Kunitz Pancreatic Trypsin Inhibitor Research Articles

The primary structure of Vipera ammodytes venom trypsin inhibitor I

The primary structure of Vipera ammodytes venom trypsin inhibitor I consists of 61 amino acid residues in the sequence: Pyr-Asp-His-Pro-Lys-Phe-Cys-Tyr-Leu-Pro-Ala-Asp-Pro-Gly-Arg-Cys-Lys-Ala-His-Ile- Pro-Arg-Phe-Tyr-Tyr-Asp-Ser-Ala-Ser-Asn-Lys-Cys-Asn-Lys-Phe-Ile-Tyr-Gly-Gly-Cys-Pro-Gly-Asn-Ala- Asn-Asn-Phe-Lys-Thr-Trp-Asp-Glu-Cys-Arg-Gln-Thr-Cys-Gly-Ala-Ser-Ala. The N-terminal group of the inhibitor is pyrrolidonecarboxylic acid. The sequential data were obtained by analysis of peptides isolated from tryptic and chymotryptic digests and by analysis of peptides derived from the hydrolysis of the aspartyl-prolyl bond of the carboxymethylated inhibitor. The primary structure of trypsin inhibitor I presented shows approximately 80% sequence homology with chymotrypsin inhibitor isolated from the venom of the same snake, and nearly 50% homology with bovine basic pancreatic trypsin inhibitor. It belongs to the Kunitz-pancreatic trypsin inhibitor family of inhibitors.

Development and evaluation of an inhibitor-releasing matrix for intrauterine devices

Antifibrinolytic agents when released into the uterine cavity decrease menorrhagia associated with IUD use. Our objective was to develop a matrix that could be incorporated onto an IUD and release anti-fibrinolytic agents. The copolymer ethylene-vinyl acetate (EVA) was selected for detailed study because it has the advantage over other materials in that it can release large molecular weight substances for more than 100 days, and allows incorporation of large amounts of anti-fibrinolytic agents with different molecular weights. Two compounds, tranexamic acid (AMCA, MW = 157) and Trasylol (Kunitz pancreatic trypsin inhibitor, MW = 6,500) were incorporated into the EVA matrix and their release rates measured. In vitro studies with AMCA showed that after the initial burst, a constant high release rate was obtained over a prolonged period of time. The in utero release rate of AMCA from the EVA matrix in rabbits was similar to that obtained in vitro . By contrast, the release rate of Trasylol decreased to low levels during incubation in vitro . The release rate of Trasylol in utero however, appeared to be higher than that in vitro .

Insulin degradation by hepatocytes in primary culture.

The mechanism by which the liver degrades insulin has not yet been completely clarified. In intact, non-"leaky" cells the primary process seems to be mediated by initial receptor binding. We now demonstrate that isolated rat hepatocytes in primary culture are suitable for examining insulin degradation. Hepatocytes did not leak degrading activity into the medium, and thus, the degradation seen was essentially exclusively cell mediated. [125I]Iodoinsulin degradation by these cells was dependent on time and cell concentration. There was a short lag time before degradation products could be detected in the medium. After incubation with the hepatocytes, three peaks of 125I-labeled material could be separated by chromatography on Sephadex G-50. The same three peaks were seen with 125I-labeled material extracted from the cells. When [3H]insulin, labeled exclusively at the B-1 phenylalanine residue, was incubated with the cells, additional peaks of labeled material were recovered from the column. These additional peaks were intermediate in size between insulin and iodotyrosine, suggesting the production of products smaller than insulin but larger than individual amino acids. In order to begin to characterize the subcellular mechanisms for insulin metabolism, the effect of various potential inhibitors on insulin degradation were examined. The most effective inhibitors were N-ethylmaleimide, bacitracin, and Kunitz pancreatic trypsin inhibitor. Chloroquine decreased degradation only 10%, and NH4Cl had no detectable effect. The effect of the inhibitors on the purified insulin-degrading enzyme, insulin protease, was also examined. The purified enzyme responded essentially identically as the intact cells to the various inhibitors. From all these data it would seem that lysosomal degradation of insulin in the hepatocyte may be a relatively minor pathway and the neutral protease may play a major role.

Biochemical Aspects of the Coagulation and Liquefaction of Human Semen

The coagulation and liquefaction process of human semen was studied in some detail. It was found that contact of seminal vesicle fluid with the other accessory sex gland secretions or spermatozoa does not seem to be essential for the formation of the coagulum, and that heparin and sodium citrate do not affect coagulum formation, indicating a difference between the seminal coagulation process and that of the blood clot. Regarding the liquefaction process of the seminal coagulum, it was shown that 1) spermatozoa do not influence liquefaction; 2) that the seminal plasminogen activator, lysozyme, α‐amylase, pepsin, and neuramini dase are not the primary liquefying factors; 3) that the liquefying agent is present in the first portion of a split ejaculate, is heat labile, is partially destroyed by freezing at −20 C but is stable to lyophilization, is precipitable with (NH4)2SO4, is not affected by serum or EDTA, and is inhibited by rabbit bile but not by the seminal plasma proteinase inhibitors, the Kunitz pancreatic trypsin inhibitor, or epsilon‐aminocaproic acid (EACA). All of these prop erties are characteristics of the seminal en zyme, seminin. A partially purified preparation of this enzyme enhanced liquefaction, and two patients who had low seminin activity pos sessed poorly lysing coagula. These findings confirm that seminin is the agent primarily re sponsible for liquefaction of the seminal coagulum of man. The data further show that liquefaction of the seminal coagulum occurs by a different mechanism than that of the lysis of the blood clot.

The preparation and properties of bovine enterokinase.

Bovine enterokinase was purified from duodenal mucosa. The purification included an initial extraction with 2% deoxycholate, ammonium sulfate fractionations, DEAE-cellulose chromatography, and affinity chromatography on basic pancreatic trypsin inhibitor (Kunitz) (PTI)-Sepharose. The purified enzyme contained 35% carbohydrate; it had a molecular weight of 150,000, with a heavy (115,000) and light (35,000) chain connected by one or more disulfide bonds. Enterokinase hydrolyzed lysine and arginine substrates and slowly reacted with the trypsin active site titrant 4-methylumbelliferyl-p-guanidinobenzoate. The enzyme activated bovine trypsinogen with kinetic parameters similar to those of other preparations of enterokinase. Bovine enterokinase was inhibited by Kunitz pancreatic trypsin inhibitor with a Kassoc of 2 X 10(8) M-1 and only weakly by other proteinase inhibitors. The amino acid composition differed from bovine enterokinase isolated from duodenal contents (Anderson, L.E., Walsh, K.A., and Neurath, H. (1977) Biochemistry 16, 3354-3360). The mucosal enzyme and the duodenal contents enzymes also differed in the size of the heavy and light chains. The mucosal enterokinase more closely resembled the properties of porcine enterokinase (Baratti, J., Maroux, S., Louvard, D., and Desnuelle, P. (1973) Biochim. Biophys. Acta 315, 147-161). The amino acid composition and size of the light chain were also similar to bovine trypsin.

Isolation of a trypsin-like enzyme from Streptomyces paromomycinus (paromotrypsin) by affinity adsorption through Kunitz inhibitor-sepharose.

A trypsin-like enzyme has been isolated from the filtrate of a Streptomyces rimosus forma paromomycinus culture. Purification involves acetone fractionated precipitation, ultrafiltration on a Diaflo UM 10 membrane and affinity adsorption on to Kunitz pancreatic trypsin inhibitor linked to Sepharose. The trypsin-like enzyme (paromotrypsin) appears homogeneous by zone electrophoresis on gelatinized cellulose acetate. Specific activity toward Tos-Arg-OMe, calculated from amino acid analysis, is about 220 mu mg-1. The overall yield in activity is about 30%. The molecular weight of the trypsin-like enzyme, determined by gel filtration, is around 22,000-25,000 daltons. Electrophoretic migration on cellulose acetate strips indicates an isoelectric point around 8. Amino acid composition has been determined; the protein comprises about 210 residues on the basis of a single histidine residue per molecule. Paromotrypsin is unstable in acidic medium and is not stabilized by calcium ions. Enzymic activity towards Bz-Argo-OEt is not increased by the addition of calcium ion in contrast to the activating effect observed on bovine trypsin. Paromotrypsin is inhbited by TLCK and NPGB; it interacts with naturally occurring bovine trypsin inhibitors such as soya bean and Kunitz pancreatic inhibitors, but not with chicken ovomucoid. Proteolytic specificity, examined by hydrolysis of oxidized Kunitz pancreatic inhibitor and characterization of resulting peptides, seems similar to that of bovine trypsin.

Inhibition spectra of the human pancreatic endopeptidases

The present work describes the effect of seven naturally occurring proteinase inhibitors on the human pancreatic endopeptidases cationic trypsin, anionic trypsin, chymotrypsin I, chymotrypsin II, and protease E (an elastase-like protease). The inhibitors tested in order of their decreasing effectiveness were alpha-1-proteinase inhibitor (alpha-1-antitrypsin), lima bean trypsin inhibitor, soybean trypsin inhibitor, Bowman-Birk (soybean) inhibitor, Kunitz pancreatic trypsin inhibitor, porcine Kazal inhibitor, and chicken ovomucoid. The human trypsins demonstrated a higher degree of susceptibility to these inhibitors than did the chymotrypsins while human protease E showed remarkably little inhibition by any of these naturally occurring proteinase inhibitors except for alpha-1-proteinase inhibitor. The contribution of each of these proteolytic enzymes to the total proteolytic activity of crude extracts was also investigated using specific active-site directed reagents. These studies revealed that the trypsins constituted approximately 35% of the proteolytic activity while the chymotrypsins represent approximately 32% of the total proteolytic activity. Human protease E and possibly human pancreatic elastase are responsible for approximately 21% of this activity as measured on crude pancreatic extracts.

Studies on ram acrosin. Isolation from spermatozoa, activation by cations and organic solvents, and influence of cations on its reaction with inhibitors.

1. A simple method is given for isolating from ram spermatozoa a water-soluble form of acrosin (a trypsin-like enzyme) which is about 25% pure. It is free from an acrosin inhibitor which is located in the spermatozoa. 2. In the hydrolysis of N-alpha-benzoyl-l-arginine ethyl ester the degree of activation of acrosin by Ca(2+), and by some other cations, is dependent on the extent of contamination by the inhibitor. In 50mm-Tris-HCl buffer (pH8.2) activation by Ca(2+) did not exceed 40%, but acrosin that is partially inhibited may be activated by up to 300%: this is due to cation-mediated protection of acrosin against the inhibitor. 3. Increasing concentrations of buffers (e.g. Tris) also activate acrosin but at above certain buffer concentrations Ca(2+) no longer exerts an activating effect and may become inhibitory. Ca(2+) is also inhibitory when added to assay systems involving anionic buffers with chelating properties. This is due to a fall in pH. 4. The above results suggest reasons for conflicting conclusions in papers dealing with the effects of Ca(2+) on acrosin activity. 5. Inhibition of acrosin by the Kunitz pancreatic trypsin inhibitor is increased on addition of Ca(2+). Inhibitions of trypsin by the acrosin inhibitor and by the Kunitz inhibitor are insensitive to Ca(2+). 6. Like trypsin, acrosin is activated, up to 60%, by 2-methyl-propan-2-ol, dimethyl sulphoxide, and some other water-miscible solvents. Effects of cations and solvents tend to be additive and a common maximum acrosin activity can be achieved with various concentrations of solvent, salts and buffer in the assay system. Activation by solvents is increased when low concentrations of the acrosin inhibitor are present. 7. Activations of acrosin by salts and by solvents are more pronounced when the substrate is N-alpha-benzoyl-dl-arginine 2-naphthylamide. 8. K(m) values for ram acrosin (about 0.2mm) are much higher than those for trypsin, and k(cat.) values are slightly higher than those for trypsin. Considerations of the influences of ions and dimethyl sulphoxide on the activities and kinetic constants of acrosin and trypsin suggest that conformational changes are the factors mainly responsible for the reported activations of acrosin. 9. The following conclusions are reached. (a) Acrosin plays a role in the penetration of the sperm cell into the egg without becoming detached from the acrosomal membrane. (b) The enzyme is a peripheral membrane protein which may be classed as a cathepsin. (c) The susceptibility of the activity of soluble acrosin to cations and solvents points to a flexible molecule, i.e. one lacking conformational restraints imposed by association (presumably ionic) with the acrosomal membrane.

Studies on ram acrosin. Fluorimetric titratiion of operational molarity with 4-methylumbelliferyl p-guanidinobenzoate.

1. Titration in sodium barbiturate buffer of acrosin, a serine proteinase from sperm acrosomes, with the ester substrate 4-methylumbelliferyl p-guanidinobenzoate gave rise to an incomplete 'burst' of 4-methylumbelliferone. Studies of the effects on the reaction of activators of acrosin (Ca2+, water-miscible solvents) showed that titrations carried out in barbiturate buffer containing 1M-CaCl2 and diluted with 0.2 vol. of dimethylsulphoxide produced a rapid quantitative burst within 4 min at 20 degrees C. 2. The net post-burst production of 4-methylumbelliferone was neglibible because (a) the acyl-enzyme was very stable, and (b) the slow post-burst formation of 4-methylumbelliferone (turnover of acyl-enzyme) was virtually equal to the slow photolytic destruction of 4-methylumbelliferone that was liberated during the burst. 3. The standard procedure permits titrations of 20-100pmol of acrosin, i.e. amounts normally taken for conventional rate assays, and with these amounts the impurities present in crude enzme fractions did not interfere. The burst was judged to be quantitative on the basis of comparisons with titrations of acrosin with p-nitrophenyl p'-quanidinobenzoate. 4. The burst reaction of trypsin with the 4-methylumbelliferyl ester was inhibited by high Ca2+ concentrations and by dimethyl sulphoxide. 5. The association and dissociation of complexes of both acrosin and trypsin with protein-type inhibitors (Kunitz pancreatic trypsin inhibitor and a spermatozoal acrosin inhibitor) are rather slow. It is thus possible, in certain cases, to use the ester to titrate both total enzyme in an inhibitor-enzyme mixture and net enzyme, i.e. the stoicheiometric excess of enzyme over inhibitor.

The two human trypsinogens. Inhibition spectra of the two human trypsins derived from their purified zymogens.

The two human anionic trypsinogens 1 and 2 were purified from human pancreatic juice by gel filtration on Sephadex G-100 and by chromatography on DEAE-cellulose. After activation of their respective zymogens by porcine enterokinase, human trypsins 1 and 2 were studied for their reaction with a wide variety of proteinase inhibitors. Kunitz pancreatic trypsin inhibitor and human pancreatic secretory trypsin inhibitor completely inhibited both human trypsins at a stoichiometric inhibitor-to-enzyme ratio of one to one. In contrast, bovine pancreatic secretory trypsin inhibitor (Kazal's inhibitor) failed to inhibit either human trypsin. The inhibition of both human trypsins by porcine pancreatic secretory trypsin inhibitor was demonstrated. The reactions of the trypsins with chicken ovomucoid, Ascaris lumbricoides (type suis), human sperm and blood plasma trypsin inhibitors were studied. The most striking difference between the two human trypsins was the reaction with soybean trypsin inhibitor (Kunitz). Trypsin 2 was completely inhibited in a one-to-one molar ratio while trypsin 1 was poorly inhibited. The presence of a prekallikrein in human pancreatic juice is discussed.

Effect of proteolytic enzyme inhibitors on steroidogenic action of corticotropin and its synthetic analogues.

The in vitro effect of Kunitz pancreatic trypsin inhibitor (KPTI) on the release of corticosterone from rabbit adrenals in the presence of three different preparations of corticotropins has been investigated. These preparations include highly purified porcine corticotropin, Pcorticotropin and Gly^a-*^ corticotropin. The effect of KPTI was found to be more marked in sustaining the action of corticotropin with shorter peptide chain than that with longer peptide chain. KPTI per se did not show any stimulatory effects on steroidogenesis. It may be concluded that KPTI protects shorter chain corticotropin against digestion by proteolytic enzymes. The above mentioned effect of KPTI was not demonstrated, however, with soybean trypsin inhibitor. (Endocrinology 90: 1652, 1972) T T HAS BEEN known that corticotropins are •*• inactivated by proteolytic enzymes. White and Gross (1) reported that plasmin inactivated corticotropins in blood. Other proteolytic enzymes, such as trypsin, pepsin and liver cathepsin have been also recognized as corticotropin inactivators (1). However, little is known as to whether corticotropins could be inactivated in the adrenal, during exerting their hormone action in adrenal. In the present report we employed proteolytic enzyme inhibitors to evaluate the possible influence of tissue proteolytic activities on the maintenance of steroidogenic action of corticotropins including synthetic analogues with shorter chain peptides. Our data suggest that the action of synthetic corticotropin analogue with 18 amino acids is markedly augmented by protecting against the degradation of the peptide. Materials and Methods A highly purified porcine corticotropin (HP corticotropin) with a potency of 120 U/mg was purchased from Calbiochem; synthetic P* corticotropin from Organon-Daiichi; Kunitz pancreatic trypsin inhibitor and soybean trypsin inhibitor from Worthington Biochemical Corp.; bovine serum albumin (Fr. V) from Armour Pharm. Co.; synthetic Gly-a-2 Received November 17, 1971, corticotropin was a gift from Dr. Otsuka of Shionogi Laboratory, Osaka. Adrenals were obtained from male rabbits weighing 2.0-2.5 kg. They were washed exhaustively by Krebs-Ringer bicarbonate buffer, pH 7.4 (KRB) and quadricepted after removal of thin fibrous capsules. Each adrenal preparation weighed 30-50 mg. Two pieces of quadricepted tissue from a pair of adrenal glands were placed in each incubation vessel containing 5 ml KRB with glucose (2 mg/ml) and bovine serum albumin (5 mg/ml) (G-KRB) in the presence or absence of trypsin inhibitors. The vessels were preincubated for 60 min in an atmosphere of 95% O2—5% CO2 at 37 C with shaking at 100 cycles/min. After preincubation, corticotropin preparations were added and incubation was continued for another 120 min under the same condition. The steroidogenic action was followed by measuring the amount of corticosterone released into the incubation medium. 0.5 ml aliquots of medium were removed at 0, 20, 40, 60 and 120 min for the assay of corticosterone which was performed by the method of Guillemin (2). Results Effect of KPTI on steroidogenic action of corticotropins The effect of three different corticotropin preparations on corticosterone production in rabbit adrenals were tested. As shown in Fig. 1A, HP corticotropin (0.04 y-g/ml), synthetic J3 NOTES AND COMMENTS 1653 FIG. 1. The effect of HP corticotropin, P' corticotropin and Gly-a-2 corticotropin on the corticosterone production in rabbit adrenals in the presence or absence of proteolytic enzyme inhibitors (KPTI or STI). Quadricepted adrenals were preincubated for 60 min with or without proteolytic enzyme inhibitors, each preparation of corticotropins was then added to incubation vessels as indicated with an arrow and incubation was continued for 120 min. Each value is the mean ± SE of four or five experiments. 12.0

Biologic action of proinsulin on ventral toad skin in vitro.

Insulin increases active sodium transport across isolated skin segments and urinary bladder of the toad, Bufo marinus, and this response can be diiectly and continuously monitored by measuring the short-circuit current. In this study proinsulin was found to stimulate sodium transport in toad skin preparations. Proinsulin and insulin were equipotent on a molar basis with concentrations from 5 × lO-10 to 10-6M, and the short-circuit response to either agent at maximally effective concentrations was similar with respect to time of onset, rate of development and time of peak effect. Preincubation with Kunitz pancreatic trypsin inhibitor resulted in inhibition of the effect of proinsulin, but did not affect the response to insulin. Although this finding lends support to the hypothesis that the biological activity of proinsulin is dependent upon its prior proteolytic conversion to insulin, it is difficult to reconcile this hypothesis with the equipotent activity and similar time-iesponse patterns of the 2 prote...

Degradation of porcine insulin and proinsulin by rat adipose tissue.

Degradation of 6.95 × 10−10 M I-125 porcine insulin and proinsulin to TCA soluble radioactivity was assessed in intact and homogenized rat fat pieces and isolated fat cells, with and without Kunitz pancreatic trypsin inhibitor (KPTI), 50μg./ml. Fat cells degraded both substances more actively than fat pieces. Homogenization increased degradation rate in fat pieces but had no effect in fat cells, suggesting the most active degrading sites were readily available to substrate in the fat cell preparation. They, therefore, could be either intimately associated with the cell membrane or, alternatively, intracellular with the fat cell of necessity permeable to insulin. Insulin and proinsulin competed for degradation, and KPTI inhibited only proinsulin degradation. These results are consistent with an initial trypsin-like cleavage of proinsulin with subsequent common degradation pathways with insulin.

Assay of insulin-like activity by the isolated fat cell method. IV. The biological activity of proinsulin.

The effect of bovine proinsulin and related factors on the glucose metabolism of isolated, rat, fat cells was studied. The dose-response curve of proinsulin was parallel to that of insulin and the action of the two proteins showed an identical time course. The activity of single chain proinsulin was about 0.5 U/mg (as estimated from its ability to increase the conversion of14C-glucose to14CO2 or to14C-glycerides by fat cells) and rose to about 17 U/mg after treatment with trypsin. — The activity of insulin was quantitatively recovered in the presence of proinsulin. Split chain proinsulin showed an activity of 5 U/mg, which rose to about 18 U/mg after treatment with trypsin. Connecting peptide did not influence the glucose metabolism in the absence or presence of insulin. — There was no conversion of proinsulin in the isolated cell incubation medium to insulin or a similar molecule with high biological activity. The activity was the same on rat epididymal fat pads as on isolated fat cells, and there was no significant suppression by Kunitz pancreatic trypsin inhibitor in either system. — The following was concluded: the biological activity of proinsulin on rat isolated fat cells and epididymal fat pads is about 2 per cent of that of insulin, and the effect is caused by the proinsulin molecule itself. The reason for the low biological activity is presumably a smaller affinity for insulin receptors.

Differential effect of porcine proinsulin on rat epididymal fat cells and fat pieces.

Proinsulin is a biosynthetic precursor of insulin which reacts with insulin antibody and has been found in the peripheral circulation. The present studies were undertaken to determine the extent of its intrinsic biologic activity as compared with insulin and to provide evidence regarding the possibility and anatomic location of its conversion to insulin or biologically active related products in peripheral tissues. The effects of single component porcine insulin and proinsulin on the conversion of glucose-l-C-14 to C-14-O2 and inhibition of the theophylline-stimulated lipolysis were studied in rat adipose tissue cells and pieces. Kunitz pancreatic trypsin inhibitor (KPTI) was used to block tissue conversion of proinsulin to insulin. Proinsulin was twentythree to twenty-seven times less effective than insulin when assayed on isolated fat cells. These results were not changed significantly by KPTI. Whereas insulin appeared less effective on fat pieces than on cells—presumably due to difficulty with diffusion into the more intact tissue—proinsulin, an even larger molecule, was as effective in fat pieces as fat cells. Thus proinsulin was only nine to eighteen times less effective than insulin when assayed on fat pieces. With KPTI, fat pieces became markedly insensitive but still responsive to proinsulin. These results suggest that the proinsulin molecule has biologic activity not necessitating conversion to insulin for expression. Intact tissues contain trypsin-like activity capable of converting proinsulin to insulin. This activity must be located between the vascular endothelium and before access to, but not including, the cell membrane.

Comparative study of the effects of porcine proinsulin and insulin on lipolysis and glucose oxidation in rat adipocytes.

Proinsulin blocked lipolysis activation by epinephrine, glucagon or theophylline. The antilipolytic effect of pro-insulin was not blocked by Kunitz pancreatic trypsin inhibitor (KPTI). The-concentrations of proinsulin and insulin required to cause 50 per cent inhibition of epinephrine-stimulated lipolysis were estimated to be approximately 1 × 10−9 M and 3 × 10−11 M respectively. Proinsulin which had been reduced and allowed to reoxidize was found to have approximately the same antilipolytic activity as native proinsulin, indicating that the antilipolytic effect was due to proinsulin and not insulin contamination. The concentrations of proinsulin and insulin required to give 50 per cent of maximal stimulation of glucose oxidation were estimated to be approximately 5 × 10−9 M and 1 × 10−11 M respectively. The effect of proinsulin on glucose oxidation did not appear to be caused by any insulin contamination since reduced-reoxidized proinsulin had approximately the same activity as native proinsulin. KPTI had no effect on proinsulin-stimulated glucose oxidation in isolated fat cells, in contrast to the marked inhibitory effect observed using intact epididymal fat pad. This suggests that the KPTI-sensitive proteolytic activity of the epididymal fat pad is not located in the fat cell but in some other type cell or extracellular space.

Studies on the biological activity of porcine proinsulin.

The biological activity of purified porcine proinsulin was investigated in rats. In vivo studies revealed that proinsulin produced a hypoglycemic response similar to insulin but of lesser magnitude. Hypophysectomized and adrenalectomized animals proved to be more sensitive to proinsulin than normal. In vitro studies with rat hemidiaphragm were consistent with the in vivo findings. No competition with insulin action could be demonstrated. Experiments were carried out to determine whether proinsulin is converted to intermediate forms or insulin as a requisite to its biological activity. Labeled proinsulin injected in vivo or incubated in vitro remained intact by a variety of techniques (Sephadex column chromatography and polyacrylamide-gel electrophoresis). An inhibitory action of Kunitz pancreatic trypsin inhibitor on proinsulin action in vitro was confirmed. No clarification of this effect could be ascertained.

Antilipolytic effect of porcine proinsulin.

Shaw and Chance have recently demonstrated that Kunitz pancreatic trypsin inhibitor (KPTI) blocks the increased conversion of labeled glucose to carbon dioxide and fatty acids in normal adipose tissue of rats in the presence of porcine proinsulin. The experiments reported here were designed to observe the antilipolytic effect of insulin and porcine proinsulin on lipolysis induced by epinephrine and glucagon in isolated fat cells and intact fat pads from rats, both in the presence and absence of KPTI and soybean trypsin inhibitor. It was found that both insulin and proinsulin are potent inhibitors of lipolysis induced by DL-arterenol and by glucagon. Neither KPTI nor soybean trypsin inhibitor blocked the antilipolytic effect of insulin or proinsulin when lipolysis was stimulated by DL-arterenol or by glucagon. The failure of KPTI to inhibit the antilipolytic effect of proinsulin on isolated fat cells may have been caused by inactivation of KPTI by collagenase used in preparation of the isolated fat cells. To test this hypothesis experiments were performed using intact fat pads. As with the isolated fat cells, KPTI did not inhibit the antilipolytic effect of insulin or proinsulin. These observations indicate that, in isolated fat cells and intact fat pads of the rat, proinsulin itself is an effective antilipolytic agent.

Effect of porcine proinsulin in vitro on adipose tissue and diaphragm of the normal rat.

The effect in vitro of porcine proinsulin and single component porcine insulin on the epididymal adipose tissue and the isolated diaphragm of the normal rat has been investigated. Both proteins enhance the oxidation of labeled glucose to carbon dioxide and its conversion to fatty acids by adipose tissue as well as glycogen formation from glucose by the hemidiaphragm. Kunitz pancreatic trypsin inhibitor (KPTI) but not the purified Kazal or soybean trypsin inhibitor block these effects of proinsulin on adipose tissue and diaphragm. KPTI has no effect on the response of these tissues to the single component insulin. Anti-insulin serum blocks the response of both adipose tissue and hemidiaphragm to proinsulin and single component insulin. The enhancement of the oxidation of carbon one of glucose to carbon dioxide by adipose tissue in the presence of proinsulin is also blocked by KPTI. It is concluded that porcine proinsulin itself cannot affect the glucose metabolism of adipose tissue and hemidiaphragm of the normal rat. Proinsulin exerts the biologic effect seen in this study because it is converted by an enzyme or group of enzymes present in adipose tissue and diaphragm muscle, to a molecule which is similar to or identical with insulin.