Effect of arginine on the activity of proteolytic enzymes in the rat brain and liver

The influence of arginine on autolysis and proteolysis was studied. Arginine at the concentration of 0.5 and 1.0 microM/ml was added to the incubation mixture. Proteolytic processes were studied in the acid, neutral and alkaline media (pH 4.5; 7.4; 8.5). Autolysis was determined by incubation of the brain and liver homogenates and proteolysis by the use of bovine serum albumin as a substrate. Autolytic and proteolytic activities were calculated as an increase of Folin positive compounds or amino nitrogen in the samples. It was established that the influence in vitro of arginine on the proteolytic processes depended on pH, type of the peptide-hydrolases, to a lesser extent, on the arginine concentration and did not depend on the tissue type. Arginine displayed its regulative action in the brain and liver by the same way. The addition of arginine had an effect on autolysis and proteolysis in the neutral and alkaline media. Determination of autolytic and proteolytic activities by Folin positive compounds has shown that arginine addition into the samples decreased autolysis and proteolysis. At the same time determination of autolysis and proteolysis by amino nitrogen in the presence of arginine has shown that autolytic and proteolytic activities increased.     

Esterolytic and proteolytic enzymes of the rat adenohypophysis

Summary Characteristics of the hydrolysis of histochemical substrates 5-bromoindoxyl acetate, naphthyl acetate, proprionate, butyrate, caprylate, laurate, myristate, and palmitate, acetyl and butyryl thiocholhie, chloroacetyl and trifluoroacetyl -naphthylamide, benzoyl-arginine -naphthylamide and proteinase substrates human hemoglobin and glycyl-phenylalanine amide by the rat pituitary tissue homogenate and DEAE-cellulose chromatography fractions were determined.In DEAE-cellulose chromatography fractions four separate activities were found splitting short and long chain carboxylic esters. The activity hydrolysing most rapidly 5-bromoindoxyl acetate was resistant to E 600 and was identified as C esterase. Three of the remaining esterase activities were sensitive to E 600 and two of them hydrolysed more rapidly short-chain fatty acid esters while one preferred long-chain fatty acid esters as substrate.One peak of activity was identified as nonspecific cholinesterase on the basis of inhibition studies and hydrolysis of thiocholine substrates. Chloroacetyl -naphthylamide was hydrolysed minimally. Hydrolysis of trifluoroacetyl -naphthylamide was ascribed to E 600 resistant enzyme with pH-optimum at 8.3 hydrolysing also the thiocholine substrates and slowly long-chain fatty acid esters.Five different proteinases hydrolysing human hemoglobin were separated, three of them with pH-optima on the acid and two on the alkaline side of pH. The activities hydrolysing benzoyl-arginine naphthylamide were cysteine activated and had pH-optima around 5.3. None of the peaks of the proteinase activities appeared to coinside with the hydrolysis peaks of any of the histochemical ester substrates in the DEAE fractions.     

Methods of determining the activity of proteolytic enzymes

The present methods for determination of the proteinases activity based on the protein, low-molecular weight, fluorescent and radioactive substrates, also active-site titration have been analyzed. The comparative evaluation of its effectiveness which depends on duration, pH range, possible errors, caused by concomitant proteinases are considered. The influence of this factors on the choice of assay for measuring the proteinases activity and its efficiency are discussed.     

The action of venom and proteolytic enzymes on the non-specific inhibitor of hyaluronidase

1. It has been shown that a number of proteolytic enzymes and snake venom, in relatively small amounts, and within a wide range of pH variation, will restore hyaluronidase activity after its inhibition by serum. 2. The known properties of the venom protease are found to be identical with those of Haas' "proinvasin I." It is concluded that the protease of the venom offers adequate explanation for the effects previously attributed to "proinvasin I." 3. Proteolytic activity is found in hyaluronidase preparations of bovine origin and is considered to be responsible for the reversal of inhibition of hyaluronidase by serum.     

Effect of proteolytic enzymes on masked insulin receptors in rat submaxillary gland microsomes

Trypsin and alpha-chymotrypsin effects on masked insulin receptors were studied. Phospholipase C treatment, incubation in a high ionic strength buffer or solubilization were used as alternative procedures for the unmasking of insulin receptors. These three methods expose receptor structures which are inaccessible to insulin in the current experimental conditions of binding assays without any significant change in binding affinity. Both exposed and masked receptors proved to be equally sensitive to trypsin and alpha-chymotrypsin degradation. At 25 degrees C, about 5 micrograms trypsin/ml for 50 min or 80 micrograms alpha-chymotrypsin/ml for 200 min were necessary in each case to cause a 50% inhibition of the binding of 125I-iodo insulin to microsomes. The results suggest that masked receptors are only nonfunctional to bind insulin but they are not located in compartments inaccessible to molecules present in the medium.     

Influence of delta-sleep-inducing peptide on the activity of proteolytic enzymes in the rat brain under hypokinesia]

Single administration of the delta-sleep-inducing peptide (DSIP) in a dose of 12 micrograms/100 g to intact animals makes the activity of neutral proteinases and cathepsin D higher in the rat brain and blood serum. Hypokinesia of different duration changes activity of neutral and acidic proteinases and induces accessibility of cathepsin D to the cytosol as a result of damage in lysosomal membrane. Injection DSIP induces a decrease A/B of cathepsin D to the control level under 1-h hypokinesia condition and normalizes the neutral proteolytic activity under 6-h hypokinesia condition.