PubMed · 329149
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B Mattiasson, P O Larsson, K Mosbach. 1977-08-11. The microbe thermistor.. https://doi.org/10.1038/268519a0
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Recombinant wild-type non-phosphorylated human beta-casein was obtained from Escherichia coli. Turbidity vs. temperature (T) without Ca(2+) showed wild-type self-association like native except for irreversibility upon T-cycling with the original pattern re-established after concentrated urea/dialysis. With Ca(2+), wild-type was more native-like. Intrinsic Trp fluorescence spectra were similar but with lowered intensity for the wild-type protein. Changes in extrinsic ANS fluorescence from 4 to 37 degrees C showed less exposure of hydrophobic surface for wild-type than native. Trp to ANS fluorescence resonance energy transfer was higher for wild-type than native at 4 degrees C but 2- to 3-fold lower at 37 degrees C. The native protein must be directed by the environment and/or a chaperone to fold into a unique, somewhat flexible, conformation, unaltered by urea during purification. Wild-type protein, with many native properties, does not spontaneously fold to the native conformation, even after solubilization with urea. T-cycling gives a stable conformation that is different from the native.
The inclusion of casein, either as the acid or sodium salt form, was found to significantly modify the release of the acidic drug diclofenac from hydroxypropylmethycellulose (HPMC, Methocel grades K100LV and K15M)-based matrices. The presence of casein in diclofenac-K100LV matrices increased the drug release rate and rendered the release profile more linear. Furthermore, sodium caseinate appeared to retard the disintegrating tendency of the higher molecular weight HPMC-drug systems, apparently by enhancing the initial gel forming ability of these systems. Surprisingly, dissolution profiles of the salt and acid forms of the drug were similar when co-compressed with sodium caseinate, and addition of HPMC retarded release of both forms of drug to the same extent. Swelling and erosion experiments indicated that the presence of casein decreased the extent of medium uptake (swelling) of the matrices and accelerated the rate of erosion, while not altering the dissolution medium infiltration rate. Phase solubility analysis indicated that the solubility of the drug was also enhanced by sodium caseinate, consistent with complex formation between the drug and casein.
During prolonged starvation, yeast cells enter a stationary phase (SP) during which the synthesis of many proteins is dramatically decreased. We show that a parallel decrease in proteasome-dependent proteolysis also occurs. The reduction in proteolysis is correlated with disassembly of 26S proteasome holoenzymes into their 20S core particle (CP) and 19S regulatory particle (RP) components. Proteasomes are reassembled, and proteolysis resumes prior to cell cycle reentry. Free 20S CPs are found in an autoinhibited state in which the N-terminal tails from neighboring alpha subunits are anchored by an intricate lattice of interactions blocking the channel that leads into the 20S CPs. By deleting channel gating residues of CP alpha subunits, we generated an "open channel" proteasome that exhibits faster rates of protein degradation both in vivo and in vitro, indicating that gating contributes to regulation of proteasome activity. This open channel mutant is delayed in outgrowth from SP and cannot survive following prolonged starvation. In summary, we have found that the ubiquitin-proteasome pathway can be subjected to global downregulation, that the proteasome is a target of this regulation, and that proteasome downregulation is linked to survival of SP cells. Maintaining high viability during SP is essential for evolutionary fitness, which may explain the extreme conservation of channel gating residues in eukaryotic proteasomes.