New reducing sugar assay for the study of cellulases

A simple assay for reducing sugars based on the production of a coloured formazan and solvent extraction has been developed and used to study the cellulase complex of a Gram-negative bacterium. This assay is more sensitive than others previously described and allows direct study of unconcentrated culture supernatants. The levels of enzyme activity in subcellular fractions were measured after growth on different carbohydrate sources.     

Isolation of a branched chain α-keto acid dehydrogenase from rabbit liver

An enzyme catalysing a series of reactions resulting in the oxidative decarboxylation of branched chain α-keto acids and production of NADH, was extracted from rabbit liver mitochondria with the aid of NaClO₄. Purification yielded a product which appeared homogeneous on electrophoresis. The enzyme is active on three substrates α-ketoisocaproate, α-keto-β-methyl valerate, and α-ketoisovalerate.     

Colony morphogenesis in a group of cellulose degrading Gram negative rods from brackish habitats

Cellulose degrading bacteria were isolated from brackish Phragmites reed beds near the Humber Estuary. Of 23 strains brought into pure culture, all developed characteristic differentiated colonies on certain media. On the basis of colour of the colonies (pseudosori) the organisms could be allocated to two groups, XMo and XMb. Related strains were isolated from a similar habitat on the Dee Estuary. Numerical taxonomy showed the group to be relatively diverse but, with a single exception, the XMo and XMb organisms appeared in separate clusters. The organisms were weakly motile or non-motile and certain cells had a polar flagellum. Pigments isolated from one strain had u.v. spectral characteristics similar to those of the xanthomonadins; the organisms may therefore be related to Xanthomonas .     

Adhesion of phospholipid vesicles to Chinese hamster fibroblasts: Role of cell surface proteins

The adhesion of artificially generated lipid membrane vesicles to Chinese hamster V79 fibroblasts in suspension was used as a model system for studying membrane interactions. Below their gel-liquid crystalline phase transition temperature, vesicles comprised of dipalmitoyl lecithin (DPL) or dimyristoyl lecithin (DML) absorbed to the surfaces of EDTA- dissociated cells. These adherent vesicles could not be removed by repeated washings of the treated cells but could be released into the medium by treatment with trypsin. EM autoradiographic studies of cells treated with[(3)H]DML or [(3)H]DPL vesicles showed that most of the radioactive lipids were confined to the cell periphery. Scanning electron microscopy and fluorescence microscopy further confirmed the presence of adherent vesicles at the cell surface. Adhesion of DML or DPL vesicles to EDTA-dissociated cells modified the lactoperoxidase-catalyzed iodination pattern of the cell surface proteins; the inhibition of labeling of two proteins with an approximately 60,000- dalton mol wt was particularly evident. Incubation of cells wit h (3)H-lipid vesicles followed by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis showed that some of the (3)H-lipid migrated preferentially with these approximately 60,000-mol wt proteins. Studies of the temperature dependence of vesicle uptake and subsequent release by trypsin showed that DML or DPL vesicle adhesion to EDTA- dissociated cells increased with decreasing temperatures. In contrast, cells trypsinized before incubation with vesicles showed practically no temperature dependence of vesicle uptake. These results suggest two pathways for adhesion of lipid vesicles to the cell surface-a temperature-sensitive one involving cell surface proteins, and a temperature-independent one. These findings are discussed in terms of current models for cell-cell interactions.