Knut Hjelmeland

Characteristics of two trypsin type isozymes isolated from the Arctic fish capelin (Mallotus villosus)

1. Two trypsin-like enzymes, assayed by their amidase activity with N-alpha-benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA) as the substrate, were isolated from the gut of the arctic fish capelin (Mallotus villosus). 2. Purification involved affinity chromatography (Benzamidine-CH-Sepharose 4B) of the 30 to 70% (NH4)2SO4 precipitation fraction of a crude extract of the gut, followed by DEAE-Sephadex chromatography, yielding two enzymes, designated Enzyme I and II. 3. Both enzymes had MW of about 28,000 as determined by SDS-electrophoresis. Their isoelectric points were 5.6-5.9 (Enzyme I) and 5.1-5.3 (Enzyme II) and they had similar amino acid composition. 4. Both enzymes were inhibited by standard trypsin inhibitors including the serine protease inhibitor phenylmethyl sulphonyl fluoride (PMSF), but not by the chymotrypsin inhibitor L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK). 5. The enzymes had a pH optimum of 8-9 and their stability was not affected by CaCl2. Low pH (2.3) caused an initial rapid loss of enzyme activity, followed by relatively slow decomposition of the activity remaining after 1 hr at 4 degrees C. 6. The enzymes had an apparent temperature optimum of 42 degrees C, resulting from rapid self digestion at higher temperatures.

The specificity of atlantic salmon antibodies made against the fish pathogen Vibriosalmonicida, establishing the surface protein VS-P1 as the dominating antigen

The specificity of salmon (Salmo salar) antibodies made against the fish pathogen Vibrio salmonicida was studied. Salmon immunized with V. salmonicida emulisified in Freunds complete adjuvant produced antibodies which preferentially bound to a 40 KD outer surface molecule (VS-P1). Moreover, the bulk of the antibodies were specific for one particular epitope on VS-P1, defined by a mouse monoclonal antibody - as detected with a blocking ELISA. The data imply that salmon B cells mainly (90%) recognize one particular determinant on V. salmonicida and thus express a limited repertoire of antibody specificities against this pathogen.

Proteinase inhibitors in the muscle and serum of cod (Gadus morhua). Isolation and characterization

Abstract 1. 1. A proteinase inhibitor present in serum and muscle of cod has been purified and characterized. 2. 2. It has a molecular weight of about 60,000 daltons, isoelectric point of 4.7–4.9 and inhibits the serum proteinases, elastase, trypsin and chymotrypsin. 3. 3. The inhibitor resembles the α-1-proteinase inhibitor of mammals, but differs in being deplete of tyrosine and methionine, and in having a higher stability at acidic pH values.

Evaluation of Radioimmunoassay as a Method to Quantify Trypsin and Trypsinogen in Fish

Abstract A radioimmunoassay of trypsin and trypsinogen in fish has been developed. The antibodies detect both trypsin and trypsinogen, and the presence of trypsin inhibitors in the assay extract does not affect the readings. The radioimmunoassay, used in combination with conventional enzyme assay, is very suitable for mechanistic studies of the production and activation of trypsin in fish.