Establishment of serum based essentiall free of proteolytic activity for the culture of mouse pancreatic islets.

The present study demonstrates that a) serum based culture medium degrades 125I inhibits its proteolytic activity leading to the recovery of more insulin secreted by islets cultured in the presence of high glucose concentration alone or with glucagon; c) aprotinin also favoured the accumulation of secreted insulin by protecting the hormone from a residual degradative capacity of the hear treated serum.     

Debriding ability of a novel multi-enzyme preparation isolated from Antarctic krill (Euphausia superba)

Summary The wound-debriding activity of various types of proteolytic enzymes and proteases from Antarctic krill (multi-enzyme system consisting of both endo- and exopeptidases) was evaluated. The results, based on the enzymatically acieved weight reduction of a necrotic animal material (excised rat skin) in vitro, clearly showed that the multi-enzyme system (krill) had a higher degrading activity than the single enzyme preparation, or that with only a few enzymes. The debriding effect of the krill enzymes was markedly related to the enzyme concentration, resulting in 70–100% substrate degradation after 24 h. The digesting capacity of trypsin reached about 50%, but an increase in concentration of this enzyme did not substantially influence its overall activity. The effect of streptokinase-streptodornase, collagenase and plasmin-desoxyribonuclease was weak (10–20% digested).     

Establishment of serum based medium essentially free of proteolytic activity for the culture of mouse pancreatic islets

Summary The present study demonstrates that a) serum based culture medium degrades¹²⁵I insulin; b) heat in-activation of serum (1 h, 56°C) inhibits its proteolytic activity leading to the recovery of more insulin secreted by islets cultured in the presence of high glucose concentration alone or with glucagon; c) aprotinin also favoured the accumulation of secreted insulin by protecting the hormone from a residual degradative capacity of the heat treated serum.This work was supported by a grant (No. 71 5 426-2) from the INSERM and by the CNRS.These results have been presented at the V International Congress of Endocrinology. Hamburg, July 18–24, 1976.Acknowledgments. We should like to thank Mrs E. Gammelgard and K. Christensen for their technical assistance.     

Increase of proteolytic activity inLactobacillus casei by X-ray irradiation

Summary A study was conducted on the effect of X-ray irradiation on proteolytic activity ofLactobacillus casei (RTS). Substantial increase (82%) in the proteolytic activity was noted among the isolates of X-ray irradiation survivors after 18,000 R.     

Die Solubilisation der Gehirnneuraminidase durch proteolytische Enzyme

Summary The release of membrane-bound brain neuraminidase from brain homogenate by proteolytic digestion with trypsin, -chymotrypsin and pronase is described. Trypsin shows the highest solubilizing effect. On the basis of the ammoniumsulphate precipitation the pronase-released neuraminidase has the lowest molecular size.     

Induction of proteolytic activity in serum by treatment with anionic detergents and organic solvents

Using casein plates as a sensitive assay for proteolytic activity, it was observed that sodium-dodecyl sulfate (SDS) and other anionic detergents induce caseinolysis when mixed with sera and plasma. Caseinolysis was dependent on the presence of plasminogen in the fluids and could be blocked by inhibitors of serine proteases and antibody to plasminogen. Similarly, organic solvents such as isopropanol induced caseinolysis after mixing with plasma, but not normal serum. Isopropanol dissociated complexes of alpha 1-antitrypsin or alpha 2-macroglobulin with trypsin preformed in vitro. As both SDS and organic solvents are widely used in biochemical investigations of biological fluids, attention should be paid to the possible induction of proteolysis.     

Fragmentation of myosin A-1 light chain of fast muscle by trypsin]

Limited proteolysis of myosin by such proteolytic enzymes as trypsin, chymotrypsin or papain produces typical fragmentation of its heavy chain. Presently evidence is given that trypsin treatment cleaves the alkali light chain A-1 (20,700 dalton) to a shorter (ca 20,000 dalton) chain. The two "essential" thiols (SH-1 and 2) of moysin were alkylated with 17-C-N-ethylmaleimide and a non-negligible amount of radioactivity was also found in the two alkali light chains. Using the specific radioactivity of alkali light chain A-1 it was possible to identify it among heavy chain fragmentation products. The molecular weight of the newly formed A-1 indicates that limited tryptic cleavage of this A-1 confers on it a closer similarity with alkali light chain A-2.     

Über die Hemmung des Gerinnungsfermentes Thrombin durch Benzamidinderivate

Summary A series of aminomethyl, guanidino and amidino derivatives of benzene were investigated for their inhibitory effect on the activity of thrombin. Amidines were generally more potent than the other compounds.p-Amidinophenylpyruvic acid, a strong trypsin inhibitor, was found also to be the strongest small-molecular inhibitor on the activity of thrombin.     

Bombesin promotes pancreatic growth in suckling rats

The pancreatic growth promoting effect of long term administration of bombesin was investigated in suckling rats. The authors showed that bombesin given in 10 micrograms/kg b.wt doses s.c. every 8 h for 10 days from the day of parturition stimulated pancreatic growth: it increased pancreatic weight, protein and DNA content, trypsin and amylase activity and trypsin/DNA ratio. Conclusion: Bombesin is an effective stimulator of pancreatic growth in suckling rats.     

Proteolytische Aktivität normaler und antigenstimulierter Makrophagen

Summary The proteolytic activity of polynuclear leucocytes of the guinea-pig was determined prior to and after incubation with the pseudorabies virus (the virus of Aujeszky disease). It was found that the proteolytic activity of leucocytes treated with the virus was increased by 34.5%. There is a brief discussion of the biological value of the increased proteolytic activity.     

Protein digestion in the sea gooseberryPleurobranchia bachei A. Agassiz (Ctenophora: Tentaculata)

Summary Peaks of proteolytic activity of pharyngeal juice occur at pH 5.75 and pH 7.5. The proteases responsible for digestion include a tryptic alkaline protease and a thiol-activated acid protease which is probably cathepsin B. Levels of proteolytic activity parallel those of other carnivorous invertebrates which feed on zooplankton.This study was supported by operating grants from the National Research Council of Canada.We are grateful to Betsy Sweeney for her technical assistance.     

Glucose does not influence the insulin-like growth factor (IGF) binding to carrier proteins (IGFBPs): Analysis of rat and human serum by western ligand blotting

The insulin-like growth factors (IGFs) circulate bound to specific proteins (termed IGFBP-1 through IGFBP-6) that modulate IGF bioactivity in tissues. The aim of this study was to analyse the effects of glucose on IGF binding to IGFBPs in rat and human serum by means of western ligand blotting. Serum samples were incubated with increasing concentrations of glucose (0 to 50 mmol/l), and EDTA (25 mmol/l) was added to inhibit protease activity. To analyse the effect of glucose on protection of IGFBPs from protease activity, serum from pregnant women (reported to be very rich in proteolytic activity against IGFBPs) was added to rat serum previously incubated with glucose. Glucose did not affect the¹²⁵I-IGF binding to rat and human serum IGFBPs. The intensity of IGFBP-3 bands decreased considerably during the incubation. This appeared to be due to endogenous protease activity, since the decrease was blocked by addition of EDTA. The incubationi of rat serum with pregnant human serum produced a marked attenuation of IGFBP-3 and disappearance of IGFBP-4 bands. In conclusion, our study shows that glucose does not influence the IGF binding to IGFBP-3 either in rat or in human serum, confirms the presence of endogenous proteolytic activity in normal non-pregnant serum, and demonstrates that glucose has no protective action against protease activity.     

Bombesin promotes pancreatic growth in suckling rats

Summary The pancreatic growth promoting effect of long term administration of bombesin was investigated in suckling rats. The authors showed that bombesin given in 10 g/kg b.wt doses s.c. every 8 h for 10 days from the day of parturition stimulated pancreatic growth: it increased pancreatic weight, protein and DNA content, trypsin and amylase activity and trypsin/DNA ratio. Conclusion: Bombesin is an effective stimulator of pancreatic growth in suckling rats.     

Ureteral contractions induced by rat urine in vitro: Probable involvement of renal kallikrein

Summary Rat urine, even at a 110 final dilution in Tyrode's solution, stimulates contraction of the ureteral musculature in vitro. This effect can be ascribed to the presence of kallikrein or a kallikrein-like enzyme in urine. Isometric contractions of ureters were prevented by previous addition of aprotinin to the organ bath. Urine also lost its activity after inactivation of enzymes by heat or acid treatment.Acknowledgments. Aprotinin (Trasylol^®) was kindly supplied by Baypropharm, Köln, W.-Germany. This work was supported by the German Research Foundation (SFB 90).     

Low-molecular-weight proteinase inhibitor in human plasma inhibiting papain and trypsin activity

A low-molecular-weight proteinase inhibitor was isolated from human plasma on the basis of its papain-inhibiting capacity. The isolated fraction demonstrated inhibitory activity against papain and trypsin activity and the molecular weight was estimated to be approximately 3200 by gel-filtration.     

Low-molecular-weight proteinase inhibitor in human plasma inhibiting papain and trypsin activity

Summary A low-molecular-weight proteinase inhibitor was isolated from human plasma on the basis of its papain-inhibiting capacity. The isolated fraction demonstrated inhibitory activity against papain and trypsin activity and the molecular weight was estimated to be approximately 3200 by gel-filtration.     

Model of the interaction between trypsin and alpha 2-macroglobulin of porcine serum]

A comparative study of the dissociation into subunits of Porcine alpha2 M, either native or bound to trypsin (Tn), has been carried out in order to determine the modifications of the alpha2 M structure due to the formation of the Tn-alpha2 M complex. Analytical ultra-centrifugation at pH 3.5 shows that the dissociation is smaller when alpha 2 M is bound to trypsin. Electrophoresis in 4% polyacrylamide gels, in presence of 0.1% SDS, of alpha2 M and Tn-alpha2 M incubated in 1% SDS leads to the same conclusion; the enzyme must stabilize the quaternay structure of alpha2 M. In presence of SDS + beta-mercaptoethanol, only a molecular weight (M.W.) 200,000 band is revealed in electrophoresis pattern of native alpha2 M. In the case of reduced Tn-alpha2 M, some other bands of M.W. 100,000, 50,000, 30,000 appear. When trypsin is inactivated by TLCK 100,000 M.W. band is present, accompanied by the 200,000 M.W. band whose intensity is function of the alpha2 M concentration. The 100,000 M.W. band appears therefore characteristic of the formation of the complex which must imply a proteolytic cleavage in the middle of the 100,000 polypeptidic chain of alpha2 M. A model of the complex is proposed in which the enzyme forms a proteic bridge between the two halves of the alpha2 M molecule.     

Modification of effects of biologically active peptides,caused by enzyme treatment,on the excitability of identifiable giant neurones of an African giant snail (Achatina fulica Férussac)

Summary Physalaemin, which excites an identifiable molluscan giant neurone (the TAN, tonically autoactive neurone), lost the effect after the trypsin treatment. Unexpectedly, this peptide shows an inhibitory effect on the same neurone after chymotrypsin treatment. Deamino-dicarba-(d-d-)oxytocin and d-d-Arg-vasotocin, which excite another identifiable neurone (the PON, periodically oscillating neurone) continue to show the effect after chymotrypsin treatment (6 h). But d-d-Arg-vasotocin losts the effect on the PON after trypsin treatment.The authors thank Dr Sadaaki Iwanaga of Osaka University and Dr Atsuo Inoue of Daiichi Pharmaceutical Co. for their helpful advice, and Miss Hiroko Tamura for her technical assistance.     

Cryo-bioorganic chemistry: molecular interactions at low temperature

Freezing of aqueous or organic solutions plays a pivotal role in enhancement of rate and/or yield of biomolecular reactions. The smooth conditions of the frozen state at low temperature can also suppress racemization and side-product formation of the reactions. Molecular interactions in liquid undercooled solutions, on the other hand, offer the possibility to study enzyme activity mechanisms in vitro and a chance for survival of organisms in vivo. This review illustrates the differences between frozen and liquid conditions on several small and large biomolecules, together with the synthetic use of freezing. In relation to the freezing effect on enzyme activity, a peculiar phenomenon is discussed: 'cryo-oscillations' are temporal motions of trypsin activity in frozen solution in the presence of Mn2+ ion. The molecular basis of cold adaptation is also discussed, which points to mechanisms evolved by organisms living at subzero temperatures. The factors involved in the freezing effect are shown; i.e. the role of freeze-concentration and frozen solvent surface is demonstrated and elucidated using several examples.     

Effects of enterally- and parenterally-administered bombesin on intestinal luminal tryptic activity and protein in the suckling rat

Because of the presence of bombesin-like immunoreactivity in milk, we investigated if enteral administration of bombesin affects the intestinal luminal content of trypsin and protein in 12-14-day-old rats. Bombesin (40 micrograms/kg), given either orogastrically or subcutaneously, produced a significant elevation in the intestinal content of trypsin activity. Thus, enterally-administered bombesin can produce acute biologic effects in suckling rats.