Shifts in metabolic parameters surrounding glucose homoeostasis resulting from tricyclic antidepressant therapy: implications of insulin resistance?

This study displayed the physiological effects the tricyclic antidepressants amitriptyline or trimipramine have on glucose homoeostasis in male Wistar rats. An insulin secreting cell line (INS-1) was also used to determine effects tricyclic antidepressants have on insulin secretion and insulin displacement. Thirty rats each received a 1 mg kg-1 dose of amitriptyline or trimipramine for a period of 14 weeks; another 14 rats served as the control group. Blood glucose, serum insulin and muscle and liver glycogen levels were determined. Kidney, liver and muscle insulin degradation was measured and compared with insulin degrading enzyme concentrations in the latter two tissues. INS-1 cells were used to determine the effect 1 microM amitriptyline has on insulin secretion. Displacement studies for [3H]glibenclamide by amitriptyline or trimipramine were undertaken on INS-1 cells. A significant increase in blood glucose (P<0.01) was found for both test groups after 6 and 14 weeks of receiving the medication, which may be related to a significant decrease in liver and muscle glycogen levels (P<0.001). Serum insulin levels remained unchanged, although a significant increase in insulin degradation was observed in the muscle, liver and kidney, which may be related to a significant increase in insulin degrading enzyme (P<0.001) that was found. A significant increase in insulin secretion was observed for the INS-1 cells treated with amitriptyline, although no significant displacement for the [3H]glibenclamide was evident for amitriptyline or trimipramine. The significant alterations in glucose homoeostasis observed, as well as the significant changes associated with insulin secretion and degradation associated with amitriptyline or trimipramine treatment, imply that prolonged use of these medicines may lead to insulin resistance and full blown diabetes.

Biochemical properties of porcine white adipose tissue mitochondria and relevance to fatty acid oxidation.

The capacity of white adipose tissue mitochondria to support a high beta-oxidative flux was investigated by comparison to liver mitochondria. Based on marker enzyme activities and electron microscopy, the relative purity of the isolated mitochondria was similar thus allowing a direct comparison on a protein basis. The results confirm the comparable capacity of adipose tissue and liver mitochondria for palmitoyl-carnitine oxidation. Relative to liver, both citrate synthase and alpha-ketoglutarate dehydrogenase were increased 7.87- and 10.38-fold, respectively. In contrast, adipose tissue NAD-isocitrate dehydrogenase was decreased (2.85-fold). Such modifications in the citric acid cycle are expected to severely restrict citrate oxidation in porcine adipose tissue. Except for cytochrome c oxidase, activities of the enzyme complexes comprising the electron transport chain were not significantly different. The decrease in adipose cytochrome c oxidase activity could partly be attributed to a decreased inner membrane as suggested by lipid and enzyme analysis. In addition, Western blotting indicated that adipose and liver mitochondria possess similar quantities of cytochrome c oxidase protein. Taken together these results indicate that not only is the white adipose tissue protoplasm relatively rich in mitochondria, but that these mitochondria contain comparable enzymatic machinery to support a relatively high beta-oxidative rate.

Purification and partial characterisation of alpha(2)-antiplasmin and plasmin(ogen) from ostrich plasma.

This study reports the isolation and partial characterisation of the ostrich serpin, alpha(2)AP, and its target enzyme, ostrich plasmin, in its active and inactive proenzyme, namely plasminogen, forms. Ostrich alpha(2)AP was purified using L-lysine-Sepharose chromatography, ammonium sulfate fractionation, and Super Q-650S and ostrich LBSI-Sepharose chromatographies. It revealed a M(r) of 84 K (thousand) and had one and two N-terminal amino acids in common with 11 of those of human and bovine alpha(2)AP, respectively. It showed the largest inhibitory effect on ostrich plasmin, followed by bovine trypsin and plasmin, respectively, and much less plasmin inhibition than bovine aprotinin, but much more so than human alpha(2)AP, DFP and EACA. Ostrich plasminogen was highly purified after L-lysine-Sepharose chromatography and showed a M(r) of 92 K, a total of 775 amino acids and its N-terminal sequence showed approximately 53% identity with those of human, rabbit, cat, and ox plasminogens. Ostrich plasmin, obtained by the urokinase-activation of ostrich plasminogen, revealed a M(r) of 78 K, a total of 638 amino acids, an N-terminal sequence showing two to four residues identical to five of those of human, cat, dog, rabbit, and ox plasmins, and pH and temperature optima of 8.0 and 40 degrees C, respectively.

Properties of porcine white adipose tissue and liver mitochondrial subpopulations.

Properties of porcine white adipose tissue heavy and light mitochondrial subpopulations were investigated so as to identify any functional heterogeneity. Liver mitochondrial subpopulations were concurrently evaluated since their properties have been studied in some detail. Mitochondrial subpopulations were isolated by means of differential centrifugation and the relative purity estimated using marker enzymes. Due to the greater contamination of the light mitochondrial fractions, mtDNA content, determined by PCR analysis, was used as a basis to demonstrate any mitochondrial heterogeneity. Enzymatic activity, electron microscopy, lipid analysis and Western blotting were used to characterise the different populations. With the exception of liver cytochrome c oxidase, the enzymatic capacity of adipose and liver heavy mitochondria ranged between approximately two- and threefold higher than the corresponding light fraction. The cardiolipin content and mean mitochondrial diameters paralleled these differences, suggesting an increased mitochondrial mass rather than a functional difference. However, the cytochrome c oxidase activity of the liver heavy mitochondria was 4.75-fold higher relative to the light fraction. A strong correlation between cytochrome c oxidase activity and the subunit I content was evident. Adipose tissue mitochondrial subpopulations would seem to possess a comparable oxidative capacity per gram mitochondrial protein, while liver heavy mitochondria possess an increased oxidative capacity and mass.

Purification and characterization of ostrich prothrombin.

The work focused on the penultimate enzyme, prothrombin, in the coagulation cascade. Prothrombin was purified and characterized from ostrich plasma. The results obtained contribute to a better understanding of blood coagulation in the ostrich and the evolution of prothrombin and the coagulation cascade. Prothrombin was purified from ostrich plasma by barium chloride precipitation, ammonium sulfate fractionation, and DEAE-cellulose and Cu(2+)-chelate Sepharose chromatography. Ostrich prothrombin exhibited a M(r) of 72,800 and a pI of 6.9 using SDS-PAGE and PAG-isoelectrofocusing, respectively. The N-terminal sequence of ostrich prothrombin showed 78 and 87% identity with human and bovine, respectively. The cDNA was isolated from ostrich liver and the predicted amino acid sequence compared with those from other species. Ostrich prothrombin shares sequence identity with chicken (84%), human (60%), bovine (59%), rat (60%), mouse (59%) and hagfish (50%) prothrombin, suggesting a common function of prothrombin in these vertebrates. Amino acid sequence identities indicate that the thrombin beta-chain (62%) and the propeptide-Gla (75%) domains are the regions most constrained for the common functions of vertebrate prothrombins. Ostrich prothrombin, therefore, shows similarity in structure to other vertebrate prothrombins.

Comparative blood coagulation studies in the ostrich.

Blood coagulation of the ostrich was compared to that of mammalian (man and sheep), avian (chicken) and reptilian (puff adder) systems. The international normalised ratio (INR), partial thromboplastin time (PTT), thrombin time and fibrin degradation were determined, as well as the various coagulation factors in venous ostrich plasma, using human physiological substrates. Thromboplastin was isolated from fresh brain tissue with the exception of the reptile for which lung tissue was used. The levels of markers of the coagulation [antithrombin III (AT), factor X (FX) and prothrombin], the fibrinolytic (alpha2-antiplasmin) and the kallikrein system were determined using chromogenic substrates. Elevated values for INR, PTT and thrombin time were obtained as compared to known human standards. It was found that factors VII, IX, X, XI and XII were absent from ostrich plasma. A study of the homologous and heterologous thromboplastin activities indicated that ostrich plasma exhibited a lower thromboplastic activity when compared to human standards, but was comparable to avian and reptilian values. Ostrich plasma revealed 42.2% FX, 72.9% AT, 35.3% prothrombin, 115.6% alpha2-antiplasmin and 19.8% plasma kallikrein, relative to human plasma. All the results suggest that the ostrich coagulatory system has not evolved to include all the complex myriad of reactions found in the human system.

Interaction of plasma lipoprotein subfractions with differentiating 3T3-L1 and human mammary preadipocytes in culture.

Differentiating 3T3-L1 preadipocytes (murine fatty fibroblasts) and human preadipocytes interact with human lipoprotein subfractions (HDL2 and LDLII/III) at all stages of the differentiation program, displaying saturable binding behavior. Both cell types interact similarly with LDLII/III as differentiation proceeds, showing increased binding affinities and capacities and maximal rates of uptake in the mature cells, as compared with the preadipocyte stage. These changes coincide with the intracellular appearance of lipid droplets. However, with regard to HDL2, a markedly different pattern of interaction is evident in both cell types. For 3T3-L1 cells, lowered binding and uptake affinities and capacities are apparent in the fully differentiated state for HDL2, as compared with LDLII/III. Human preadipocytes displayed two distinct affinity binding sites for HDL2 during the early stages of differentiation (days 2 and 3), as compared with a single affinity site for LDLII/III at all stages. However, in the fully differentiated human cells, only a single affinity site, indistinguishable from the high-affinity site present on day 2, is evident, and probably represents the only binding site of physiological significance in these cells. All the cellular developments appear to be largely unaffected by exposure of both preadipocyte types to added lipoproteins (HDL + LDL) in the medium during the early stages of the conversion process.

The isolation and partial characterization of precursor forms of ostrich carboxypeptidase.

Ostrich carboxypeptidases A and B were recently purified and characterized. The aim of this study was to isolate and purify, and partially characterize in terms of molecular weight, pI, amino acid composition and N-terminal sequencing, the precursor forms of carboxypeptidases from the ostrich pancreas. Inhibition studies with soybean trypsin inhibitor and activation studies with three proteases (bovine trypsin, bovine chymotrypsin and porcine elastase) were performed on crude ostrich acetone powder and the carboxypeptidase A and B activities were determined. SDS-PAGE was carried out after every incubation to monitor the rate and degree of conversion of a M(r) 66K component to procarboxypeptidase and carboxypeptidase A and B. The precursor forms were purified by Toyopearl Super Q and Pharmacia Mono Q chromatography. All three proteases converted the M(r) 66K component to procarboxypeptidases and carboxypeptidases over a set time interval, with carboxypeptidase A and B activities being detected in the acetone powder. Chymotrypsin was the preferred protease since it exhibited a more controlled activation of the procarboxypeptidases. The amino acid composition of procarboxypeptidase A revealed 525 residues. The N-terminal sequence of procarboxypeptidase A showed considerable homology when compared with several other mammalian sequences. M(r) and pI values of 52K and 5.23 were obtained for procarboxypeptidase A, respectively. This study indicated that ostrich procarboxypeptidase A is closely related to other mammalian procarboxypeptidase A molecules in terms of physicochemical properties.

The influence of plasma lipoprotein subfractions on 3T3-L1 and human preadipocyte differentiation in cell culture.

3T3-L1 and human preadipocyte differentiation was significantly (P < 0.001) enhanced by HDL2, LDLII/III and LDLIV. The concentrations of lipoproteins required for maximal differentiation in human preadipocytes were not achieved over the concentration range 50-150 micrograms lipoprotein protein ml-1, whereas maximal differentiation in 3T3-L1 preadipocytes was achieved for all lipoprotein subfractions at approximately 75 micrograms lipoprotein ml-1, a level almost double that required for complete HDL and LDL fractions in 3T3-L1 cells. Despite the enhanced extent of differentiation caused by certain lipoprotein subfractions, the time needed for the conversion process was unaffected. GPDH activity development in both cell types was most pronounced in response to LDLIV, with HDL2 resulting in the lowest activity. In both cell types, the enhancement of differentiation was only evident when the cells were exposed to lipoproteins during the early stage of the program, i.e. before visible formation of lipid droplets.

Ostrich intestinal glycohydrolases: distribution, purification and partial characterisation.

Intestinal glycohydrolases are enzymes involved in assimilating carbohydrate for nutrition. The avian forms of these enzymes, in particular the maltase-glucoamylase complex (MG), are not well characterised. This study encompassed characterisation of these enzymes from ostrich intestines, and the first kinetic analysis of an avian MG. Proteolytically solubilised MG from ileal brush border membrane vesicles was purified by Sephadex G-200 gel filtration and Tris-affinity-chromatography, while jejunal sucrase-isomaltase (SI) and MG were purified by Toyopearl-Q650 and phenyl-Sepharose chromatography. Amino acid sequences and compositions of enzyme subunits, resulting from SDS-PAGE, were determined. Kinetics of hydrolysis of linear oligosaccharides was studied. Ostrich MG and SI showed the highest activity in the jejunum, followed by the ileum and duodenum. No lactase or trehalase activity could be detected. The jejunal MG and SI, resulting from brush-border membrane vesicles, could not be separated during purification. However, a minor form of ileal MG was purified using Sephadex G-200 chromatography. Ileal MG contained three subunits of M(r) 145,000, 125,000 and 115,000. Although the N-terminal amino acid sequences bear no homology to SI, the M(r) 115,000 subunit shows homology to porcine MG in both sequence and amino acid composition. The pH optimum of maltose-, starch- and isomaltose-hydrolysing activity was 6.5 and that of sucrose-hydrolysing activity 5.5. The glycohydrolases were most active at 58 degrees C, but were quickly denatured above 60 degrees C. Sucrose- and starch-hydrolysing activities were more thermostable than maltose- and isomaltose-hydrolysing activities. Kinetic parameters (K(m), kcat and kcat/K(m)) for the hydrolysis of maltooligosaccharides, starch and glycogen are reported for ileal MG. Maltotriose and maltotetraose displayed partial inhibition of ileal MG. The study revealed large similarities between ostrich SI and MG in charge, size, shape and hydrophobicity, based on their inseparability by several methods. Measurement of the specificity constants for maltooligosaccharide hydrolysis by ileal MG revealed less efficient hydrolysis of longer substrates as compared to maltose and maltotriose.

An alternative PCR assay for quantifying mitochondrial DNA in crude preparations.

A method is described for the quantification of mitochondrial DNA present in crude biological preparations. A known copy number of a standard is amplified in the presence of inactivated target DNA so as to determine the overall efficiency of the PCR process in a particular sample. In this way any inhibitory and/or stimulatory substances present in sample preparations can be taken into account. To reduce tube-to-tube variations product DNA quantification is limited to small cycle numbers. Using this method quantitations of DNA amounts in different crude preparations can be compared.

Effect of Chemical and Physical Dry-curing Parameters on Cathepsins B, H and L from Ostrich Muscle.

The effects of curing agents (NaCl, nitrate, ascorbic acid and glucose) and processing parameters (pH, temperature and cooking temperatures) on cathepsins B, H and L activities were investigated. NaCl, nitrate, ascorbic acid and glucose exhibited different influences on ostrich cathepsin B, B+L and H activities. In the range 20-60gl(-1), NaCl inhibited cathepsin B+L and H activities. All three cysteine proteinase activities were inhibited by up to 8g ascorbic acid l(-1). With the exception of cathepsin B activity, which was inhibited by glucose, nitrate and glucose had very little effect on cathepsin B, B+L and H activities. Cathepsins B and D were active at 65 and 69°C and might play an important degradative role during the cooking of meat and meat products. Cathepsins B, B+L and H were optimally active at temperatures of 40-45°C and 50°C, and were still quite active at the low temperatures used in the dry-curing process; they showed maximum activity in the pH range 5·5-7. A simulation of the three stages of the dry-curing process of hams revealed that cathepsins B and B+L might play an important role throughout the complete process, whereas cathepsin H could only participate in the middle and at the end of the dry-curing process. Although ostrich cathepsins show many properties similar to those from other species, the present study also revealed some interesting distinguishing features.

Ovine cardiac Na,K-ATPase: isolation by means of selective solubilization in Lubrol and the effect of 1 alpha,2 alpha-epoxyscillirosidin on this enzyme.

The inhibition of cardiac Na,K-ATPase by 1 alpha,2 alpha-epoxyscillirosidin is the principal cause of poisoning of cattle by the tulip, Homeria pallida. The ultimate goals of this study were to study the interaction between 1 alpha,2 alpha-epoxyscillirosidin and ovine Na,K-ATPase by means of inhibition and displacement binding studies. Ovine cardiac Na,K-ATPase was isolated in membrane-bound form by means of deoxycholate treatment, high-speed ultracentrifugation, NaI treatment and selective solubilization in Lubrol. The inhibition of ovine cardiac and commercial porcine cerebral cortex Na,K-ATPase by 1 alpha,2 alpha-epoxyscilirosidin and ouabain was studied using a discontinuous Na,K-ATPase assay. The binding of 1 alpha,2 alpha-epoxyscillirosidin, ouabain and digoxin to the above enzymes was compared using a displacement binding assay with [3H] oubain. The Lubrol-solubilized ovine cardiac Na,K-ATPase showed a specific activity of 0.3 U/mg with no ouabain insensitive activity. I50 values of 2.1 x 10(-8) and 2.7 x 10(-8) were obtained for the inhibition of this enzyme by 1 alpha,2 alpha-epoxyscillirosidin and ouabain, respectively. 1 alpha,2 alpha-Epoxyscillirosidin has a much higher KD value (1.5 x 10(-7) M), however, than ouabain (9.5 x 10(-9) M) and digoxin (1.7 x 10(-8) M) in displacement binding studies with [3H]ouabain. 1 alpha,2 alpha-Epoxyscillirosidin is a potent inhibitor of ovine cardiac Na,K-ATPase and is a slightly stronger inhibitor of the enzyme than ouabain. The anomalous result for the displacement of 1 alpha,2 alpha-epoxyscillirosidin from its receptor is either a result of different affinities that K+ has for the enzyme ouabain and enzyme-1 alpha,2 alpha-epoxyscillirosidin complexes or because of different complex stabilities of these complexes.

Purification and partial characterization of an ostrich alpha 1-antichymotrypsin-like serum inhibitor.

alpha 1-Antichymotrypsin, a member of the serpins, is the predominant plasma inhibitor of neutrophil cathepsin G. The aim of this study was to purify ostrich alpha 1-antichymotrypsin and to compare its biochemical properties with those of other species. Ostrich alpha 1-antichymotrypsin was purified from serum by ammonium sulphate fractionation, QAE-Sephadex C-50 and phenyl-Toyopearl chromatography. N-terminal sequence, amino acid composition, molecular mass, isoelectric point and reaction with cathepsin G, elastase and chymotrypsin were determined. SDS-PAGE revealed a M, of 55,000 for ostrich alpha 1-antichymotrypsin and pI values of 6.8 and 4.1-4.3 were obtained. The amino acid composition revealed 444 residues and the N-terminal sequence of the first 20 residues revealed a homology of 30% when compared with several other alpha 1-antichymotrypsin sequences. Total inhibition of cathepsin G by ostrich alpha 1-antichymotrypsin was found at a 4:1 molar ratio of inhibitor to enzyme which was similar to that found for commercial alpha 1-antichymotrypsin. Immunological studies highlighted the lack of cross-reactivity between ostrich and human alpha 1-antichymotrypsin. The study indicated that ostrich alpha 1-antichymotrypsin-like molecule exhibited similar properties to human alpha 1-antichymotrypsin although there were notable differences.

Purification and characterization of proteasome from ostrich liver.

The proteasome (EC 3.4.99.46) is a high molecular mass (approximately 700 kDa) multisubunit enzyme complex which is the focus of worldwide research in order to identify the structure, mechanism of action and specificity of the complex. The purpose of the present study was to investigate the tryptic, chymotryptic and peptidylglutamyl-peptide hydrolysing (PGPH) activities of ostrich liver proteasome. The proteasome was purified from ostrich liver by employing ammonium sulphate fractionation, followed by three sequential chromatographic steps on Toyopearl Super Q-650 S, Sephadex G-150 and phenyl-Toyopearl columns. Temperature and pH optima were examined and the effect of inhibitors, detergents, fatty acids and cations on the peptidase activities was determined. Ostrich proteasome exhibited a relative M(r) of approximately 665,000 using non-denaturing gradient PAGE and dissociated into the characteristic "ladder" associated with the proteasome subunits during SDS-PAGE. The pH optima for the peptidase activities were found to be slightly alkaline (tryptic activity) and neutral (chymotryptic-like and PGPH activities). Ostrich liver proteasome was found to be activated in terms of the PGPH activity by fatty acids and SDS, whereas the chymotryptic and tryptic-like activities were differentially inhibited. Ostrich proteasome, in its inhibition by monovalent cations, was similar to the proteasomes extracted from other sources. The specificity of the proteasome appears to be very broad, although it lacks aminopeptidase activity. The yield compared favourably with similar extraction procedures which have been reported. On the basis of the physicochemical and kinetic properties which ostrich liver proteasome exhibited, it can be safely concluded that it corresponds well with the proteasomes isolated from many other sources.

Effect of lipoproteins on the differentiation of 3T3-L1 and human preadipocytes in cell culture.

High-density and low-density lipoprotein (LDL) stimulated 3T3-L1 and human preadipocyte differentiation in vitro. In both cell types, LDL exhibited the greatest stimulatory effect. LDL suppressed the development of catecholamine-stimulated lipolysis in differentiating 3T3-L1 and human preadipocytes when present during preadipocyte proliferation and early stages of differentiation. The effect of lipoproteins on development of human preadipocyte (but not 3T3-L1) lipolysis may involve beta-adrenoceptors.

The effects of Ca ions, EGTA and storage time on myofibrillar protein degradation, levels of Ca(2+)-dependent proteases and cathepsins B, H, L and D of ostrich skeletal muscle.

The effect of Ca ions and ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) on myofibrillar protein degradation showed that when ostrich iliotibialis lateralis muscle was incubated with 10 mM EGTA at 2-4 °C for 24 hr, the activity of extracted cathepsin H was unchanged compared with a buffer-incubated sample. Ca(++) had no effect on extracted cathepsin H activity, while that of Ca(2+)-dependent protease (CDP) decreased significantly (p < 0.05). Ca(2+)-treatment enhanced post-mortem changes observed in myofibrillar protein patterns (production of fragments around 30 K) that were not observed in EGTA-incubated myofibrils. The effect of storage time on shear force, CDP activity, cathepsin B, D, H and L activities and the SDS-PAGE pattern of myofibrils showed a time-dependent reduction in CDP activity. Of the cathepsins studied only cathepsin H showed a reduction (40%) in activity. The most prominent component appearing on storage at 2-4 °C had a M(r) of 27 K. The incubation of myofibrils with CDP mimicked the post-mortem changes. CDP may be responsible for some of the post-mortem changes observed, although shear force measurements suggest these changes do not lead to significant tenderisation.

Optimisation of calcium-dependent protease and cathepsin D assays in ostrich muscle and the effect of chemical and physical dry-curing parameters.

The best conditions for the assay of cathepsin D and Ca(2+)-dependent pro tease (CDP) activity in ostrich muscle was established in order to have a simple, rapid and reliable method for its determination. Measurements of A(280nm) of TCA-soluble peptides and amino acid digests of casein and haemoglobin were used for measuring proteolytic activity in muscle extracts. The best conditions for the reliable determination of cathepsin D activity were found to be the incubation of an enzyme extract for 1 hr at 55 °C in a reaction mixture containing 0.9% (w v ) haemoglobin in 50 mM sodium formate buffer, pH 3.7. Characterization of the assay system for CDPs, obtained after phenyl-Sepharose chromatography, indicated that proteolytic degradation of casein by CDPs was linear with time up to 30 min at 30 °C and up to 0.1 units of activity. The effect of NaCl, KCl, nitrate, ascorbic acid, phosphate, glucose and sucrose on ostrich muscle CDP and cathepsin D activities has been studied. Salt (NaCl and KCl) acts as a strong inhibitor of proteolytic activity. Sodium and potassium nitrates (in the range 0-1000 mg l(-1)) affected activity to varying degrees. CDP activity was enhanced by sodium nitrate concentrations below 700 mg l(-1) and unchanged by potassium nitrate. Cathepsin D activity was inhibited to some extent by sodium nitrate above 200 mg l(-1) and completely by potassium nitrate. Results showed that phosphate is an inhibitor of both activities. High concentrations of ascorbic acid (above 6 g l(-1)) inhibited cathepsin D activity. Glucose (up to 2g l(-1)) activated cathepsin D activity and inhibited CDP activity (up to 1 g l(-1)). Sucrose activated enzyme activities at very low concentrations (1 × 10(-3) M) and inhibited activities above 1 × 10(-3) M.

Purification and characterization of ostrich pancreatic secretory trypsin inhibitor.

Ostrich pancreatic secretory trypsin inhibitor was isolated and purified using acid extraction, salt fractionation. SP-Sephadex C-50 and QAE-Sephadex A-25 chromatography and RP-HPLC. The amino acid sequence of ostrich PSTI showed it is a single peptide chain containing 69 amino acid residues with the highest homology between ostrich and chicken PSTI. The molecular weight, as determined by electronspray mass spectrometry and from amino acid sequence data, is 7650 Da. The isoelectric point of ostrich PSTI was found to be 5.7. Ostrich PSTI specifically inhibited ostrich and commercial bovine trypsin with Ki values of 8.0 x 10(-9) and 2.4 x 10(-7) M, respectively, while no inhibitory effects were observed with other serine proteases.

Ostrich (Struthio camelus) carboxypeptidase B: purification, kinetic properties and characterization of the pancreatic enzyme.

Carboxypeptidase B has been isolated from numerous mammalian and invertebrate species. In contrast, very little is known about carboxypeptidases of avian origin. To provide information for a comparative study, we have undertaken an investigation of the kinetic and physical properties of ostrich carboxypeptidase B. Carboxypeptidase B from the pancreas of the ostrich was purified by water extraction of acetone powder and aminobenzylsuccinic acid affinity and hydroxylapatite chromatography. The effects of pH and temperature on CPB activity were examined. K(i)-values for numerous inhibitors (PCI, ABSA, hipp-D-lys, epsilon-aminocaproic acid, D-arg and 3-phenylproprionic acid) and kinetic parameters (K(m), k(cat) and k(cat)/K(m)) for several substrates (hipp-arg, hipp-lys, FAAA, FAAL and hipp-AA) were determined. N-terminal sequencing and amino acid analysis were also performed. Purified ostrich carboxypeptidase B was assessed to be homogeneous by SDS-PAGE with a M(r) value of approx. 35,000. For ostrich carboxypeptidase B the K(m) values for the different substrates were of the same order as those reported for other species, whereas the k(cat) values were 8- to 21-fold lower than the reported values. FAAA and hipp-AA were the preferred substrates. PCI was the most effective inhibitor, with a K(i) in the nM region, and no inhibition was shown with 3-phenylpropionic acid. The N-terminal sequence showed a high degree of homology when aligned with CPB from other species. Amino acid analysis showed significantly lower levels of Asx and Cyh and higher levels of Trp and Leu when compared with other species. Ostrich carboxypeptidase B would appear to show many physical, chemical and kinetic properties similar to those of other known carboxypeptidases.