Emilio Muñoz

Membrane ATPase of Escherichia coli K 12 Selective solubilization of the enzyme and its stimulation by trypsin in the soluble and membrane-bound states☆

Abstract ATPase (EC 3.6.1.3) of Escherichia coli has been solubilized from two morphologically distinct membranes (vesicles and “ghosts”). Maximum ATPase release is attained with 3 mM EDTA in NH4HCO3, pH 9.0, and depends on protein concentration. After solubilization, the total enzyme activity is increased by 300% with respect to the membrane-bound enzyme. The released soluble ATPase accounts for more than 90% of this activity. Its specific activity is at least 10 times higher than the original value. Membrane treatment with buffers of various ionic strengths without EDTA and detergents is less selective. The molecular sieving properties (gel electrophoresis and Sephadex G-200 filtration) confirm the soluble nature of the preparation. A molecular weight close to 300 000 has been estimated for it. The membrane-bound ATPase is stimulated by trypsin by 70–100%. Most of the soluble ATPase maintains a trypsin activation of the same order. Exceptions are the preparations obtained at high protein dilution and extracted with sodium dodecyl sulphate and deoxycholate. The soluble ATPase is more labile than the membrane-bound enzyme. Its sensitivity to different temperatures depends upon protein concentration and pH during storage. Inactivation seems to result from dissociation and/or proteolysis. We suggest an ATPase link to the membrane through ionic divalent cation bridges. We also suggest that the enzyme possesses self-regulatory properties which would account for trypsin stimulation.

Membrane adenosine triphosphatase of Micrococcus lysodeikticus. Isolation of two forms of the enzyme complex and correlation between enzymatic stability, latency and activity.

SummaryTwo new forms of the plasma membrane ATP-ase ofMicrococcus lysodeikticus NCTC 2665 were isolated from a sub-strain of the microorganism by polyacrylamide gel electrophoresis. One of them had a mol.wt of 368,000 and a very low specific activity (0.80 µ mol.min−1.mg protein−1) that could not be stimulated by trypsin. This form has been called BI (strain B, inactive). If the electrophoresis was carried out in the presence of reducing agents (i.e., dithiothreitol) and the pH of the effluent maintained at a value of 8.5 another form of the enzyme was obtained. This had a mol.wt of 385,000 and a specific activity of 2.5–5.0 µ mol.min−1.mg protein−1 that could be stimulated by trypsin to 5–10 µ mol.min−1.mg protein−1. This preparation of the ATPase has been called form BA (strain B, enzyme active). The subunit composition of both forms has been studied by sodium dodecyl sulphate and urea gel electrophoresis and compared to that of the enzyme previously purified from the original strain (form A). The three forms of the enzyme had similarβ and δ subunits, with mol.wt of about 50,000 and 30,000 dalton, respectively. They also had in common the component(s) of relative mobility 1.0, whose status as true subunit(s) of the enzyme remains yet to be established. However, subunitα, that had a mol.wt of about a 52,500 in form A (Andreuet al. Eur. J. Biochem. (1973) 37, 505–515), had a mol.wt similar toβ in form BI and about 60,000 in form BA. Furthermore BA usually showed two types of this subunit (α′ andα′′) and an additional peptide chain (ε) with a mol.wt of about 25,000 dalton. This latter subunit seemed to account for the stimulation by trypsin of form BA.Forms BA could be converted to BI by storage and freezing and thawing. Conventional protease activity could not be detected in any of the purified ATPase forms and addition of protease inhibitors to form BA failed to prevent its conversion to form BI. The low activity form (BI) was more stable than the active forms of the enzyme and also differed in its circular dichroism. These results show thatM. lysodeikticus ATPase can be isolated in several forms. Although these variations may be artifacts caused by the purification procedures, they provide model systems for understanding the structural and functional relationships of the enzyme and for drawing some speculations about its functionin vivo.

Glycoprotein nature of energy-transducing ATPases. Chemical characterization of glycopeptides isolated from bacterial and chloroplast coupling factors.

Evidence to support the notion that the ATPases or F, coupling factors from bacteria and organelles are glycoproteins has been presented [ 1,2]. The significance of this finding with regard to the peripheral location of these membrane proteins as well as to their role as phosphorylating coupling factors have been discussed [2]. A further characterization of the glycoprotein nature of these proteins has been attempted by extensive digestion with pronase and isolated glycopeptides by Sephadex filtration. This report describes the quantitative and qualitative characterization of these glycopeptides from Micrococcus lysodeikticus and chloroplast ATPase . The results further support to the idea that these coupling factors are glycoproteins.

Isolation and partial characterization of the two major subunits of the BF1 factor (ATPase) from Micrococcus lysodeikticus and evidence for their glycoprotein nature

Purified bacterial ATPases (BF1 coupling factors) resemble the soluble coupling factors, F1 and CF1, of mitochondria and chloroplasts [l-3] by their mol. wt. [4-7 ] , amino acid composition [4,6] and presence of two major subunits ((u and fi) of mol. wt. about 55 000 [4-91. The isolation and characterization of the subunits of different ATPase systems may help in further elucidating the functional and evolutionary significance of these energy-transducing proteins. The subunits of mitochondrial F1 ATPase were isolated in preparative scale and their molecular weights and amino acid compositions determined [lo] . Nelson et al. [ 1 I] isolated limited amounts of the CF1 subunits, characterized them chemically and prepared antisera against the isolated subunits. Recently Kanner et al. [ 121 prepared antisera against the three major subunits of Escherichia coli ATPase. In this paper, we describe for the first time the isolation in preparative scale of the two major subunits (a and 0) of a bacterial F1 factor and their partial characterization. We also present evidence suggesting the glycoprotein nature of the ATPase of M. lysodeikticus and their (11 and 0 subunits and discuss it in relationship to the microheterogeneity and sensitivity to alkali of this BFI factor [13,14].

Does Peer Review Predict the Performance of Research Projects in Health Sciences

Peer review is a basic component of the scientific process, but its performance has seldom been evaluated systematically. To determine whether pre-approval characteristics of research projects predicted the performance of projects, we conducted a retrospective cohort study of all 2744 single-centre research projects financed by the Spanish Health Research Fund since 1988 and completed before 1996. Peer review scores of grant applications were significant predictors of performance of funded projects, and the likelihood of production was also higher for projects with a basic research component, longer duration, higher budget or a financed research fellow. Funding agencies should monitor their selection process and assess the performance of funded projects to design future strategies in supporting health sciences research.

Molecular organization in bacterial cell membranes: I. Sodium dodecyl sulphate solubilization and fractionation of the components of a depleted membrane from Micrococcus lysodeikticus

Abstract A method is presented for the solubilization and fractionation of Micrococcus lysodeikticus membrane after it has been subjected to mild washing procedures (i.e. depleted membrane). The method involves the solubilization of the membrane, treatment with iodoacetamide and sodium dithionite and the action of sodium dodecyl sulphate on the membrane extracted with n-butanol. Polyacrylamide electrophoresis and Sephadex G-200 filtration in the presence of dodecyl sulphate result in the separation of at least eleven protein components whose molecular weights range from 17000 to 75000. Residual lipid and carbohydrate are found as components of the depleted membrane, and fractionated as a glycolipoprotein complex. A component tentatively identified as ribonucleic acid is also found associated with the membrane although its association with other membrane components appears to be broken by the detergent.

Micrococcus lysodeikticus membrane ATPase. effect of trypsin on stimulation of a purified form of the enzyme and identification of its natural inhibitor

A soluble purified form of Micrococcus lysodeikticus ATPase (form BAT, from strain B, active, trypsin-stimulated) was stimulated 100% by trypsin and this stimulation was inhibited by preincubation of the protease with phenyl methyl sulphonylfluoride. This form of the enzyme was also stimulated 125-150% by filtration on Sephadex G-200. Analysis by sodium dodecyl sulphate-gel electrophoresis showed that stimulation of this form of M. lysodeikticus ATPase was always accompanied by the disappearance of a subunit of mol. wt. 25000 (epsilon subunit). It suggests that this subunit is the natural inhibitor of M. lysodeikticus ATPase. In the case of ATPase stimulation by trypsin, a partial and limited degradation of the alpha subunit was also observed. The interaction between the epsilon subunit and the rest of the ATPase complex was reversibly affected by pH, suggesting its non-covalent nature.

Scientific Organizations in Spain (1950-1970): Social Isolation and International Legitimation of Biochemists and Molecular Biologists on the Periphery

Through a study of Spanish scientific organizations and the involvement of Spanish scientists in international ones, the beginnings of biochemistry and molecular biology in Spain are explored. The centre-periphery dichotomy is useful in explaining the progress of the establishment of these disciplines, together with the political context, both national and international, in which such establishment took place. It is suggested that international ties and national support are both necessary, but not sufficient conditions to overcome the distance between the Spanish scientific community and the centres of development and productivity in biochemistry and molecular biology. The interplay between both national and international factors has proved to be crucial. As a tool to study the whole scientific community of biochemists and molecular biologists, scientific societies allow us to analyze the role of that interplay in Spanish scientific development.

Biotechnology Development in Spain: A Change of Paradigm?

This article presents a quantitative and qualitative analysis of the Spanish biotechnology sector, including its scientific, business, policy and social aspects. The study differentiates two sub-sectors, considered to be the most representative: Biopharma and Agro-food. The results show that biotechnology development in Spain has entered a new phase. Traditionally, research and development in Spain followed an unstructured path built around regional spaces and driven by factors and influences external to Spain. However, the emergence of a fast-growing sector of spin-off companies from the public research system, specifically in the Biopharma sector, points to a change of model of development of the industry, on the national as well as the regional level. The data also show the key role which public policy plays in creating a suitable framework for innovation and point to the need of analyzing the influence of social and cultural factors on the development of the biotechnology sector.

Substructure of F1-ATPase (BF1 factor) from Micrococcus lysodeikticus

SummaryDimethyl suberimidate and dithiobis (succinimidyl propionate) have been used to explore the nearest neighbor relationship of the subunits (α, β, γ and δ by decreasing molecular weight) of F1-ATPase or BF1 factor of Micrococcus lysodeikticus. Cross-linking with the two diimido esters inhibited the ATPase activity but this inhibition never exceeded 50% of the initial value. The cross-linking pattern of this BF1 factor, as revealed by sodium dodecyl sulfate gel electrophoresis, shows a relative low proportion of high molecular weight aggregates which move slowly than the heaviest subunit (α). They are resolved as three components of molecular weights 200,000, 130,000 and 100,000 in 5% acrylamide gels, plus an additional component (mol. wt 80,000) identified in 10% acrylamide gels. The other aggregate bands represent cross-linking products of the smaller subunits (γ and δ) that may travel to the conventional position of the heavier subunits.The subunit composition of the aggregate bands has been determined through the reversion of dithiobis (succinimidyl propionate) cross-linking of the BF1 factor by dithiothreitol and analysis in second dimension by gel electrophoresis. The results indicate that γ subunit can cross-link with itself and with each of the other subunits except β. The α subunit is also able to cross-link with itself and with the other subunits although to a minor extent than γ, and that δ2 aggregates are present. These results represent a specific pattern of cross-linking for this BF1 factor as compared to other F1 coupling factors. It suggests a certain asymmetry in the spatial organization of the major subunits of M. lysodeikticus F1-ATPase where the γ subunit must play a central role. A subunit stoichiometry α3 β3 γ2 δ2 is proposed for whole F1-ATPase which leads to a molecular weight 440,000 consistent with the 430,000 value estimated by sedimentation equilibrium at low speed. A tentative structural model of M. lysodeikticus BF1 factor is derived from these data. The significance of the results in relation to the possible generalization of the molecular architecture of F1 factors is discussed.