A B $ T R A C T A latent ATP-dependent Ca storage system is enriched in preparations of pinched-off presynaptic nerve terminals (synaptosomes), and is exposed when the terminals are disrupted by osmotic shock or saponin treatment. The data indicate that a fraction of the Ca uptake (measured with ~Ca) is associated with the intraterminal mitochondria; it is blocked by ruthenium red, by FCCP, and by azide + dinitrophenol + oligomycin. There is, however, a residual ATP-dependent Ca uptake that is insensitive to the aforementioned poisons; this (nonmitochondriai) Ca uptake is blocked by tetracaine, mersalyl and A-23187. Moreover, A-23187 rapidly releases previously accumulated Ca from these (nonmitochondrial) storage sites, whereas the Ca chelator, EGTA, does not. The proteolytic enzyme, trypsin, spares the mitochondria but inactivates the nonmitochondrial Ca uptake mechanism. Chemical measurements of total Ca indicate that the ATP-dependent Ca uptake at the nonmitochondrial sites involves the net transfer of Ca from medium to tissue fragments. This system can sequester Ca when the ambient-ionized Ca 2+ concentration (buffered with EGTA) is <0.3 v,M; brain mitochondria take up little Ca when the ionized Ca 2+ level is this low. Preliminary subfractionation studies indicate that the nonmitochondrial Ca storage system does not sediment with synaptic vesicles. We propose that this Ca storage system, which has many properties comparable to those of skeletal muscle sarcoplasmic reticulum, may be associated with intraterminal smooth endoplasmic reticulum. This Ca-sequestering organelle may help to buffer intracellular Ca.
The large majority of proteins of alkaliphilic Bacillus pseudofirmus OF4 grown at pH 7.5 and 10.5, as studied by two-dimensional gel electrophoresis analyses, did not exhibit significant pH-dependent variation. A new surface layer protein (SlpA) was identified in these studies. Although the prominence of some apparent breakdown products of SlpA in gels from pH 10.5-grown cells led to discovery of the alkaliphile S-layer, the largest and major SlpA forms were present in large amounts in gels from pH 7.5-grown cells as well. slpA RNA abundance was, moreover, unchanged by growth pH. SlpA was similar in size to homologues from nonalkaliphiles but contained fewer Arg and Lys residues. An slpA mutant strain (RG21) lacked an exterior S-layer that was identified in the wild type by electron microscopy. Electrophoretic analysis of whole-cell extracts further indicated the absence of a 90-kDa band in the mutant. This band was prominent in wild-type extracts from both pH 7.5-and 10.5-grown cells. The wild type grew with a shorter lag phase than RG21 at either pH 10.5 or 11 and under either Na ؉ -replete or suboptimal Na ؉ concentrations. The extent of the adaptation deficit increased with pH elevation and suboptimal Na ؉ . By contrast, the mutant grew with a shorter lag and faster growth rate than the wild type at pH 7.5 under Na ؉ -replete and suboptimal Na ؉ conditions, respectively. Logarithmically growing cells of the two strains exhibited no significant differences in growth rate, cytoplasmic pH regulation, starch utilization, motility, Na ؉ -dependent transport of ␣-aminoisobutyric acid, or H ؉ -dependent synthesis of ATP. However, the capacity for Na ؉ -dependent pH homeostasis was diminished in RG21 upon a sudden upward shift of external pH from 8.5 to 10.5. The energy cost of retaining the SlpA layer at near-neutral pH is apparently adverse, but the constitutive presence of SlpA enhances the capacity of the extremophile to adjust to high pH.Bacillus species have been a major component of the extremely alkaliphilic bacterial flora isolated both from highly selective environments such as alkaline lakes and from ostensibly unselective environments such as conventional soils (21,24,26). While many studies have focused on useful products of alkaliphilic bacilli (21), others have focused on the basis for alkaliphily itself (19,26,28). Among the questions that immediately arise are how can those membranous and protein structures that are exposed to the alkaline medium function, and how can cells growing above pH 10 maintain a cytoplasmic pH that is well below the external pH? With respect to the first question, recent structural studies of extracellular enzymes from extreme alkaliphiles and numerous deduced protein sequences of alkaliphile proteins have begun to indicate properties that may correlate with the ability to function at extremely high pH (26). The adaptations, moreover, appear to depend upon whether a high net charge is important to the function of the molecule or molecular segment. When that is the case, the...
Two-dimensional electrophoresis in combination with Coomassie Blue staining was refined for use as a quantitative method. Microcomputer software was developed for use with the IBM AT and compatible computers for analyzing the gels. To test the refined method to determine its usefulness in simultaneous measurements of 28 human serum proteins, we measured each protein relative to a single standard (bovine serum albumin) polymerized at different concentrations in a calibration scale, rather than using 28 individual standards. All samples were analyzed in triplicate. We evaluated calibration, linearity of response, recoveries, units, within-run CV, and between-run CV. The five isoforms of apolipoprotein A-I were analyzed in samples from 16 healthy donors and the isoform ratios determined. The method as presented here should prove useful for diagnosis of non-urgent disease states and for analysis for protein isoforms in relation to disease; it should also be applicable to assays of proteins in other fluids and tissues.
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