Inhibition of modulation of thymus-leukemia antigens by concanavalin A.

When incubated at 37 °C in medium containing antibodies specific for thymus-leukemia (TL) antigens, viable cells bearing these antigens become resistant to the cytolytic effects of guinea pig complement, a process termed antigenic modulation. Antibody-induced membrane redistribution of the TL antigens, detected by indirect immunofluorescence, occurs with a similar pace. When high concentrations of concanavalin A (Con A) were included with antibodies in the incubation medium, TL antigenic modulation as well as antigen patching and capping were markedly inhibited, similar to effects of Con A on membrane immunoglobulin redistribution with murine spleen cells. Colchicine antagonized the inhibition by Con A suggesting the involvement of microtubules. In parallel experiments high concentrations of Con A failed to alter the quantity of TL antigen expression or its rate of change with time during incubation in cognate antisera. These results support the hypotheses that (a) generalized alterations in membrane receptor mobility may be induced by ligand binding to the cell membrane, and (b) under certain conditions stable interactions occur between normally independent cell surface antigens.     

Shared chemical properties of different murine thymus-leukemia antigens.

Immunochemical studies of murine thymus-leukemia antigens (TLA) have confirmed that the subunit structure consists of a 45,000-dalton heavy chain and a beta 2 microglobulin (beta 2m) light chain. Similar structural features are exhibited by the TLA from thymocytes of Tlaa, Tlac, Tlad, and a leukemia cell derived from C57BL/6, a Tlab strain. In addition to the similar subunit structure from the four haplotypes, each TLA shows a similar pattern of trypsin proteolysis. This procedure yields a major heavy chain cleavage product of approximately 37,000 daltons that remains associated with beta 2m and retains most or all of the antigenic determinants of the intact TLA. Evidence is presented that TLA do not exhibit Fc receptor properties, nor do they adsorb to murine leukemia virus antigens under the conditions of isolation for analysis on polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). Taken together these findings strongly support the hypothesis that TLA comprise a family of chemically similar antigens belonging to a structurally and genetically related group that includes H-2D, H-2K, and Qa-2,3.     

Limited proteolysis of p-chloromercuribenzoate-dissociated hemoglobin by trypsin.

p-Chloromercuribenzoate-treated hemoglobin was digested by trypsin. The hydrolysate was subjected to gel-filtration on Bio-Gel P-4 and Sephadex G-50 columns, ion-exchange chromatography on CM-Sephadex and DE 52 columns, and paper electrophoresis. Peptides obtained by this procedure were analyzed for amino acid compositions and amino-terminal amino acid sequences. The results showed that p-chloromercuribenzoate-treated hemoglobin was hydrolyzed to a limited extent by trypsin at the bonds involving the carboxyl group of a lysine or arginine residue in planes A--E in the parent hemoglobin, which represent the external region of the parent tetramer. It is concluded therefore that the slight modification of hemoglobin enhances the susceptibility of the protein to proteases and that the hydrolysis of the modified protein is limited.     

Human inter-alpha-trypsin inhibitor. Limited proteolysis by trypsin, plasmin, kallikrein and granulocytic elastase and inhibitory properties of the cleavage products.

The acid-labile inter-alpha-trypsin inhibitor is cleaved enzymatically in vivo, liberating a smaller acid-stable inhibitor. The molar ratio of native inhibitor to this smaller inhibitor in plasma is significantly changed in some severe cases of inflammation and kidney injury. To clarify this observation on a molecular basis, the action of four different types of proteinases (trypsin, plasmin, kallikrein and granulocyte elastase) on the inter-alpha-trypsin inhibitor was studied. The initial rate of cleavage of the inter-alpha-trypsin inhibitor by a 1.3-fold molar excess of proteinase over inhibitor was found to be 4375 nM x min-1 with granulocyte elastase, 860 nM x min-1 with trypsin, 67 nM x min-1 with plasmin, and 0.3 nM X min-1 with kallikrein. Obviously, of the enzymes studied so far, the granulocyte elastase known to be released during severe inflammatory processes is by far the most potent proteinase in the transformation of the inter-alpha-trypsin inhibitor. The inter-alpha-trypsin inhibitor and its cleavage products inhibit bovine trypsin very strongly (Ki = 10(-9)--10(-11) M), porcine plasmin much less strongly, human plasmin very weakly and pancreatic kallikrein practically not at all.     

Limited MHC polymorphism in whales

Little is known about disease and genetic variation in aquatic mammalian species such as whales. In this paper human HLA class I and class 11 probes were used to study major histocompatibility complex (MHC) genes from two species of whale: Fin (Balaenoptera physalus) and Sei (B. borealis). Stronger signals were obtained on whale than on equivalent concentrations of mouse DNA. Evidence was obtained for severalDRB-related genes, aDNA gene, oneDQA gene, and multiple class I genes in whales. Interestingly, the whale genes, from the small panel studied, were less polymorphic than those of humans or mice. The aquatic environment of this mammalian species may be a unique factor in shaping its immune response through the MHC.     

Modification of Escherichia coli DNA ligase by cleavage with trypsin.

Limited treatment of Escherichia coli DNA ligase with trypsin results in rapid loss of DNA joining activity. However, the ability to react with DPN to form the covalent enzyme-AMP intermediate is unaffected. The cleaved enzyme is also unable to catalyze the formation of DNA-adenylate, the second covalent intermediate in the ligase-catalyzed reaction. These findings demonstrate that portions of the DNA ligase molecule that are required for phosphodiester bond formation are not required for at least one of the partial reactions catalyzed by this enzyme.     

Limited trypsin proteolysis of photoreceptor GTP-binding protein. Light- and GTP-induced conformational changes

The amphibian photoreceptor rod outer segment contains a guanine nucleotide-binding complex which consists of a 39,000-dalton polypeptide that binds guanine nucleotides (G protein), a 36,000-dalton polypeptide (H protein), and an approximately 6,500-dalton polypeptide. Sensitivity to trypsin proteolysis was utilized as a probe of structure-function relationships for these polypeptides. Digestion of the H protein generated fragments of 26,000 and 15,000 daltons whose proteolytic susceptibility was not altered by guanosine triphosphates, light, or membranes. The approximately 6,500-dalton polypeptide was not trypsin sensitive. When the G protein was eluted from illuminated membranes by GTP, trypsin proteolysis cleaved a terminal 1,000-dalton fragment (G1) to yield a 38,000-dalton fragment (G38). With increased digestion time, a 6,000-dalton fragment (G6) was removed from G38 to yield a 32,000-dalton fragment (G32). G32 was subsequently digested to fragments of 23,000 and 12,000 daltons. However, when the G protein was eluted from illuminated membranes by hydrolysis-resistant analogues of GTP, G32 was protected from further digestion. This is consistent with a GTP-induced conformational change in the G protein which is altered by GTP hydrolysis. Proteolysis of the G protein after covalent labeling with a photoaffinity analogue of GTP demonstrated that the analogue is bound to first G38 and then G32, indicating the GTP-binding site is associated with G32. Fragment G6 was cleaved when the G protein was soluble or bound to unilluminated membranes. However, when bound to illuminated membranes, fragments were generated reflecting the loss of 7,500, 9,000, or 11,000 daltons from the G protein. This light-induced alteration in proteolytic susceptibility indicates there is a light-induced conformational change in the G protein. Fragment G1 was not removed from the G protein when it was membrane bound, suggesting G1 is involved in binding to a membrane structure. These data suggest that the light-induced binding of the G protein to illuminated membranes and the reversal of this binding by GTP are mediated through conformational changes in the G protein and that three conformations exist: 1) a basal, inactive conformation; 2) a primed conformation induced by binding to photolyzed rhodopsin, with a high affinity for GTP; and 3) an active conformation, induced by binding of GTP, which activates the catalytic complex of light-activated phosphodiesterase.     

Formation and cleavage of thiohydantoin ring catalysed by trypsin

The intramolecular dehydratation of phenyl thiocarbamyl amino acid to the corresponding thiohydantoin and the reverse hydrolytic reaction are specifically and separatelycatalyzed by trypsin at two different pH values when l-Arg derivatives are concerned. These substrates disclose the ability of trypsin to catalyze unexpected synthetic and hydrolytic reactions.     

Short communications. The effect of trypsin on cell surface antigens.

The effect of trypsin on cell surface antigenic determinants was measured in a mouse plasmacytoma model. While loss of some antigenic determinants could be detected within 1 min, relevant residual antigens remained even after 1 hr. For solid plasmacytoma of such mice, trypsin could be safely used to prepare single-cell suspensions for similar studies.     

Expression of MHC antigens by mouse thymic dendritic cells.

Thymic epithelial cells express MHC antigens in several different patterns. I-A is present throughout the thymic cortex on dendritic cells. The remainder of the I region and H-2K/D are expressed on dendritic cells apparently only variably in the cortex (at least in some haplotypes). All MHC antigens tested are present in the medulla on epithelial cells; expression on medullary lymphocytes cannot be evaluated. Monoclonal anti-MHC antibodies confirm these results. The significance of these findings to T cell maturation is discussed.     

Limited MHC polymorphism in the southern elephant seal: implications for MHC evolution and marine mammal population biology.

Genes of the major histocompatibility complex (MHC) are highly polymorphic in most terrestrial mammal populations so far studied. Exceptions to this are typically populations that lack genome-wide diversity. Here I show that two populations of the southern elephant seal (Mirounga leonina) have low DNA restriction fragment length polymorphism at MHC loci when compared with terrestrial mammals. Limited studies on MHC polymorphism in two cetacean species suggest this is a feature of marine mammal populations in general. MHC polymorphism is thought to be maintained by balancing selection, and several types of disease-based and reproductive-based mechanisms have been proposed. For the three marine mammal species examined, the low MHC polymorphism cannot be explained by low genome-wide diversity, or by any reproductive-based selection pressure. It can, however, be explained by diminished exposure to pathogenic selection pressure compared with terrestrial mammals. Reduced exposure to pathogens would also mean that marine mammal populations may be susceptible to occasional pathogen-induced mass mortalities.     

Limited caspase cleavage of human BAP31

Human BAP31 was cleaved at both of its two identical caspase cleavage sites in two previously reported models of apoptosis. We show here that only the most carboxy-terminal site is cleaved during apoptosis induced in HeLa cells by tunicamycin, tumor necrosis factor and cycloheximide, or staurosporine. Similar results were obtained in HL-60 cells using Fas/APO-1 antibodies, or cycloheximide. This limited cleavage, which is inhibited by several caspase inhibitors, removes eight amino acids from human BAP31 including the KKXX coat protein I binding motif. Ectopic expression of the resulting cleavage product induces redistribution of mannosidase II from the Golgi and prevents endoplasmic reticulum to Golgi transport of virus glycoproteins.     

Limited cleavage of human lactoferrin with pepsin

• 1.1. Human lactoferrin in the iron-saturated form was treated with pepsin at pH 3.0, and a fragment with a single iron-binding site was isolated in a 90% pure state.
• 2.2. Its mol. wt was near 33,000.
• 3.3. Its N-terminal residue was alanine, and its iron-binding properties were identical to those of lactoferrin.
• 4.4. The curve showing iron incorporation from lactoferrin and its fragment into reticulocytes showed a biphasic character.
• 5.5. It is concluded that lactoferrin consists of two largely independent parts, which may have arisen by a gene duplication process.