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Unequivocal demonstration of the incorporation of [14C]cyanate into brain proteins as a carbamyl derivative rather than via decomposition to CO2 is presented; this is based on the isolation of radioactive hydantoins and on the loss of radioactivity on hydrolysis of the hydantoins. The influence of conditions on the extent of carbamylation of brain proteins in vitro by cyanate are presented. The optimum pH is about 7 and the carbamylation is dependent on temperature, concentration and time. There was no detectable difference in the rate and/or extent of cyanate incorporation in vitro between normal and previously carbamylated animals. Experiments in vivo show a linear increase in the carbamylation of brain proteins with intraperitoneals injection of cyanate up to the highest concentration tested (2μmol/g). The extent of carbamylation increases progressively with repeated injections for about 3 days. After this time with further injections there is increased incorporation, but at a lower rate. Experiments in vivo show that animals who have been previously carbamylated with non-radioactive cyanate incorporate about 255 less cyanate than control animals when given a pulse of [14C]cyanate. Fractionation of the major cell components revealed cyanate incorporation into all fractions but a higher incorporation in the supernatant fraction. When brain was fractionated by differential extraction there was incorporation in all the fractions, including the water, salt and solvent-insoluble residue. Although some differences were detected, additional studies are needed to determine whether carbamylation affects proteins turnover. The higher sensitivity of experiments in vivo for discrimination of protein carbamylation is discussed in relation to the present work and to recent work from others. [ABSTRACT FROM AUTHOR]

Amino groups of a series of model compounds and proteins are methylated by reductive condensation with formaldehyde and borohydride to provide observable resonances for proton magnetic resonance experiments. The extent of methylation varies from 92% for ribonuclease A to 66% for bovine serum albumin. As observed by PMR difference spectroscopy, the conformation of most of these proteins is unaltered by the modification. Retention of native conformation by methylated lysozyme is also shown by enzymatic activity measurements and PMR observation of thermal denaturation. Resonances corresponding to each of the six Nε-dimethyllysine residues in methylated lysozyme are resolved at 270 MHz and titrated to obtain individual pK values. A correction factor of 0.6 pH units obtained from model compound studies is applied to obtain apparent pK values ranging from 10.1 to 10.7 for the ε-amino groups of lysozyme. Assignment of resonances to specific residues is accomplished using the selective broadening effect of Gd3+. These results indicate the utility of the N-dimethyl group for probing the environment of lysine residues in proteins. [ABSTRACT FROM AUTHOR]

1. High-molecular-weight fractions were isolated from extracts of the cortex and the nucleus of calf lenses. By chromatography of this material on large-pore agarose gels a separation into two populations, with S20,w-values ranging from 100 to 190 S and from 30 to 55 S was obtained. 2. It is shown that the high-molecular-weight fraction is identical to α-crystallin in poly-peptide chain composition. 3. For the α-crystallin fractions from the nucleus a subunit structure different from that of cortical α-crystallin fractions is found. 4. The quaternary structure of the α-crystallin fractions is not constant during the life span of the animal. The variety in subunit structure and state of aggregation of the α-crystallin molecule is supposed to be due to aging processes. [ABSTRACT FROM AUTHOR]

Highly purified rabbit IgM anti-Forssman antibody, after reduction in dilute solution with 2-mercaptoethanol and alkylation with iodoacetamide, showed no diminution and often some increase in haemagglutination of sheep erythrocytes. The capacity to cause complement dependent lysis was, however, destroyed. It is shown that despite reduction and alkylation reaggregation of the subunits occurs to a variable extent producing non-covalently bound aggregates with S values ranging from 7 to more than 20. The aggregates can bind specifically to and agglutinate erythrocytes with greater effectiveness as their size increases. This spontaneous re-aggregation is not prevented by the presence of gelatin, but fails to occur when the IgM antibody is reduced in the presence of a variety of other proteins. The effect of extraneous proteins is evident only when they are present at the time of reduction or are added within 10–15 minutes. It is suggested that following rupture of the interchain disulphide bonds, the IgM molecule can undergo a complex, time-dependent rearrangement into new stable forms which retain combining site activity but do not involve covalent bonds. [ABSTRACT FROM AUTHOR]