Purification and properties of the methanol dehydrogenase from Methylophilus methylotrophus.

1. A dye-linked methanol dehydrogenase, resembling many others from a variety of methylotrophic bacteria, was purified to homogeneity from extracts of methanol-grown Methylophilus methylotrophus. 2. The enzyme was very stable in the presence of methanol; in the absence of methanol it had a half-life of 1-2 days at 4 degrees C. 3. The value of A1% 1cm,280 was 17.5. 4. The enzyme retained bound methanol after passage through Sephadex G-25. This tightly-bound methanol slowly exchanged with free [14C]-methanol from a value of 0.27 mol of [14C]methanol/mol of enzyme after 48 h incubation at 4 degrees C to a limiting value of approx. 2.5 mol of [14C]methanol/mol of enzyme after 3 weeks incubation at 4 degrees C. 5. One mol of this enzyme reduced 89.4 mol of 2,6-dichlorophenol-indophenol (via phenazine methosulphate) in the absence of any additional methanol in the assay mixture. The source of the electrons involved in this reduction is not known.     

A model for the temperature dependence of membrane excitability

Based on a decorated Ising model, the possible existence of multiple phase transitions in biological membranes is explored in this paper. Attempt is made to relate the stimulus-response behavior of membranes with structural order in different temperature regions.     

Isolation of non-myelin plasma membranes unique to white matter

A procedure is described for isolating two membrane fractions from rabbit spina-cord white matter enriched with 5′-nucleotidase, a nonspecific plasma membrane marker, 2′, 3′-cyclic nucleotide phosphohydrolase, an oligodendroglial plasma membrane marker, and acetylcholinesterase, an axonal plasma membrane marker. While the two membrane fractions exhibited similar enrichments with respect to cyclic nucleotide phosphohydrolase, enrichments of 5′-nucleotidase and acetylcholinesterase were significantly greater in the heavier membranes were not detected. Moreover, gray matter did not yield homologous membrane fractions in the gradient when subjected to the identical procedure, indicating that the two membrane fractions were unique to white matter. While electronmicroscopic examination revealed that both membrane fractions were contaminated with myelin, the heavier fraction was least contaminated and exhibited a fair degree of homogeneity with respect to single membrane vesicular profiles. It was concluded that both membrane fractions were enriched with oligodendroglial and axonal plasma membranes, with the heavier fraction containing significantly more axolemma.     

X-Ray radiography for studying diffusion characteristics of sucrose in plaster of paris matrix containing yeast cells

Summary X-Ray radiography was employed to monitor the diffusion of sucrose into plaster of Paris matrix containing 20% yeast cells. It was observed that the depth of penetration of tracer P_b detected by radiography matched with the substrate penetration detected by chemical test. However electron probe microanalysis (EPMA) did not yield any conclusive evidence regarding the movements of tracer P_b and substrate to the same extent.     

Ca2(+)-dependent noradrenaline release from permeabilised PC12 cells is blocked by botulinum neurotoxin A or its light chain

Permeabilisation of PC12 cells with digitonin allowed a direct study of the intracellular action of botulinum neurotoxin A, one of a group of dichain proteins produced by Clostridium botulinum that causes the fatal neuroparalytic condition, botulism. Release of [3H]noradrenaline from these permeabilised cells could be evoked by Ca2+ and this was inhibited specifically by the neurotoxin in a dose-dependent manner (half-maximal dose approximately 2 nM under the conditions used). Inclusion of the reducing agent dithiothreitol (up to 10 mM) had no effect on the level of inhibition. Moreover, electrophoretic analysis showed that this treatment of the toxin in the native state caused negligible reduction of inter-chain disulphide bonds. Toxin-induced blockade of neurotransmitter release was incomplete and could not be overcome by increased Ca2+ concentration (100 microM). The observed toxin-insensitivity of the release from intact PC12 cells must result from inefficient toxin uptake, relative to that in peripheral cholinergic neurones. Refolded light chain alone inhibited exocytosis to the same degree and with similar potency to that of the intact neurotoxin, an effect not altered by the heavy chain. This inhibitory activity of the light chain in PC12 cells accords with observations made in permeabilised chromaffin cells [(1989) J. Biol. Chem. 264, 10354-10360; (1989) FEBS Lett. 255, 391-394] but contrasts with invertebrate neurones, where intracellular injection of the same preparations of both chains were necessary for inhibition of quantal release of acetylcholine [(1988) Proc. Natl. Acad. Sci. USA 85, 4090-4094]. These collective findings may signify an interesting difference in the release process in such diverse systems or denote a dissimilarity in the transport or processing of the toxin when applied into intact neurones or cells permeabilised by detergent or streptolysin.     

A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite.     

Acid pH-induced fusion of cells by herpes simplex virus glycoproteins gB an gD

Enveloped animal viruses enter host cells either by direct fusion at neutral pH or by endocytosis. Herpes simplex virus (HSV) is believed to fuse with the plasma membrane of cells at neutral pH, and the glycoproteins gB and gD have been implicated in virus entry and cell fusion. Using cloned gB or gD genes, we show that cells expressing HSV-1 glycoproteins gB or gD can undergo fusion to form polykaryons by exposure only to acidic pH. The low pH-induced cell fusion was blocked in the presence of monoclonal antibodies specific to the glycoproteins. Infection of cells expressing gB or gD glycoproteins with HSV-1 inhibited the low pH-induced cell fusion. The results suggest that although the glycoproteins gB and gD possess fusogenic activity at acidic pH, other HSV proteins may regulate it such that in the virus-infected cell, this fusion activity is blocked.