biochemical activities intrinsic to the virus-encoded large tumor antigen (T antigen). We report the preliminary biochemical characterization of the T antigens encoded by three SV40 mutants, 5030, 5031, and 5061, each of which have altered residues within or near the ATP binding pocket. All three mutants are defective for viral DNA replication in cultured cell lines. However, while 5030 and 5031 can be complemented in vivo by providing a wild-type T antigen in trans, 5061 exhibits a strong trans-dominant-negative phenotype. In order to determine the basis for their replication defects and to explore the mechanisms of trans dominance, we purified the T antigens encoded by each of these mutants and examined their activities in vitro. The 5061 T antigen had no measurable ATPase activity and failed to hexamerize in response to ATP, and its affinity for the SV40 origin of DNA replication (ori) DNA was not increased by ATP. In contrast, the 5030 and 5031 T antigens exhibited at least some ATPase activity and both readily formed hexamers in the presence of ATP. These mutants differed in that 5030 was very defective in an ori-dependent unwinding assay while 5031 retained significant activity. Both the 5030 and 5031 T antigens bound to ori-containing DNA, but the binding was less efficient than that of wild-type T antigen and was not affected by the presence of ATP. These results suggest that 5030 and 5031 are defective in some aspect of communication between the ATP binding and DNA binding domains and that the ability of ATP to induce T-antigen hexamerization is distinct from its action to increase the affinity for ori. Finally, all three mutants were defective for the ability to support SV40 DNA replication in vitro. Both the 5031 and 5061 T antigens inhibited wild-type-T-antigen-stimulated replication in vitro, while the 5030 T antigen did not. The fact that the 5031 T antigen was trans dominant in the in vitro assays but not in vivo indicates that the in vitro system does not accurately reflect events occurring in vivo.