Crude Pancreatic Extract Research Articles

Investigation of lipase-ligand interactions in porcine pancreatic extracts by microscale thermophoresis.

The evaluation of binding affinities between large biomolecules and small ligands is challenging and requires highly sensitive techniques. Microscale thermophoresis (MST) is an emerging biophysical technique used to overcome this limitation. This work describes the first MST binding method to evaluate binding affinities of small ligands to lipases from crude porcine pancreatic extracts. The conditions of the MST assay were thoroughly optimized to successfully evaluate the dissociation constant (Kd) between pancreatic lipases (PL) and triterpenoid compounds purified from oakwood. More precisely, the fluorescent labeling of PL (PL*) using RED-NHS dye was achieved via a buffer exchange procedure. The MST buffer was composed of 20mM NaH2PO4 + 77mM NaCl (pH6.6) with 0.05% Triton-X added to efficiently prevent protein aggregation and adsorption, even when using only standard, uncoated MST capillaries. Storage at -20°C ensured stability of PL* and its fluorescent signal. MST results showed that crude pancreatic extracts were suitable as a source of PL for the evaluation of binding affinities of small ligands. Quercotriterpenoside-I (QTT-I) demonstrated high PL* binding affinity (31nM) followed by 3-O-galloylbarrinic acid (3-GBA) (500nM) and bartogenic acid (BA) (1327nM). To enrich the 50 kDa lipase responsible for the majority of hydrolysis activity in the crude pancreatic extracts, ammonium sulfate precipitation was attempted and its efficiency confirmed using capillary electrophoresis (CE)-based activity assays and HRMS. Moreover, to accurately explain enzyme modulation mechanism, it is imperative to complement binding assays with catalytic activity ones.

Use of insulin in diabetes: a century of treatment.

Insulin is a key player in the control of hyperglycaemia for patients with type 1 diabetes mellitus and selected patients with type 2 diabetes mellitus. There have been many advances in insulin drug delivery from its first administration as a crude pancreatic extract till today. The traditional and most predictable method for administration of insulin is by subcutaneous injection. Currently available insulin delivery systems include insulin syringes, infusion pumps, jet injectors, and pens. The major drawback of insulin therapy is its invasive nature. Non-invasive delivery of insulin has long been a major goal for the treatment of diabetes mellitus. Although there have been improvements in insulin therapy since it was first conceived, it is still far from mimicking the physiological secretion of pancreatic β-cells, and research to find new insulin formulations and new routes of administration continues. This article reviews the emerging technologies, including insulin inhalers, insulin buccal spray, insulin pill, islet cell transplant, and stem cell therapy, as treatment options for diabetes mellitus.

PRE-CLINICAL MODELS OF SEPSIS

Just over 75 years ago, a small town Canadian surgeon, Dr. Frederick Banting, and a graduate student in physiology, Charles Best, administered a crude pancreatic extract to a pancreatectomized dog. The animal became normoglycemic and appeared healthy, providing the two scientists with the ammunition necessary to administer their diabetes-reversing extract to a 14 year old boy, Leonard Hutchinson. He too improved, and within two years of initiating their studies, Banting and Best were Nobel laureates for their discovery of insulin, a discovery that has fundamentally transformed the care of diabetic patients. At the turn of the millennium, the situation for researchers seeking to prevent or reverse the terrible toll of human sepsis is much more sombre. Literally thousands of animal studies have been published, reporting dozens of differing strategies in scores of different models, and many differing animal species. The sheer volume of these has increased over time, and especially over the past ~ve years (Figure 1). Most of these have shown that manipulation of one or more components of the endogenous in_ammatory cascade can alter survival or improve vital function in one or more organ systems. Yet none of these has been able to provide the unequivocal evidence of bene~t in human studies that would lead to licensure, and the release of new antiin_ammatory therapies into clinical practise. Much has been written on the possible reasons for the disappointing results of clinical trials of novels mediator-targeted therapies [1–3]; less attention has been paid to the preclinical studies that lay the foundation for these trials. In November 1997, a group of researchers met in Toronto Canada for a roundtable symposium organized to address the challenges of pre-clinical models of sepsis (for a list of participants, see Appendix A). Papers from that meeting comprise this issue of Sepsis.

Polymerized liposome as ligand carrier for affinity precipitation of proteins

A polymerized liposome (PLS) was prepared using a synthesized phospholipid with a diacetylene moiety in the hydrophobic chain and an amino group in the hydrophilic head. The PLS was used as a novel ligand carrier for affinity precipitation of proteins because it showed a reversibly precipitable property on salt addition and removal. Soybean trypsin inhibitor (STI) was easily immobilized on the PLS by a one-step carbodiimide reaction. The PLS showed no nonspecific adsoprtion of proteins. It had a large ligand coupling capacity, and then a large adsorption capacity for trypsin after STI immobilization. The PLS with immpbilized STI was recycled three times for the purification of trypsin from a crude pancreatic extract. Although the degree of purification was compromised by the impurity of the STI employed, in each run the purification factor reached about 6 and more than 80% of trypsin activity was recovered. The results indicated that the PLS was a potential ligand carrier for affinity precipitation of proteins. (c) 1995 John Wiley & Sons, Inc.

Isolation and Structural Characterisation of Herring Gull (Larus argentatus) Pancreatic Polypeptide

Pancreatic polypeptide (PP) has been isolated from pancreatic extracts of the herring gull (Larus argentatus) using a radioimmunoassay employing an antiserum (PP 221), generated to the conserved C-terminal hexapeptide amide of mammalian PP. Gel permeation and reverse-phase HPLC fractionation of crude pancreatic extracts resolved a single molecular form of gull PP in each case. Purification of gull PP to homogeneity indicated that the mammalian PP antiserum employed was only 4% cross-reactive with this peptide. When radioimmunoassay data were corrected for this, the herring gull pancreas was found to contain 5 nmol PP/g wet wt. The molecular mass of gull PP was determined as 4237 ± 2 Da by 252Cf plasma desorption mass spectroscopy (PDMS). Gas-phase sequencing established unequivocally the entire primary structure of a 36-amino-acid residue peptide as Gly-Pro-Val-Gln-Pro-Thr-Tyr-Pro-Gly-Asp-Asp-Ala-Pro-Val-Glu-Asp-Leu-Val-Arg-Phe-Tyr-Asn-Asp-Leu-Gln-Gln-Tyr-Leu-Asn-Val-Val-Thr-Arg-His-Arg-Tyr. The computed molecular mass of this peptide (4235.6 Da) was in close agreement with that derived by PDMS. The primary structure of herring gull PP differs from that of the chicken in four residues at positions 3 (Val/Ser), 18 (Val/IIe), 22 (Asn/Asp), and 23 (Asp/Asn). Avian PP thus appears to be a highly conserved regulatory peptide.

Design of novel cationic ligands for the purification of trypsin-like proteases by affinity chromatography.

A number of new cationic ligands have been designed and synthesized for the selective resolution and purification of the trypszin-like proteases. A series of ligands based on 4-[2'-methyl-4'-(2'',4''-dichloro-1'',3'',5''-triazin-6-ylamino ) phenylazo]benzamidine were able to bind to trypsin and the trypsin-like proteases, thrombin and urokinase, but bound pancreatic kallikrein only weakly. Ligands possessing a second cationic group (either 4-aminophenyltrimethylammonium or 4-aminobenzamidine) substituted onto the triazine ring displayed higher affinities than the parent compound for trypsin in solution but bound the enzyme weakly or not at all after immobilization. In contrast, these bis-cationic ligands bound pancreatic kallikrein in solution and following immobilization. The presence of the second cationic group was crucial, since its replacement by neutral or anionic groups led to loss of affinity for pancreatic kallikrein. One of the bis-cationic ligands was used to purify pancreatic kallikrein 9.5-fold from a crude pancreatic extract in 79% yield, to generate a product 99.9% free of contaminating trypsin activity.

Design of novel affinity adsorbents for the purification of trypsin-like proteases.

A number of ligands for the selective purification by affinity chromatography of the trypsin-like protease, porcine pancreatic kallikrein, were designed de novo by computer-aided molecular design. The ligands were designed to mimic the side-chains of a number of arginyl dipeptides and included a benzamidine moiety substituted on a triazine ring. The ligands displayed inhibitory activities against pancreatic kallikrein which mirrored the specificity constants of the dipeptides they were designed to mimic. The ligand with the highest affinity for the enzyme, an analogue of a Phe-Arg dipeptide, when immobilized to Sepharose CL-4B via a hexamethylene spacer arm, purified pancreatic kallikrein 110-fold in one step from a crude pancreatic acetone extract.

Esterification in non-aqueous media: Activity and selectivity of porcine pancreas carboxylesterase depending on the structure of the alcoholic substrate

Enzymatic esterification in n-hexane was carried out using various unsaturated and aromatic (R,S)-alcohols, dodecanoic acid and a crude pancreatic extract. A “model” was developed describing reactivity and selectivity of the enzymatic catalysis. These parameters were found to depend on the structural features of the side chains at the reactive chiral center. In several homologous series of substrates reversal of configuration of the preferred enantiomer was observed. The evaluated “model” will be a helpful tool to establish future synthetic strategies.

Effect of insulin, proinsulin and pancreatic extract on myelination and remyelination in organotypic nerve tissue in culture

The effect of insulin, proinsulin and crude pancreatic extract was studied in organotypic nerve tissue cultures, principally in relation to the development of myelin. Cultures were exposed to media supplemented with these substances beginning on the first day of explanation. By 4 days in vitro, there was a good neuritic outgrowth from all the fragments. That from the insulin and pancreatic extract-fed were more profuse and extended further than from the control group. By 8–12 days in vitro it was also possible to observe more myelinated axons in these treated groups. The pattern of changes in the myelin associated enzyme activity, 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) paralleled the differential increase in myelination. Insulin-fed cultures showed a more rapid increase in CNPase activity, which, after 21 days in vitro reached a plateau about 30–50% over that of the controls. Cultures treated with pancreatic extract showed a similar pattern of increased activity, while in proinsulin-treated explants the activity was only significantly higher after 21 days in vitro. To study the effect of these substances on remyelination, well myelinated cultures were completely demyelinated by exposure to anti-white matter antiserum and were subsequently exposed to the same normal control or supplemented media. The amount of myelin and concomitantly the CNPase activity increased rapidly and in the same proportion between the various groups as was observed previously during primary myelination. Insulin as well as crude pancreatic extract and, to some extent, proinsulin demonstrated a marked effect on the time of onset and principally on the total amount of myelin developed by treated cultures as compared to those maintained in normal nutrient medium.

Membrane Biochemistry By Edith Sim, and: Dynamics of Biological Membranes: Influence on Synthesis, Structure and Function By Miles D. Houslan and Keith K. Stanley (review)

velopments of the technique for improving the concentration of the insulin extract was invaluable but was probably regarded by the Nobel Committee as a technological refinement. Perhaps this was an unjust decision. Macleod was die guiding professor. Although the original idea was not his, he contributed important guidance and was, in fact, die only scholar in the entire group who knew a great deal about carbohydrate metabolism. The many details of the intense work and the working relations of the foursome—Banting, Best, Collip and Macleod—during 1921 and 1922 uncovered the fact that, in terms of certain personal qualities, our heroes did indeed have clay feet. Apparently the rivalries for credit became bitter and, in retrospect, since there was enough honor to be passed around, inexcusable. Banting was jealous of his own great ideas and accomplishment and wanted credit for this. His driving zeal apparently annoyed some ofthe others. After all, he was not a sophisticated scientist. Perhaps Bliss decided that the description of the quibbling and arguing of the principal participants made diem real people; to this reviewer, much of this was uninteresting. One thing was clear and admirable . There was no rivalry for financial gain; none was sought, and diere was none except, appropriately, for the University ofToronto. The few stories ofthe effects of insulin in patients starving to deadi to live longer on Allen's starvation diet are poignant and require no literary embellishment. Excerpts from die very articulate letters of Elizabeth Hughes, a 15-year-old daughter of Charles Evans Hughes, are remarkable. It does appear that Bliss as a nonphysician was unaware of the great courage required of a physician in making die decision to administer the crude pancreatic extract to patients for the first time. This is certainly an interesting aspect of the story in the light of today's necessity for protocol review by peers and outsiders before proceeding with clinical investigative programs. The Dwovery of Insulin deserves wide readership. The excitement of one of medical science's greatest advances could not be suppressed by this radier pedantic presentation. For physicians and interested laymen and perhaps particularly young people considering a career in biology and medicine, Bliss has provided an important historical document. Richard L. Landau Department of Medicine University of Chicago Membrane Btochermstry. By Edith Sim. London: Chapman & Hall, 1982. Pp. 80. $6.50 (paper). Dynamics ofBiological Membranes: Influence on Synthesis, Structure and Function. By Miles D. Houslan and Keith K. Stanley. Chichester: John Wiley & Sons, 1982. Pp. 330. $54.95. The researcher who wishes to keep up with the rapid developments in the field ofbiological membranes has available to him many original articles and scholarly reviews. However, these are generally narrowly focused and require that the Perspectives in Biology and Medicine, 27, 2 ¦ Winter 1984 | 317 reader have an in-depth knowledge ofdie specialized areas under discussion. As a consequence, such accounts are of lesser value to diose beginning dieir investigative career or to students who will find textbooks too scanty in details and up-to-date information. The filling ofthis information gap is not easily achieved and, in fact, represents a challenge. In this context, two recent books, one by E. Sim (Department of Biochemistry, Oxford University) on membrane biochemistry and the other by M. D. Houslan (Department of Biochemistry, University of Manchester) and K. K. Stanley (European Molecular Biology Laboratory , Heidelberg), with emphasis on membrane dynamics, attempt to meet this challenge each in its own way. Sim's account, which is in the form of a guidebook, attempts to put together conceptual and practical information on membrane biochemistry. Remarkably concise and informative, this monograph is aimed at those desiring to gain a general overview ofdie diverse structural expressions and functions ofbiological membranes. One soon finds that the early concepts favoring the universality of membrane structure (i.e., lipid bilayer and fluid mosaic models) have now been challenged by new developments pointing to the occurrence of numerous variations on a unifying structural theme. These variations are likely to be a reflection of the multiple functions which biomembranes possess. The basic information is presented with objectivity and clarity, although it is perhaps too condensed in some areas. This potential deficiency is offset...

Pancreatic content of insulins I and II in laboratory rodents. Analysis by immunoelectrophoresis.

The purpose of these experiments was to determine the relative proportion of insulins I and II in the pancreas of inbred strains of mice and rats, and whether this proportion would be altered by conditions known to affect insulin biosynthesis. Rapid analysis of multiple samples was accomplished by an immunoelectrophoretic method in which insulin can be detected in crude pancreatic extracts without further purification. Insulin I was found to account for 65–76% of total pancreatic content in C3H/HeJ, C57BL/6J, and CD2F1 mice. Glucose injection produced a significant increase in insulin content, which was completely accounted for by insulin I, although fasting for 2 days had no effect. In all three strains there was either no change or a decrease in content of insulin II, while insulin I increased 77–83% (P < 0.05) of the total pancreatic insulin. To determine the effects of chronic hyperglycemia, the content of insulins I and II was measured in the pancreas of obese hyperglycemic mice and compared with that of lean littermates. Insulin content in pregnant rats was also evaluated, since pregnancy is characterized by insulin resistance and hyperinsulinemia. The results of these studies indicate that insulin I predominates in the pancreas of a variety of strains of rats and mice. More important is the observation that conditions that increase insulin production preferentially increase insulin I relative to II, suggesting independent regulation of the two genes.

Human ribonucleases. Quantitation of pancreatic-like enzymes in serum, urine, and organ preparations.

Antibodies against pure human pancreatic ribonuclease (RNase) were used to study ribonuclease levels in human tissues and body fluids. The antibodies completely inhibit the activity of purified RNase as well as ribonuclease activity in crude pancreatic extracts. RNase activity is inhibited by 70-80% in serum and urine, indicating that a significant proportion of the RNases in these preparations are structurally like the pancreatic enzyme. In contrast, inhibition of RNase activities from spleen (8%) and liver (30%) was inefficient suggesting that most of the RNases in these tissues are structurally unlike the pancreatic enzyme. A competitive binding radioimmunoassay (RIA), sensitive in the range of 1-100 ng of RNase, was developed to quantitate the pancreatic like enzymes. The RIA of crude tissue preparations and samples fractionated by gel filtration was compatible with inhibition results. Enzymes structurally like pancreatic RNase could be quantitated despite the presence of other RNase activities. Immunological quantitation of pancreatic like RNases was also found to be much more simple and precise than enzymatic assays comparing RNA and polycytidylate substrates. We suggest the immunological assays will be useful in the quantitation and definition of tissue of origin of RNases in serum of patients with pancreatic carcinoma.

Polycytidylate hydrolysing ribonuclease variants of human pancreas

Human pancreatic ribonuclease (RNase) comprises three variants with isoelectric points at pH 4.8, pH 9.3 and pH 9.7. These variants are not artifacts of the purification procedure, since they can be demonstrated in crude pancreatic extracts obtained with mild procedures (4). Although these RNase variants differ in their isoelectric points, they are similar in all the other properties so far studied. With polycytidylic acid as a substrate, their activities are optimal in 0.05M sodium phosphate buffer at pH 6.5. Of the several buffers tested, sodium phosphate buffer yielded maximal enzyme activity, but the concentration of phosphate is highly critical. Borate concentrations of 0.05M and 0.1 M respectively brought about 25% and 50% reductions in enzyme activity. Presence of phosphate in the reaction mixture obviates to a great extent the borate inhibition. These RNase variants are highly specific for the secondary phosphate esters of cytidine 3'-phosphate. They have no measurable activity on polyadenylic and polyguanylic acids and their activity on polyuridylic acid is less than 2% of that on polycytidylic acid. These RNase variants could serve as distinctive biochemical markers for the pancreas.

The kinetic properties of beef pancreatic l-Asparagine synthetase

1. 1. Beef pancreatic l-asparagine synthetase was resolved into its α-(113,000) and β-(57,000) forms. 2. 2. Kinetic properties of a crude pancreatic extract and the enzyme resolved into its components are described. 3. 3. K m values for aspartate, ATP and glutamine for the α-form are 1.1, 1.0 and 0.4 mM and for the β-forms are 0.6, 25.0 and 3.1 mM, respectively. 4. 4. These results are discussed in terms of the regulation of tumor and normal cell asparagine biosynthesis.

Affinity chromtography of proteases on hydroxyalkyl methacrylate gels with covalently attached inhibitors

The efficient isolation of trypsin and chymotrypsin from a crude pancreatic extract was achieved by affinity chromatography on specif adsorbents prepared by coupling of both naturally occuring protease inhibitors and also synthetic low-molecular-weight protease inhibitors to hydroxyalkyl methacrylate gels. Specific sorbents prepared with synthetic inhibitors are stable and are suitable for the isolation of chymotrypsin and trypsin even on a large scale.

Hypersensitivity to pancreatic extracts in parents of patients with cystic fibrosis

Because immediate hypersensitivity reactions can occur in individuals exposed to powdered pancreatic extracts, 36 patients with cystic fibrosis and 51 parents of such patients were studied for evidence of sensitization. Sensitivity to the extracts as evidenced by history and skin testing was infrequent in the children with cystic fibrosis. However, skin testing for immediate hypersensitivity with either crude pancreatic extracts or inactivated trypsin correlated well in their parents with a history of clinical symptoms. IgE mediation of these reactions in sensitized individuals was demonstrated by antigen-induced histamine release from leukocytes, passive transfer studies, and immediate response to inhalation challenge.

Affinity chromatography on hydroxyalkyl methacrylate gels III. Adsorption of chymotrypsin to poly(hydroxyalkyl methacrylates) with covalently bound benzyloxycarbonyl-glycyl- d-phenylalanine and - d-leucine as function of pH and ionic strength

Chymotrypsin is specifically adsorbed at low ionic strength and alkaline pH to hydroxyalkyl methacrylate gels with N- benzyloxycarbonylglycyl- d-phenylalanine or N- benzyloxycarbonylglycyl- d-leucine attached through 1,6-hexanediamine. Chymotrypsin is not adsorbed either to the unmodified gel (Spheron) or to the gel with attached 1,6-hexanediamine (NH 2-Spheron). The adsorption of chymotrypsin to Z-Gly- d-Phe-NH 2-Spheron was investigated as a function of pH and ionic strength. Trypsin is not adsorbed to this gel. Chymotrypsin isolated from a crude pancreatic extract by affinity chromatography on Z-Gly- d-Phe-NH 2-Spheron had the same activity as the enzyme isolated on a column of Spheron, to which the naturally-occurring trypsin inhibitor had been coupled.

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.

Elastolytic enzymes and elastin in the aging rat. I. Pancreatic elastolytic enzymes.

Since improved methods make it possible to determine both elastoproteinase and elastomucase activity in crude pancreatic extracts, the effect of age on enzyme titer in the pancreas was studied in virgin Wistar rats of both sexes. In males, the elastoproteinase concentration (in elastoproteinase unit [EU] per gram acetone-dried pancreas) decreased with age over the whole period of 0–24 months; the elastomucase Em-I and Em-S concentrations (in elastomucase unit [Em-U] per gram acetone-dried pancreas) rapidly decreased with age up to 12 months (Em-S) and 18 months (Em-I), remaining nearly constant after that age period. In female rats, the elastoproteinase concentration remained constant during the 1st year of life, increased rapidly between 15 and 18 months, then decreasing to the 15-month level at an age of 27 months. The titer of both elastomucases fell during the growth of the females, remaining constant between 6 and 15 months of age and showed a rapid increase in enzyme level between 15 and 18 months. Although there was a tendency for the enzyme concentration to decrease after 18 months, the fall was not statistically significant. The data obtained with rat pancreas were compared with those for human pancreatic enzymes and for human serum ‘elastoproteinase’ reported previously. Similar age-relationships were found.

Nonsuppressible insulin-like activity: Immunochemical and physicochemical evidence against its pancreatic origin

Rat serum contains several substances that exhibit insulin-like activity when biologically assayed. One of these is insulin; the others, collectively referred to as antibody nonsuppressible insulin-like activity (NSILA), represent larger proteins of uncertain origin. To assess the possibility that pancreatic islets secrete these nonsuppressible insulin-like components of serum, rat pancreatic islets were isolated and incubated in glucose containing buffer; the resulting insulin-like activity released into the incubation medium was measured by the rat fat pad bioassay following immunochemical and physicochemical characterization. Guinea pig antiinsulin serum suppressed the insulin-like activity of the incubation medium. Dowex 50 chromatography, at pH 7.4, of islet incubation medium, pancreatic homogenates, and crude pancreatic extracts revealed no cationic ILA, only anionic ILA. Gel filtration (G-75) of the incubation medium resulted in the isolation of suppressible ILA in the same distribution volume as purified rat insulin. No larger molecular weight substance possessing NSILA was isolated. Based upon these physicochemical findings, it is concluded that the pancreas is not the source of circulating NSILA in the rat.