Jorma E. Fräki

Human skin tryptase: purification, partial characterization and comparison with human lung tryptase.

Human skin tryptase was isolated using stepwise low- and high-salt extraction and further purified 448-fold with 33% yield using octyl-Sepharose CL-4B hydrophobic affinity chromatography, Sephacryl S-200 gel filtration and finally octyl-Sepharose CL-4B or cellulose phosphate ion exchange chromatography. The skin tryptase, which has an apparent Mr of 120,000 by gel filtration in high-salt buffer, consisted of polypeptide chains of Mr 34,000 and 38,000 when resolved on SDS gels. Both polypeptide chains, labelled with [3H]diisopropyl fluorophosphate, indicated that they were representative of subunits and that the native proteinase was an aggregate of subunits. However, in some preparations only one band with Mr 34,000 was seen. In low-salt buffer the enzyme was labile and at least 1.4 M KCl was needed to keep the enzyme stabile when incubated at 37 degrees C for 30 min. Heparin glycosaminoglycan partially stabilized the tryptase but addition of protein (e.g. albumin, 80 micrograms/ml) to the tryptase-heparin mixture was needed to keep the enzyme stabile. Tryptases purified by exactly the same method from human lung tissue and from human skin had identical molecular size in gel filtration and in SDS-polyacrylamide gel electrophoresis. They also revealed identical enzyme kinetic parameters with several synthetic peptide substrates. The inhibition profile was identical for both enzymes, and they also crossreacted completely in immunodiffusion plates. These studies strongly indicate that mast cells found in skin as well as lung contain closely related, possible identical trypsin-like proteinases.

Transplantation of Psoriatic Skin onto Nude Mice

Involved psoriatic epidermis maintains its histologic appearance, increased labeling index, and increased level of plasminogen activator after grafting onto athymic nude mice. Epidermis from clinically uninvolved psoriatic skin develops an increase in plasminogen activator activity after grafting for 6 weeks on nude mice and demonstrates an increased labeling index. Normal control skin maintains its low level of plasminogen activator and labeling index after grafting. These results indicate that psoriatic skin can maintain and develop markers of psoriatic skin independent of the host.

Optimization of histamine radio enzyme assay with purified histamine-N-methyltransferase

The radio enzyme assay for histamine based on the transmethylation with purified histamine-N-methyltransferase and utilizing [3H-methyl]-S-adenosylmethionine as the methyl donor has been optimized to measure low histamine concentrations, for example in plasma. The pH-optimum for the assay is pH 8.3 in Tris-glycine buffer at 20 degrees C. An incubation time of 90 min is necessary using an enzyme concentration of 5.8 micrograms/ml. EDTA and dithiothreitol were included in the assay to keep the histamine-N-methyltransferase active as agents that oxidize -SH groups were found to be inhibitory to the reaction. The present assay is sensitive to about 0.5 nmol/l of histamine in a sample volume of 50 microliter (about 3 pg/sample).

Purification and identification of two serine class proteinases from dog mast cells biochemically and immunologically similar to human proteinases tryptase and chymase

Serine class proteinases with trypsin-like and chymotrypsin-like specificity were purified from dog mastocytoma tissue. An antiserum was produced against the chymotrypsin-like proteinase. The antiserum reacted with mast cells in skin sections prepared from normal dogs consistent with the proteinase being a mast cell constituent. The antiserum also cross-reacted with the major chymotrypsin-like proteinase isolated from normal dog skin and partially cross-reacted with human skin chymase. No cross-reaction was detected with rat chymase. The trypsin-like proteinase from dog mastocytoma tissue was similar to tryptase isolated from human skin. It had a similar subunit structure, was not inhibited by many protein proteolytic enzyme inhibitors, bound to heparin, and reacted strongly with antiserum against human tryptase. Antiserum against human tryptase also reacted with mast cells in skin sections prepared from normal dog skin. No immunocytochemical labeling of rat skin mast cells was observed with anti-human tryptase. These studies establish the presence of a trypsin-like and chymotrypsin-like proteinase in dog skin mast cells and provide immunological evidence which suggests that both proteinases are more closely related to human than rat mast cell proteinases. These immunological and biochemical relationships are important when comparing the roles of these proteinases in different animals.

Purification and partial characterization of rat kidney histamine-N-methyltransferase

Histamine-N-methyltransferase (EC 2.1.1.8) was purified 1700-fold with a yield of 9% from rat kidney. Purification included ammonium sulfate precipitation, linear gradient DEAE-cellulose chromatography and S-adenosylhomocysteine affinity chromatography. The purified enzyme preparation showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 35000. The isoelectric point of the enzyme was at pH 5.2. The purified enzyme preparation did not contain detectable amounts of histamine. The purified enzyme was totally inhibited in 100 microM parahydroxymercuric benzoate and in 10 microM iodoacetamide, and it was found to be stabilized with dithiothreitol (1 mM), suggesting that the enzyme has an SH-group in the active center. The Km values for histamine and S-adenosylmethionine were 6.0 and 7.1 microM, respectively. 50% inhibition of histamine-N-methyltransferase was obtained at 28 microM S-adenosylhomocysteine and 100 microM methylhistamine. The purified enzyme was slightly inhibited in 1 mM methylthioadenosine. Histamine in concentrations higher than 25 microM caused substrate inhibition.

The allosteric effect of salt on human mast cell tryptase

The inhibitory effect of potassium chloride and ammonium sulphate on purified human skin tryptase and bovine trypsin was studied enzyme-kinetically, using Z-Gly-Pro-Arg-pNA, Z-Gly-Pro-Arg-AMC, benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME) as substrates. With increasing salt concentrations, the curve of reaction velocity vs. substrate concentration changed from hyperbolic to sigmoidal when anilide substrates (Z-Gly-Pro-Arg-pNA or -AMC) were used. Only the Km value increased, while the Vmax value remained unchanged. The trend was similar with BAEE or TAME as the substrates. However, the effect of salt on the hydrolysis of these ester substrates was not as strong as on the hydrolysis of anilide substrates, and sigmoidal kinetics were not observed even at the highest KCl concentration (0.7 M) used. Heparin, used as a stabilizer, had no influence on this phenomenon, but it did slightly decrease the apparent Km and Vmax values in low-salt conditions. By comparison, trypsin was not as strongly affected by salt as tryptase, and the inhibition type was mixed competitive and non-competitive. The present results indicate that the salt acts on tryptase as an allosteric effector, and this should be carefully considered when enzyme kinetic parameters and enzyme activity of skin tryptase are measured.

Effect of Human Mast Cell Tryptase on Human Plasma Proenzymes

The effect of human skin mast cell tryptase on human plasma proenzymes (prothrombin, coagulation factor XII, complement C1s, protein C and plasminogen) was investigated. Tryptase had no effect on these proenzymes, when incubated with them at 37 degrees C for up to 90 min, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by the ability to hydrolyze specific peptide p-nitroanilide substrates. After prolonged treatment with tryptase, proenzymes could be fully activated with their specific activators. The results indicate that tryptase neither activates these plasma proenzymes nor inactivates the corresponding active enzymes. As a positive control, the tryptase preparation was also incubated with human fibrinogen and rat thymus histones. Prolonged treatment with tryptase increased the thrombin-induced clotting time of fibrinogen. Tryptase also efficiently hydrolyzed histone H1 from rat thymus. Histones H3/H2B and H2A were hydrolyzed less efficiently than H1, and no hydrolysis of histone H4 by tryptase was detected under the experimental conditions.

Effect of drugs on histamine radio-enzyme assay

The effect of over 200 drugs and other compounds on histamine radio-enzyme assay was studied. Some muscle relaxants (e.g. alcuronium), some sympathomimetics (e.g. dopamine, isoxsuprine, tyramine and possibly phenylethylamine), antimalarial drugs, procaine, procainamide, Berenil and serotonin were potent compounds to interfere with this assay. In some special cases still potentially inhibitory drugs seemed to be some muscle relaxants (e.g. vecuronium, pancuronium and tubocurarine), antidepressants, antihistamines (e.g. cimetidine, ranitidine and diphenhydramine), chinidin, disopyramide, tolazoline and salazosulfapyridine.