Estimation of the stability of globular proteins

The interpretation of ΔG (the free energy change for the reaction, globular conformation ? randomly coiled conformation, in the absence of denaturant), in terms of the free energies of transfer of various parts of the protein molecule from water to denaturant solution, is unsatisfactory because the latter are assumed to be identical to the transfer-free energies of similar groups attached to smaller model compounds. We have made empirical adjustments to transfer-free energy theory that make possible linear extrapolation of the free energy of denaturation of a protein from transition region to zero denaturant concentration. The modified theory, used to analyze the denaturation of proteins by guanidine hydrochloride and urea, allowed us to calculate reasonable values for Δα, the average change in accessibility to solvent of the component groups of protein.     

Endogenous production of prothymocyte differentiating activity by phytohemagglutinin-stimulated T-cell-depleted human marrow

The PHA responsiveness of marrow T-cell precursors remains a matter of controversy. We have investigated the capacity of human marrow to proliferate under phytohemagglutinin (PHA) stimulation following extensive removal of mature T cells by complement-dependent cytotoxicity with MBG6 and RFT8 monoclonal antibodies. PHA-induced thymidine uptake by marrow cells occurred with a peak on Days 6-8 of incubation instead of Day 3 for PBL. This peak was observed 48 hr earlier in the presence of PHA-stimulated T-depleted marrow cell supernatants. These supernatants can also promote the growth of mature T-cell colonies from MBG6-, RFT8-, T11-, T3- marrow. However, full colony development requires exogenous interleukin 2 (IL-2). IL-2 could be detected in marrow supernatants but only at very low levels and beyond Days 3 and 4. In contrast Days 1-6 marrow supernatants were equally effective in promoting MBG6-RFT8- marrow cell responsiveness to PHA. We conclude that marrow T-cell precursors are not PHA responsive and that PHA induces the production by marrow non-T cells of a prothymocyte-differentiating activity (PTDA); PTDA can differentiate marrow T-cell progenitors into PHA-responsive T cells; following activation by PHA, these cells undergo limited proliferation induced by IL-2 endogenously released from de novo differentiated T cells. It is suggested that this mechanism may account for extrathymic differentiation of the T-cell lineage in heavily irradiated marrow transplantation recipients.     

Complexities in the denaturation of horse metmyoglobin by guanidine hydrochloride

The denaturation of horse metmyoglobin by guanidine hydrochloride was studied at pH 6.4 and 25 degrees C. Measurements of both the peptide circular dichroism and the absorbance in the Soret region suggest that the extent of renaturation strongly depends on the time interval during which the protein is exposed to concentrated solutions of the denaturant. From the equilibrium measurements of the absorption in the Soret region, it is concluded that the unfolding of metmyoglobin is complex. This is further supported by kinetic studies of denaturation which suggest the occurrence of the least four species in the reaction.     

Utilization of carbon and nitrogen sources and acid/alkali production by cowpea rhizobia

Summary Sixteen slow-growing strains of rhizobia (15 cowpea rhizobia and oneR. japonicum) were examined to determine the effects of carbon and nitrogen sources on acid/alkali production in culture media. We found that the pH changes of the medium were more influenced by nitrogen sources than carbon sources (with the exception of ribose). When ammonium sulphate was used as a nitrogen source, all the cowpea rhizobia strains produced acid. When yeast-extract was used as a nitrogen source, however, a heterogenous pattern for acid/alkali production was found. The majority of the strains produced alkali from nitrate, glutamate and urea irrespective of carbon sources and acid from ribose irrespective of nitrogen sources.     

Thermodynamic stability of proteins in salt solutions: A comparison of the effectiveness of protein stabilizers

The denaturation of proteins by guanidine hydrochloride was studied in the presence of different concentrations of stabilizing salts, namely potassium phosphate, ammonium sulfate, and potassium acetate. The denaturation transition was followed by observing changes in the peptide circular dichroism atpH 7.0 and 25°C. From these results the free energy of stabilization for the process native denatured was determined. It was found that the stabilizing power of the anions increased in the order acetate < sulfate < phosphate, in agreement with the anionic lyotropic series. Ribonuclease A, which is known to have a site that can bind either a phosphate or a sulfate ion, showed a larger stabilization by these anions than that for lysozyme, pepsinogen, and myoglobin.     

9-Hydroxydodecanoic acid,an acid from Blepharis sindica seed oil

A hitherto unknown hydroxy acid has been isolated from Blepharis sindica seed oil has been characterized as 9-hydroxydodecanoic acid by IR, NMR and mass spectral studies. The structure of this acid was further supported by its chemical transformations.     

Partial characterization of the phosphotransferase system of human central-nervous-system myelin.

The phosphotransferase system of human central-nervous-system myelin was investigated. Evidence obtained indicated the presence of at least two different phosphotransferase systems (cyclic nucleotide-dependent and -independent) in myelin, which were found to be firmly associated with the membrane. The cyclic AMP-dependent kinase of myelin and white-matter cytosol preferentially phosphorylated certain histone fractions and displayed only modest activity with basic protein as substrate. On the other hand, the cyclic nucleotide-independent system showed specificity toward basic protein. Its activity was not only dependent on Mg2+ but it was greatly enhanced by this bivalent cation. Whereas the cyclic nucleotide-dependent kinase could be extracted with buffers containing Triton X-100, the bivalent cation-regulated kinase resisted solubilization from myelin under these conditions.     

Fluorouracil and the isolation of mutants lacking uridine phosphorylase in Escherichia coli: location of the gene

Summary A selective technique is described for the isolation of mutants of Escherichia coli lacking uridine phosphorylase and the location of the gene specifying this enzyme on the bacterial chromosome is determined. Using strains with appropriate lesions it is shown that there are three routes via which 5-fluorouracil can be converted to compounds which inhibit cell growth.     

On the carbohydrate—protein linkage group in vitreous humor hyaluronate

Fractional molar ratios of serine, threonine and aspartic acid to neutral sugars in the purified bovine vitreous humor hyaluronate, and a 4–5-fold increase in the percentage of these amino acids and the absence of sugar alditols in hyaluronate reduced with NaBH₄---PdCl₂ after alkali treatment indicated the absence of a carbohydrate—protein linkage. Gel filtration behavior, a decrease in intrinsic viscosity of reduced hyaluronate to about one-half and a significant decrease in its specific rotation suggested that the two antiparallel chains of the hyaluronate double helix may come apart upo reduction. The vitreous humor hyaluronate contained 109.2 ppm of “bound” silicon. It is suggested that the bound silicon may bridge the two antiparallel chains through the neutral sugars and/or through the hydroxyl group of the uronic acid moiety.