Milton R.J. Salton

Biosynthesis of cardiolipin in the membranes of Micrococcus lysodeikticus

Abstract An enzyme which catalyzes the conversion of phosphatidyl glycerol to cardiolipin (diphosphatidyl glycerol) has been found to reside solely in the membrane of Micrococcus lysodeikticus . This enzyme has been tentatively designated as “cardiolipin synthetase”, since its reaction mechanism has not been defined. It appears to synthesize one molecule of cardiolipin from two molecules of phosphatidyl glycerol and the conversion has been followed by using an assay employing 32 P-labelled phosphatidyl glycerol as the only added substrate. Higher than 90% conversion of phosphatidyl glycerol to cardiolipin has been achieved with isolated membranes as the enzyme source and by stimulation with Triton X-100. A low-density, small particle fraction bearing “cardiolipin synthetase” activity has been released from whole membranes. Such preparations exhibit activity in the absence of detergent and are devoid of phosphatidic acid cytidyltransferase activity found in whole membranes. In contrast to several other phospholipid synthesizing enzymes reported 1–4 , the synthesis of cardiolipin by this enzyme(s) is not dependent upon added Mg 2+ or K + . Cardiolipin synthesis proceeds in the absence of detectable CDP-diglyceride and without significant degradation of phosphatidyl glycerol to phosphatidic acid.

Solubility characteristics of Micrococcus lysodeikticus membrane components in detergents and chaotropic salts analyzed by immunoelectrophoresis.

In order to evalute the effectiveness and selectivity of various reagents in the solubilization of bacterial membranes, membranes of Micrococcus lysodeikticus were treated with detergents and chaotropic agents. The composition of the extracts so obtained was analyzed by rocket and two-dimensional immunoelectrophoretic techniques. Recoveery of succinate-, malate-, and reduced nicotinamide adenine dinucleotide- (NADH) dehydrogenases, ATPase, succinylated lipomannan and cytochromes in the extracts was measured. Treatment with a variety of non-denaturing detergents produced extracts that were generally qualitatively uniform although quantitative differences were observed. The degree of extraction of various components was correlated with the hydrophile-lipophile balance. Several chaotropic agents were also evaluated as reagents for membrane solubilization. These agents were less effective in extraction of bulk protein, but produced extracts enriched in some membrane components.

The effects of low concentrations of glutaraldehyde on Micrococcus lysodeikticus membranes: Changes in the release of membrane-associated enzymes and membrane structure

Abstract The manner in which enzymes are associated with the plasma membrane of Micrococcus lysodeikticus can be modified in certain instances by the action of an aldehyde cross-linking agent. Enzymes such as ATPase (EC 3.6.1.3), NADH dehydrogenase (EC 1.6.99.3) and polynucleotide phosphorylase (EC 2.7.7.8) can normally be selectively released from the membrane by controlled washing with buffers. This release phenomenon is prevented when membranes are first treated with 0.5% (v/v) glutaraldehyde. Under these conditions the enzymes become more strongly attached to the membrane while retaining activity. In the case of NADH dehydrogenase it can be shown that the total activity is unchanged after its association with the membrane has been strengthened by this treatment. The inhibition of enzyme release is supported by the results of polyacrylamide gel electrophoresis and by the fact that glutaraldehyde treated membranes constitute a larger fraction of total cell protein than untreated cell membranes. Electron microscopy reveals that the selective release of enzymes from untreated membranes is associated with a progressive reduction in granular substructure on the membrane surface. The smooth surfaced residue which remains, contains cytochromes and succinate dehydrogenase and is resistant to further release by washing. Membranes treated with glutaraldehyde retain the granular substructure despite the washing procedures.

Subunit structure and properties of two forms of adenosine triphosphatase released from Micrococcus,lysodeikticus membranes

Abstract The Ca2+-activated ATPase of M. , lysodeikticus membrane has been released in two forms: (i) a “shock wash” form which is trypsin stimulated, capable of rebinding to ATPase-depleted membranes in the presence of Mg2+ and possesses additional associated protein(s) giving rise to 1–3 minor protein bands in addition to the two major subunits detected by SDS-polyacrylamide gel electrophoresis: (ii) a form released into the aqueous phase on extracting the membranes with n -butanol (referred to as “ n -butanol type”), which is not stimulated by trypsin, dose not rebind to depleted membranes and contains only two major protein subunits. The two protein subunits of both types have molecular weights of 62, 000 and 60, 000 daltons and reduction followed by alkylation or performic oxidation does not appear to significantly alter the behavior of these subunits. It is suggested that the additional proteins(s) of the shock wash ATPase have attachment and regulatory functions.

A succinylated mannan in the membrane system of Micrococcus lysodeikticus

Abstract A mannan has been isolated from membranes of Micrococcus lysodeikticus (NCTC 2665) grown in the presence of [1, 4 14 C] succinic acid. Hydrolysis of this polysaccharide with 0.1 N NaOH yielded a compound soluble in both diethyl ether and distilled water. This component was identified as [ 14 C] succinic acid by paper chromatography of both the free acid and of its corresponding hydroxamic acid derivative. Semiquantitative analysis indicated an ester-linked succinic acid content for mannan of approximately 2.5%.

Purification of a hemagglutinin from Limulus polyphemus by affinity chromatography.

Summary A. hemagglutinin has been isolated from the hemolymph of the common horseshoe crab ( Limulus polyphemus ) using affinity chromatography as the primary purification procedure. The hemagglutinin specifically binds to a bovine submaxillary mucin (BSM) affinity gel and is subsequently released upon the addition of Na citrate to the elution buffer. The purity of the final product was attested to by polyacrylamide gel electrophoresis, electron microscopic observation and by a greater than 1500-fold increase in specific activity as compared to the starting material. The purified hemagglutinin was able to give a precipitin reaction in immunodiffusion gels against purified BSM but not against desialated BSM.

Localization and distribution of Microccus lysodeikticus membrane ATPase determined by ferritin labeling

Antiserum to Ca2+-activated ATP phosphohydrolase (EC 3.6.1.3) isolated and purified from membranes of Micrococcus lysodeikicus was prepared in rabbits and guinea pigs. The γ-globulin fractions of these antisera reacted with and inhibited ATPase activity in isolated membranes but failed to absorb to intact protoplasts or purified mesosome fractions. ATPase activity was not detectable in the purified mesosomal preparations and trypsin treatment and sonication failed to release any activity. Ferritin conjugated to the γ-globulin fractions of the antiserum reacted with the ATPase particles on the membrane as visualized in negatively stained preparations examined in the electron microscope. Labeled membranes showed a distribution of ferritin very similar to the patterns observed for ATPase particles on untreated membranes. No significant labeling occurred when the ferritin conjugate was reacted with intact protoplasts or mesosome fractions. Thin sections of ferritin-labeled membranes established the asymmetric disposition of the ATPase, with the conjugate visible on only one side of the membrane. The results indicate that the ATPase protein occurs on the inner face of the membrane. All labeling experiments were verified immunologically. When ferritin-labeled membranes were subjected to the selective release procedure used in releasing the ATPase-like particles from the membranes, a complex of ferritin-conjugate associated with the ATPase particles was released. The selective release of ferritin-antibody-enzyme complexes from the membrane opens up a new way of studying the molecular architecture of cell membranes.

Electron transport components localized in a lipid-depleted sheet isolated from Micrococcuslysodeikticus membranes by deoxycholate extraction☆

Extraction of isolated membranes from Micrococcuslysodeikticus with 1% deoxycholate in 0.05M tris buffer yields an insoluble residue which accounts for 15% of the dry weight of the membrane. This fraction is composed largely of protein and contains 3–5% residual lipid compared to 23–26% for the initial membrane lipid and consequently has a higher density than the membranes on gradient centrifugation. These lipid-depleted residues are in the form of a membranous sheet when examined in the electron microscope. Cytochromes a, b and c and succinic dehydrogenase activity were localized almost exclusively in this deoxycholate insoluble fraction of the membrane.

Effects of diphenylamine on carotenoids and menaquinones in bacterial membranes.

Abstract When Micrococcus lysodeikticus was grown in the presence of 12.5, 25 and 50 μg diphenylamine per ml medium, the formation of the more conjugated carotenoids in the membranes was inhibited to the extent of 66–97%; the corresponding percentage inhibition values for Sarcina lutea membranes ranged from 83% to 98%. Menaquinone-9 was identified in the membranes of M. lysodeikticus and S. lutea and the contents of this compound in the isolated membranes were 7.8–12.1 μmoles/g and 3.8–4.4 μmoles/g, respectively, thus constituting up to about 4% of the membrane lipid. Bacillus licheniformis, Bacillus megaterium and Bacillus subtilis possessed menaquinone-7 in their membranes and the contents were 3.2, 8.4 and 10.0 μmoles per g membrane, respectively. At diphenylamine concentrations of 25–50 μg per ml medium, which reduced the carotenoid contents by 90% or more, the effects on the menaquinone contents of M. lysodeikticus and S. lutea membranes were variable, showing a reduction of 3–23% at a diphenylamine concn. of 50 μg per ml while at the 12.5 μg diphenylamine per ml level the contents were 12–34% higher than those of the control preparations. With the non-pigmented B. megaterium , diphenylamine concentrations of 12.5 and 25 μg per ml medium reduced the content of menaquinone-7 by about 10%. Diphenylamine was taken up by both growing cells and washed cell suspensions and was detected in the membrane lipid fraction. Diphenylamine was specifically localized in the membrane fractions isolated from cells grown in its presence and from washed cells incubated in buffer containing diphenylamine. B. megaterium grown in the presence of 25 μg diphenylamine per ml contained up to 1 mg diphenylamine per g membrane.

Submicrogram quantitation of an acidic polysaccharide by rocket immunoelectrophoresis and rocket affinoelectrophoresis

Abstract Rocket immunoelectrophoresis has been used to quantitate an acidic polysaccharide (a succinylated lipomannan) isolated from the membranes of the bacterium, Micrococcus lysodeikticus . This procedure is superior in sensitivity to standard colorimetric assays for carbohydrate and allows the detection of as little as 10 ng of mannan. The observed relationships between rocket height and both antibody and antigen concentrations follow those described by Weeke for protein antigens [ Weeke, B. (1973) Scand. J. Immunol. 2 , Suppl. 1, 37–46]. Furthermore, the lectin concanavalin A could be used as an alternative to immune serum in the quantitation of this polysaccharide by rocket electrophoresis. However, this modification (rocket affinoelectrophoresis) appeared to be less sensitive than rocket immunoelectrophoresis, allowing the detection of about 25 ng of mannan.