J Armstrong

Abstract Three different methods are described for separating hemoglobins from carbonic anhydrases in hemolysates from human erythrocytes. The preferred method involves adsorption on diethylaminoethyl Sephadex at pH 8.7 followed by selective elution of the carbonic anhydrases. Carbonic anhydrases A, B, and C are subsequently separated from each other on DEAE-Sephadex by elution with 0.05 m Trischloride buffer at pH 8.7. Complete amino acid analyses are reported for carbonic anhydrases B and C, with results in good accord with those of preparations obtained in other laboratories by different procedures. The partial specific volume of carbonic anhydrase B is found to be 0.731 ml per g and its intrinsic viscosity is 2.76 ml per g. Molecular weights from sedimentation, diffusion, and sedimentation equilibrium are close to 28,000 for both enzymes; estimates from the amino acid analyses are 28,730 for Enzyme B, and 30,000 for Enzyme C. The s020, w values are close to 2.75 S for both enzymes. The values for both s and D are lower than others previously reported, by a factor close to 1.2, but the resulting molecular weights are in good agreement with others. Studies of optical rotatory dispersion between 330 and 600 mµ are reported for the native and the acid-denatured carbonic anhydrases B and C, and also for solutions containing the substrate bicarbonate ion and, in other experiments, the inhibitor acetazolamide. The data in all these solvents give a good fit to the simple Drude equation, with λc values that lie below 210 mµ for native Enzyme B and average close to 210 mµ for Enzyme C. Analysis of the data in terms of the Moffitt-Yang and Shechter-Blout equations is also reported, but a computer analysis indicates that the basis for applying the former equation is dubious. There appears to be a small amount of helix (10 to 20%) in the acid-denatured proteins; the complexity of the optical rotatory dispersion spectra of the native proteins makes inferences concerning helix content uncertain (see following paper). Studies on the esterase activity of Enzyme B, with p-nitrophenyl acetate as substrate, are reported. Esterase activity rises with increasing pH between 7 and 9.5. Acetazolamide is a powerful inhibitor of the esterase activity, with Ki = 0.3 µm at pH 7, 0.9 µm at pH 8, and approximately 2.8 µm at pH 9. The inhibition appears to be noncompetitive.

Abstract Studies of optical rotatory dispersion (ORD) and circular dichroism (CD) are reported on human carbonic anhydrases B and C in the spectral region below 320 mµ. Most of the observed CD spectrum of each enzyme could be described in terms of three principal Gaussian bands: a strong negative band near 216 mµ, a weaker negative band near 270 to 275 mµ, and a positive band intermediate between the other two in position. Above 260 mµ, all CD values are negative; there is clear evidence of fine structure above 280 mµ, involving at least two additional CD bands. The ORD spectra above 260 mµ show several peaks and troughs, which are characteristic for each enzyme. The principal troughs at shorter wave lengths lie at 222 mµ for Enzyme B and at 226 mµ for Enzyme C. At still shorter wave lengths, the ORD values rise to low peaks, at 204 mµ, with [m'] = -300 for Enzyme B and +2750 for Enzyme C. These patterns are very different from those characteristic of either α-helical or β structures. Asymmetrical interactions involving the aromatic side chains almost certainly make important contributions to ORD and CD at the shorter wave lengths, as they clearly do at the longer wave lengths, above 250 mµ. On acid denaturation, the longer wave length Cotton effects vanish, and the ORD and CD spectra alter in such a way as to suggest the presence of 10 to 20% α-helix in the acid-denatured proteins. The changes on exposure to high pH are more complex. Near pH 11, the negative CD band near 270 to 275 mµ becomes less intense and shifts to longer wave lengths; the positive band at 232 mµ in Enzyme B also shifts to longer wave lengths. It is tentatively suggested that these bands arise in part from interactions of tyrosine residues. The fine structure pattern between 280 and 310 mµ undergoes changes, but in Enzyme B it still persists even at pH 13. Interactions involving tryptophan may be involved. The positive CD band at 249 mµ in Enzyme C disappears at pH 11.5, and the ORD spectrum of this enzyme also indicates more drastic structural alterations at high pH than those found in Enzyme B. The ORD spectra of both enzymes at pH near 13 show two troughs, one near 231, the other near 210 mµ, the former being deeper for Enzyme B, the latter for Enzyme C. The CD bands of Enzyme B are still observed in 2 m guanidine-HCl, although the long wave ultraviolet bands are weaker than in the native protein; in 4 m guanidine-HCl they are abolished. ORD studies of the two enzymes in 1 m guanidine-HCl indicate that the Cotton effects due to the aromatic absorption bands disappear rapidly in this solvent in solutions of Enzyme C, whereas Enzyme B is much more stable. ORD spectra have been calculated from the CD data for the native enzymes by the use of a Kronig-Kramers transform with a computer program. Comparison with the observed ORD data shows good general agreement with the pattern of the observed troughs and peaks, especially for Enzyme C. The computed spectra are displaced to more positive [m'] values than the observed spectra, indicating that there must be large negative contributions to the circular dichroism at wave lengths below 200 mµ.

Disseminated infection with herpes simplex virus type 2 was identified in two patients 20 days after they had received kidney transplants from the same organ donor. Neither patient had neutralizing antibody to herpes simplex virus before transplantation, and both had herpes simplex virus isolated from surveillance cultures of urine before the onset of clinical symptoms. A clear focus of primary infection was not found in either patient. Analysis of the patients' isolates by DNA restriction endonuclease analysis strongly suggested that the strains were identical. These data implicate the allografts as the source of the viral infection.

BACKGROUND: Despite intensive research and novel adjuvant therapies, there is currently no cure for metastatic melanoma. The chemokine receptor CXCR4 controls metastasis to sites such as the liver; however, the therapeutic blockade with the existing agents has proven difficult. METHODS: AMD11070, a novel orally bioavailable inhibitor of CXCR4, was tested for its ability to inhibit the migration of melanoma cells compared with the commonly described antagonist AMD3100. RESULTS: AMD11070 abrogated melanoma cell migration and was significantly more effective than AMD3100. Importantly for the clinical context, the expression of B-RAF-V600E did not the affect the sensitivity of AMD11070. CONCLUSION: Liver-resident myofibroblasts excrete CXCL12, which is able to promote the migration of CXCR4-expressing tumour cells from the blood into the liver. Blockade of this axis by AMD11070 thus represents a novel therapeutic strategy for both B-RAF wild-type and mutated melanomas.