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Sequence similarity between aB-crystallin and small heat shock proteins (HSPs) has prompted us to investigate whether aB-crystallin expression is induced by heat shock. Indeed, accumulation of aB-crystallin was detected immunologically in NIH 3T3 cells after incubation at elevated temperatures and after addition of Cd2+ or sodium arsenite to these cells. Two-dimensional gel electrophoresis revealed identity between aB-crystallin from eye lenses and from heattreated fibroblasts. The promoter of the aB-crystallin gene was fused to the bacterial chloramphenicol acetyltransferase gene and was shown to confer heat inducibility on this reporter gene in transient transfection assays. A perfect heat shock element within the promoter region is likely to mediate this response. Small HSPs and aB-crystallin were shown to share the following two physical properties: (i) they form supramolecular structures with sedimentation values around 17 S and (ii) they are associated with the nucleus at high temperatures and are localized in the cytoplasm under normal conditions. We conclude that aB-crystallin has to be considered a member of the class of small HSPs.Heat shock and numerous other stress conditions lead to the rapid induction of several genes whose protein products are collectively called heat shock proteins (HSPs) (for recent reviews see refs. 1-3). The HSPs have been grouped into several classes on the basis of their size and sequence homology. Members of the class of small HSPs have molecular masses in the range 15-40 kDa. All analyzed organisms possess at least one small HSP gene. In mammals, birds, and yeast this class of HSPs is represented by a single member (4-9), whereas in Drosophila melanogaster and plants there appear to be multiple small HSPs (10, 11). Small HSPs aggregate to form characteristic ring-shaped structures called heat shock granules (4,(12)(13)(14)(15). These structures resemble prosomes or proteosomes but are distinct entities (16). Their biochemical function is unknown. Under heat shock conditions the small HSPs associate with the nucleus. Following a heat shock they slowly relocalize to the cytoplasm (17)(18)(19). It is still a matter of debate whether the small HSPs are actually transported into the nucleus at high temperatures or whether they are entrapped by the intermediate filaments, which, under heat shock conditions, collapse onto the nucleus (for a review see ref.3). The amino acid sequences of the small HSPs from different organisms are only poorly conserved. However, striking sequence similarities exist between vertebrate a-crystallins and small HSPs (5, 20-23). a-Crystallins were originally found in eye lenses, where they are among the most abundant proteins (24, 25). There exist two forms of a-crystallins, aA and aB, which are closely related (26). Considerable amounts of aB-crystallin, but not aAcrystallin, are present in many nonlenticular tissues (27-31). Moreover, aB-crystallin gene expression has been observed in various diseased cells, including astrocytes of patients s...

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Bloodstream forms of Trypanosoma brucei have been screened for the presence of enzymes that could serve as markers for the plasma membrane, flagellar pocket, lysosomes, endoplasmic reticulum and Golgi apparatus in order to study the subcellular organization of the digestive system of the parasite.Acetylesterase, acid DNase, acid phosphatase, acid phosphodiesterase, acid proteinase, acid RNase, alanine aminotransferase, galactosyl transferase, a-glucosidase, inosine diphosphatase and a-mannosidase were partially characterized and their assays optimized for pH-dependent activity, linearity of reaction with respect to incubation time and enzyme concentration, and the effect of inhibitors and activators. The association of these enzymes with particulate material and the presence of structural latency were investigated.Acid proteinase and a-mannosidase are particle-bound and latent in cytoplasmic extracts ; they can be activated and solubilized in part by Triton X-100. Similar results were obtained for acid phosphatase, acid phosphodiesterase and inosine diphosphatase. Neutral a-glucosidase, though partly sedimentable, does not show latency and is readily solubilized by the detergent. Galactosyl transferase is firmly membrane-bound even in the presence of 0.1 % Triton X-100, Cell fractionation by differential centrifugation and density equilibration on sucrose gradients revealed that both a-mannosidase and acid proteinase are associated with organelles that band at a density of about 1.20 g/cm3. Inosine diphosphatase, galactosyl transferase, acid phosphatase and acid phosphodiesterase sediment predominantly as microsomal constituents equilibrating at densities between 1.13 and 1.15 g/cm3. In addition, inosine diphosphatase and galactosyl transferase exhibit considerable activity at higher densities (1.18 -1.25 g/cm3). Neutral a-glucosidase is mainly recovered in the nuclear and microsomal fraction; its particulate part equilibrates as a single band at e = 1.22 g/cm3. Acetylesterase and acid DNase are largely soluble, whereas acid RNase does not produce distinct sedimentation and banding profiles.In intact cells, neutral a-glucosidase and acid phosphatase appear to be highly accessible to their substrates.It is tentatively concluded that (a) acid proteinase and a-mannosidase are lysosomal enzymes, (b) acid phosphatase and acid phosphodiesterase are associated with the flagellar pocket and part of the former enzyme probably with the endoplasmic reticulum, (c) galactosyl transferase is a constituent of the Golgi apparatus, and (d) a-glucosidase may serve as a marker for the plasma membrane. Inosine diphosphatase may also be derived from the latter structure.