Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2

An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.     

Amino acid requirements and peptidase activities of Streptococcus salivarius subsp. thermophilus

The aim of this work was to investigate the relationship between amino acid requirements and peptidase enzyme systems in three Streptococcus salivarius subsp. thermophilus strains. A synthetic medium without nitrogen components and a milk (RD milk) without its non-protein nitrogen fraction were prepared with different mixtures of amino acids. The strains showed different amino acid requirements. Some amino acids proved to be essential, some were required, while others did not affect growth. In the synthetic medium, only leucine and glutamic acid were essential for growth. In RD milk, the amino acid requirements were found to be lower, with only the absence of glutamic acid causing complete inhibition of growth. Relationships between aminopeptidase activities of the strains and their amino acid requirements were observed. Strains with higher amino acid requirements were also found to express a wider range of peptidases.     

Asymmetric bilayers mimicking membrane rafts prepared by lipid exchange: Nanoscale characterization using AFM-Force spectroscopy

Sphingolipids-enriched rafts domains are proposed to occur in plasma membranes and to mediate important cellular functions. Notwithstanding, the asymmetric transbilayer distribution of phospholipids that exists in the membrane confers the two leaflets different potentials to form lateral domains as next to no sphingolipids are present in the inner leaflet. How the physical properties of one leaflet can influence the properties of the other and its importance on signal transduction across the membrane are questions still unresolved. In this work, we combined AFM imaging and Force spectroscopy measurements to assess domain formation and to study the nanomechanical properties of asymmetric supported lipid bilayers (SLBs) mimicking membrane rafts. Asymmetric SLBs were formed by incorporating N-palmitoyl-sphingomyelin (16:0SM) into the outer leaflet of preformed 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Cholesterol SLBs through methyl-β-cyclodextrin–mediated lipid exchange. Lipid domains were detected after incorporation of 16:0SM though their phase state varied from gel to liquid ordered (Lo) phase if the procedure was performed at 24 or 37 °C, respectively. When comparing symmetric and asymmetric Lo domains, differences in size and morphology were observed, with asymmetric domains being smaller and more interconnected. Both types of Lo domains showed similar mechanical stability in terms of rupture forces and Young's moduli. Notably, force curves in asymmetric domains presented two rupture events that could be attributed to the sequential rupture of a liquid disordered (Ld) and a Lo phase. Interleaflet coupling in asymmetric Lo domains could also be inferred from those measurements. The experimental approach outlined here would significantly enhance the applicability of membrane models.     

Mechanical characterization of 3D printed mimic of human artery affected by atherosclerotic plaque through numerical and experimental methods

Biomechanics and Modeling in Mechanobiology - This paper proposes a novel experimental investigation based on 3D printing to validate numerical models for biomechanics simulations. Soft elastomeric...     

Prime Ricerche sulla Fisionomia Enzimatica Caratteristica della Fotosintesi

Summary

The rate of oxidation of glucose-6-phosphate, ribose-5-phosphate, fructose-1,6- phosphate, iso-citrate and malate in extracts from green and etiolated pea leaves was determined, using the triphenyltetrazolium technique.

Glucose-6-phosphate, ribose-5-phosphate, and fructose-1,6-phosphate, in the presence of added TPN, where oxidized at a rate about twice higher in the extracts from green than in the extracts from etiolated leaves. Iso-citrate, in the presence of TPN, fructose-1,6-phosphate, in the presence of DPN, and malate, in the presence of either TPN or DPN, were oxidized at about the same rate in the two types of extracts.

These data seem to indicate a preferential synthesis of enzymes involved in the metabolic cycle of phosphorylated sugars during the transition of the leaf from the etiolated to the photosynthetising physiognomy. They seem also favourable to the view assigning to this metabolic system a primary importance in the anabolic pathway of photosynthesis.     

Vie di ossidazione della frazione extramitocondriale dei coenzimi piridinici: I. - Sull'Efficienza In Vivo del sistema ascorbato/deidroascorbato

Abstract

OXIDATION PATHWAYS OF EXTRAMITOCHONDRIAL PIRIDINE COENZYMES. I. - ON THE « IN VIVO » EFFICIENCY OF THE ASCORBATE-DEYHDROASCORBATE SYSTEM. — An evaluation of the efficiency in vivo of the AA-DHA couple as an electron carrier system has been attempted, by measuring after short time of anaerobiosis the rate of the increase of AA and of the dicrease of DHA in etiolated pea internode segments and in potato tuber disks. The changes of reduced glutathione (GSH) contents as induced by anaerobiosis or by the addition of DHA to the incubation medium were also followed.

In the pea segments anaerobiosis induced a significant increase of AA and a corresponding decrease of DHA. These changes were almost completed after 10 minutes from starting anaerobiosis. The value (extrapolated to 0 time) of the initial rate of DHA desappearance under anaerobiosis was taken as representing the rate of DHA reduction to AA « in vivo », under aerobic conditions. As this rate — in a steady state situation — corresponds to that of the inverse process of oxidation of AA to DHA, this value should give and indication on the « in vivo » efficiency of the AA-DHA system as an electron carrier in respiration. As some AA was probably reoxidized to DHA in the very short period required to kill the tissue, the value of the AA DHA turnover thus calculated is probably somewhat lower than the real one.

According to the present work, the oxidative turnover of the AA-DHA system would results of 0,7 micromoles/g. fr. weght/h. for the pea internode tissues and of 0,9 micromoles/g. fr. weght^?h for the potato tuber (aged disks). These values would account for 5% of total oxygen uptake, in the former, and for 3% in the latter material.

The very high AA/DHA ratio usually prevailing in living cells suggests that the contents in DHA (and thus the activity of the AA oxidizing systems) is a limiting factor for the efficiency of the AA-DHA system as an electron carrier. This view is supported also by experiments in which DHA (at pH 5) was fed to pea internode segments and to potato tuber disks : as the presence of DHA into the medium induced — under anaerobiotic conditions — a rapid increase of the level of AA in both types of materials. In aerobiosis uptake and reduction of DHA to AA was evident in the potato tuber tissue, while it appeared very scarce in the pea internodes. As an interpretation of this behaviour it is suggested that, in aerobiosis, the very active and probably surface localized ascorbic acid oxidase of the pea tissue re-oxidises the AA formed from reduction of the DHA fed; an accumulation of DHA into the cells would follow, and this excess of DHA would inhibit the enzyme GSH-DHA reductase. This enzyme, in fact, appears, from « in vitro » experiments, to be strongly inhibited by DHA when the DHA/GSH ratio becomes higher than 1. On the other hand, the same hypothesis is also supported by the finding that the addition of DHA to the medium induces a significant drop in the GSH level (probably due to its oxidation to GSSG) only under those conditions in which DHA is absorbed and reduced to AA; that is, in the pea internodes, under anaerobiosis, and in the potato disks, under both anaerobiosis and aerobiosis. These results are also taken as confirming the indication from the enzymatic data that GSH is acting, in vivo as a reducing agent for DHA. The results of this investigation are thus interpred as showing that a comparatively small, but by no means negligeable fraction of respiration is mediated, in higher plant tissues such as those of the pea stem and the potato tuber, by and electron transfer system including glutathione and the ascorbate-dehydroascorbate couple. The efficiency of this system in the materials investigated appears to account for 3–5% of the total 0₂ uptake (minimum value). As enzyme systems transferring electrons from TPNH to ox. glutathione are widely distributed and generally very active in higher plant tissues, it is suggested that the sequence TPNH-GSH-AA/DHA - O₂ is probably of considerable importance in mediating the reoxidation of extramitochondrial trophosphoridine nucleotide and thus in permitting the operation of the TPN requiring pentose phosphate pathway of respiration.     

Reduction of arsenic content in a complex galena concentrate by <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

Background  

Bioleaching is a process that has been used in the past in mineral pretreatment of refractory sulfides, mainly in the gold, copper and uranium benefit. This technology has been proved to be cheaper, more efficient and environmentally friendly than roasting and high pressure moisture heating processes. So far the most studied microorganism in bioleaching is Acidithiobacillus ferrooxidans. There are a few studies about the benefit of metals of low value through bioleaching. From all of these, there are almost no studies dealing with complex minerals containing arsenopyrite (FeAsS). Reduction and/or elimination of arsenic in these ores increase their value and allows the exploitation of a vast variety of minerals that today are being underexploited.     

Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis by SDS-PAGE of cell-wall proteins

AIMS: In the present study, a method based on SDS-PAGE fingerprinting of surface layer proteins was developed to identify Lactobacillus delbrueckii subsp. bulgaricus and subsp. lactis dairy isolates. METHODS AND RESULTS: The two subspecies, identified by species-specific PCR, were characterized by different SDS-PAGE cell-wall protein profiles; subspecies bulgaricus showed one band of about 31 kDa which, in some cases, was observed at a doublet, and subspecies lactis showed one band of about 21 kDa or 18 kDa. CONCLUSION: The sensitivity of this procedure for discriminating between the two subspecies was very high. The different types of SDS-PAGE profile for cell-wall proteins of the strains studied in this work did not seem to be correlated to the different dairies of origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The method appears to be an efficient taxonomic tool. It has the advantage of easy gel interpretation over fingerprinting of whole-cell protein extracts, and may be used as an alternative to established PCR-based techniques which, though rapid and safe, require expensive instruments and reagents.     

Lactobacillus helveticus heterogeneity in natural cheese starters: the diversity in phenotypic characteristics

The study of wild strains from natural habitats is a useful means of understanding better the heterogeneity within a species of biotechnological importance, and of obtaining atypical isolates with unknown capabilities. In the present research carried out on different Lactobacillus helveticus strains isolated from natural cheese starters, it was observed that several biotechnologically important characteristics can differ greatly between strains. Biotypes were found which differ in terms of fructose, maltose and trehalose fermentation, acidifying activity, proteolytic and peptidase activity, and antibiotic and lysozyme resistance. The possibility of choosing Lact. heleveticus strains with specific biotechnological profiles will influence the quality and the variety of dairy products.