Frits C. Stevens

The distribution of the Ca++-dependent protein activator of cyclic nucleotide phosphodiesterase in invertebrates

Summary Animal species representative of major phyla were found to contain activating factors similar to the Ca ++ binding protein activator of 3′:5′ — cyclic nucleotide phosphodiesterase isolated from bovine heart. These factors are heat stable, non-dialysable and susceptible to proteolysis. All activate bovine heart cyclic nucleotide phosphodiesterase to a comparable extent and all activations are Ca ++ dependent and reversible. The results suggest a ubiquitous distribution and fundamental importance for this Ca ++ binding protein.

Stoichiometry of the interaction of lima bean protease inhibitor with trypsin and/or chymotrypsin

Naturally occurring protease inhibitors are proteins which inhibit proteolytic enzymes by the formation of specific enzyme-inhibitor complexes [ 11. Recently it has been observed in several laboratories [2-51 that many protease inhibitors of plant origin exist in solution as dimers, trimers and even tetramers. This aggregation phenomenon has complicated the molecular weight determination of these inhibitors and a fortiori the determination of the stoichiometry of inhibition. Lima bean protease inhibitor (LBI) strongly inhibits both trypsin and chymotrypsin through independent combining sites [6,7] . Based on both osmotic pressure measurements [8] and amino acid analysis [6, 91 its molecular weight is estimated at approximately 9000. However, ultracentrifugation studies [6, 81 suggest a particle size of nearly twice that value. In this study an effort was made to establish the stoichiometry of interaction between LB1 and bovine trypsin and chymotrypsin. The results clearly indicate that the monomer of LB1 (M.W. ‘L 9000) can form 1: 1 molar complexes with either enzyme and also a tertiary complex with one mole of each of these two enzymes.

The trypsin and chymotrypsin inhibitory capacity of human and bovine α-crystallin

The existence of proteolytic enzymes in mammalian lens tissue is fairly well documented and proteinases from bovine [ 1,2] and also from human senile cataractous lenses [3] have been purified and partially characterized. At present the exact role of these enzymes is not well understood; it has been suggested, but not proven, that the loss of protein in advanced cataractous human lenses might be due to increased proteolysis [3,4]. 2.1. Materials Bovine trypsin (TRL 2DA) and chymotrypsin (CD1 11C) were obtained from Worthington (Freehold, N.J.). Calf lenses were purchased from Pel-Freeze Biochemicals (Rogers, Ark.) and human cataractous lenses were obtained from the Winnipeg General and St. Boniface hospitals. Bovine albumin (Cohn fraction V) was obtained from Sigma Chemical Co. (St. Louis, MO.); all other chemicals were reagent grade or better. During our own attempts at isolating proteases from bovine and also from human cataractous lenses we discovered that crude lens homogenates were able to inhibit trypsin and to a lesser extent chymotrypsin. The present paper describes this finding and also shows that the inhibitory activity is associated with the a-crystallin fraction of the lens. Aggregates of urea denatured a-crystallin or of its acidic (oAr , (YAZ) or basic (aB, , aBz) components ]5,6] inhibit trypsin at least five times as strongly as does native a-crystallin. The chymotrypsin inhibitory activity of a-crystallin is much weaker than its trypsin inhibitory activity and can not be increased by urea treatment or separation into the component acidic and basic polypeptide chains. To our knowledge this is the first report of a protease inhibitory activity associated with eye lens proteins. The inhibition of chymotrypsin by aqueous humor has been previously reported by other workers 1731. 2.2. Assays for inhibitory activity Assays for protease inhibitory activity were based on the method of Anson [9] for determining proteolytic activity. Trypsin or chymotrypsin (1 S-20 pg) and a sample eye lens protein (o-2000 lug) were preincubated for 30 min at 37°C in 200 ~1 of 0.006 M Tris-HCl, pH 7.6. The substrate (1 ml of 2% bovine albumin, pH 7.6) was then added and the mixture incubated for a further 20 min at 37°C. After precipitation of the proteins with trichloracetic acid (2 ml of a 5% aqueous solution) and centrifugation, the unprecipitated split products were estimated with phenol reagent [9]. For each sample a blank was carried out in an identical fashion except that the trichloracetic acid was added immediately after the addition of substrate. Protein concentrations were determined by the method of Lowry [IO] .

Lima bean protease inhibitor: Comparative study of the trypsin and chymotrypsin inhibitory activities of four chromatographically pure variants.

Extensively purified commercial preparations [l] of lima bean protease inhibitor (LBI) can be further separated into at least four biologically active variants by ion exchange chromatography [2, 31. These variants have similar but not identical amino acid compositions and tryptic peptide maps [2-41. Previous studies [3-8] on the structure, biological activity and mode of action of LB1 have been performed using either the mixture of variants or individual variant preparations. In the present study we provide the first comprehensive comparison of the inhibitory activities of four variants of LBI. For the sake of clarity it should be recalled that LB1 can inhibit both trypsin and chymotrypsin through the formation of equimolar complexes with these enzymes and that the binding sites for trypsin and chymotrypsin on LB1 are distinct and independent [4-61. We have previously identified a Lys-Ser peptide bond in the anti-trypsin site of LB1 [4, 7, 81; incubation of LB1 with catalytic amounts of trypsin at acid pH results in an equilibrium mixture of native and trypsin-modified inhibitor (LBI;) in which this Lys-Ser peptide bond has been hydrolysed [7]. LBI; combines with trypsin slower than does LB1 and therefore it appears less active than LB1 in an assay system which does not involve preincubation between enzyme and inhibitor. Removal of the newly formed carboxyterminal lysine residue with carboxypeptidase B results in complete loss of trypsin inhibitory actirity. Incubation of LBI; with near molar amounts of trypsin at neutral pH results in eventual complex