Identification of regions in interleukin-1 alpha important for activity.

Saturation mutagenesis of the mature human interleukin-1 alpha (IL-1 alpha) gene has been performed. Following expression in Escherichia coli, the biological and receptor binding activities of the mutant proteins were examined. Most of the molecule could be altered with little effect on either function. More than 3,500 mutants were examined, and only 23 unique amino acid sequences were identified which resulted in an altered ratio of biological to binding activity when compared with wild-type IL-1 alpha. These proteins possessed mutations at 38 of the 159 amino acid residues in IL-1 alpha. Random mutagenesis at several of these positions identified further substitutions that affected activity. Examination of a model for IL-1 alpha localized most of the residues which altered activity along one face of the molecule. This region appears to be distinct from areas of IL-1 which have been postulated to make contact with IL-1 receptor.

Maternal autoantibodies and fetal disease.

We have examined the relationships between maternal connective tissue disease (CTD), fetal and neonatal health, and the presence of specific autoantibodies: antinuclear antibodies (ANA), anti-Ro, antiLa, anti-Sm, anti-RNP, anti-DNA, and anticardiolipin (ACL) in 27 mothers with CTD (Group A), and 10 asymptomatic mothers of babies with complete congenital heart block (CCHB), or cardiac arrhythmias (Group B). Compared to 100 normal pregnant controls, autoantibodies were significantly more common in both Group A (96.3%, p less than 0.0005) and Group B (70%, p less than 0.0005), although the prevalence of autoantibodies in the Group A mothers having abnormal babies compared with those who had normal babies did not differ. Anti-La was present only in mothers with abnormal babies (7 of 17 compared to 0 of 10, p less than 0.025). Anti-La did not occur without anti-Ro, but anti-Ro occurred in 9 mothers without anti-La. Anti-Ro was present in the serum of all mothers of infants with CCHB and occurred alone in 3 of 4 instances. Titers of anti-Ro did not differ between abnormal and normal infants or their mothers. ACL occurred alone in the serum of 10 of 38 mothers, and was present in 7 mothers who had infants with cardiac abnormalities other than CCHB.

Interaction of carboxypeptidase A with carbamate and carbonate esters.

The carbamate ester N-(phenoxycarbonyl)-L-phenylalanine binds well to carboxypeptidase A in the manner of peptide substrates. The ester exhibits linear competitive inhibition toward carboxypeptidase A catalyzed hydrolysis of the amide hippuryl-L-phenylalanine (Ki = 1.0 X 10(-3) M at pH 7.5) and linear noncompetitive inhibition toward hydrolysis of the specific ester substrate O-hippuryl-L-beta-phenyllactate (Ki = 1.4 X 10(-3) M at pH 7.5). Linear inhibition shows that only one molecule of inhibitor is bound per active site at pH 7.5. The hydrolysis of the carbamate ester is not affected by the presence of 10(-8)-10(-9) M enzyme (the concentrations employed in inhibition experiments), but at an enzyme concentration of 3 X 10(-6) M catalysis can be detected. The value of kcat at 30 degrees C, mu = 0.5 M, and pH 7.45 is 0.25 s-1, and Km is 1.5 X 10(-3) M. The near identity of Km and Ki shows that Km is a dissociation constant. Substrate inhibition can be detected at pH less than 7 but not at pH values above 7, which suggests that a conformational change is occurring near that pH. The analogous carbonate ester O-(phenoxycarbonyl)-L-beta-phenyllactic acid is also a substrate for the enzyme. The Km is pH independent from pH 6.5 to 9 and has the value of 7.6 X 10(-5) M in that pH region. The rate constant kcat is pH independent from pH 8 to 10 at 30 degrees C (mu = 0.5 M) with a limiting value of 1.60 s-1. Modification of the carboxyl group of glutamic acid-270 to the methoxyamide strongly inhibits the hydrolysis of O-(phenoxycarbonyl)-L-beta-phenyllactic acid. Binding of beta-phenyllactate esters and phenylalanine amides must occur in different subsites, but the ratios of kcat and kcat/Km for the structural change from hippuryl to phenoxy in each series are closely similar, which suggests that the rate-determining steps are mechanistically similar.

Ability of specific monoclonal antibodies and conventional antisera conjugated to hematoporphyrin to label and kill selected cell lines subsequent to light activation.

A variety of cell lines have been prepared by fusion of the murine WEH1 3B cell line with peripheral blood leukocytes from a patient with chronic granulocytic leukemia. Fusion products were selected for their ability to produce a leukemia-associated antigen (CAMAL) previously described. One such line which originally produced CAMAL subsequently lost this ability and was used as a negative control. A number of antibodies were conjugated to hematoporphyrin (HP) and tested for their ability to bind to cell lines as detected by either fluorescence or by their ability to kill cells after light activation. The antibodies used were: rabbit anti-Hu (a conventional rabbit antiserum raised to membrane preparations from normal human peripheral blood leukocytes which served as a positive control); CAMAL-1 (a monoclonal gamma 1 antibody with specificity for the CAMAL antigen); and L1210 (an irrelevant monoclonal gamma 1 antibody). HP was conjugated to the antibodies by a carbodiimide procedure. When labeled cells were examined by fluorescence microscopy, it was apparent that both the rabbit antibody and CAMAL-1:HP showed positive labeling. The ability of the antibody:HP conjugates to kill labeled cells following light activation was tested. It was shown that rabbit anti-Hu:HP and CAMAL-1:HP conjugates were capable of killing significant numbers of cells when HP concentrations were as low as 1.2 ng/10(6) cells, whereas similarly treated cells exposed to either L1210:HP or HP alone did not exhibit significant killing until concentrations reached 240 and 120 ng/10(6) cells, respectively. Further experiments in which other cell lines were tested, all at HP concentrations of 12 ng/10(6) cells, demonstrated that those lines producing CAMAL were killed, whereas negative lines were not.

Identification of human sperm antigens to antisperm antibodies.

We have successfully applied SDS (sodium dodecyl sulfate) gel/protein blot radioimmunobinding method to identify the molecular size of sperm antigens that elicit antisperm antibodies from patients with unexplained infertility. Following the transfer of renatured proteins from SDS gel of human sperm extract onto nitrocellulose strips, the radioimmunobinding was performed by incubating the strips with patients' sera at 1:100 dilution and then with I125-labeled goat antihuman immunoglobulin G (IgG) or protein A as detecting probes. Unique sperm antigens that reacted with some patients' sera were identified following the autoradiography of the incubated paper strips. Among the fifty-nine standard serum samples from the Reference Bank of the World Health Organization, about one-fourth of them were found to react predominantly with a sperm protein band having the reference value (Rf value) of 0.2 and the approximate molecular weight of 90,000 dalton. A similar analysis was also performed with serum samples from vasectomized patients. Some of them also revealed a specific binding with the sperm antigen(s) of similar molecular weight. The results of this analysis were also compared with those of conventional tests for sperm antibodies as well as those of microplate radioimmunoassays and enzyme-linked immunoassays. This study suggests that SDS gel/protein blot radioimmunobinding method can be a useful tool for the molecular identification of unique human sperm antigen(s) that elicit naturally occurring antisperm antibodies in patients with unexplained infertility.