Purification and functional characterization of pancreatic insulin from camel (Camelus dromedarius)

Large-scale production of insulin still represents the key step in helping diabetic patients throughout the world. Many species and approaches have been used for the production of insulin. In this study, we purified and characterized for the first time pancreatic insulin from the Arabian camel (Camelus dromedarius) using a modified acid-alcohol extraction method. After extraction insulin was purified using a one-step gel filtration on a Sephadex G-50 column leading to a purification yield of 80 mg/kg (20%) of camel pancreas. The purity of camel insulin was assessed by SDS–PAGE and HPLC using insulin from human, bovine and porcine as standards. Molecular weight was determined for purified camel insulin as 5800 Daltons and its amino acid composition is similar to that known for other species. The functional characterization of purified crude camel insulin was demonstrated in vitro by positive competition by radioimmunoassay and in vivo showing camel insulin inducing acute hypoglycaemia in mice. Together, our study reports for the first time the successful purification of functional insulin from camel pancreas with similar properties compared to other insulin species. This is of great interest given that the camel represents considerable economic worth in many countries.     

Carbohydrases in camel (Camelus dromedarius) pancreas. Purification and characterization of glucoamylase

The present study analyzed the existence of carbohydrases in camel pancreas compared to some other ruminants. Disaccharidases (maltase, cellobiase, lactase, trehalase and sucrase), glucoamylase and alpha-amylase were detected in pancreas of camel, sheep, cow and buffalo. Enzyme levels in sheep were lower than in the other ruminants. The highest level was detected for alpha-amylase (EC 3.2.1.2). Moderate activity levels were detected for glucoamylase (EC 3.2.1.3) and maltase (EC 3.2.1.20), while other disaccharidases showed very low activity. The results suggested that, in addition to alpha-amylase, glucoamylase and maltase may be synthesized and secreted from pancreas to the small intestine in ruminants. Camel pancreatic glucoamylase was purified and characterized. The purification procedure included glycogen precipitation and chromatography on DEAE-Sepharose and Sepharose 6B. The molecular mass was 58 kDa for native and denatured enzyme using gel filtration and SDS-PAGE, respectively. The enzyme had a pH optimum at 5.5 and a Km of 10 mg starch/mL with more affinity toward potato soluble starch than the other carbohydrates. Glucoamylase had a temperature optimum at 50 degrees C with heat stability up to 30 degrees C. The effect of different cations and inhibitors was examined. The camel pancreatic glucoamylase may possess an essential thiol.     

Purification and partial characterization of goose ceruloplasmin

The preparation and properties of ceruloplasmin from goose blood plasma are described. Ammonium sulfate was used to precipitate the crude protein followed by adsorption and elution from DEAE-Sephadex A-50. Further treatment with an ethanol-chloroform mixture and Sephadex G-200 yielded an intensely blue protein possessing a high degree of chemical purity and biological activity. Goose ceruloplasmin, existing in two forms, appears to be a single polypeptide, apparent Mr121,300, with an A610/A280 ratio of 0.07. Copper represented 0.32%, which corresponded to six atoms of copper per protein molecule. Although the amount of EPR-detectable copper was the same as in mammalian ceruloplasmins there were some differences in EPR parameters, mainly in A parallel. Goose ceruloplasmin's amino acid composition, although similar in many residues to human ceruloplasmin, was lower in tyrosine, cystine/cysteine, and acidic amino acids. Valine was found as the N-terminal amino acid. Hexose, hexosamine, sialic acid, and fucose accounted for 6.65% of the weight. Goose protein contained only half the sialic acid of human ceruloplasmin. Two values for Km using either p-phenylenediamine (0.64 and 0.053 mM) or o-dianisidine (0.76 and 0.15 mM) were evaluated from Lineweaver-Burk plots. EPR studies on reactions with water radiolysis products at cryogenic temperatures allowed us to discover that goose ceruloplasmin, like human and bovine ceruloplasmins, possesses superoxide dismutase activity.     

Purification and characterization of cationic chymotrypsin from the pancreas of the Arabian camel (Camelus dromedarius)

This study reports on the purification and characterization of a cationic enzyme with chymotryptic activity from camel pancreas. The enzyme was purified 52-fold in a 48% yield by a three-step chromatographic procedure consisting of anion-exchange, cation-exchange and affinity chromatographies. The purified enzyme was homogeneous on gel isoelectric focusing and on SDS gel electrophoresis. Its isoelectric point was estimated to be 7.3 and its molecular mass was found to be 23,600 Da. The enzyme was identified as a cationic chymotrypsin according to its physiochemical properties, substrate specificity and susceptibility to inhibition. It was active towards esters of aromatic amino acids but much less active towards a leucine ester. In all cases, the k_cat values of the camel enzyme were less than the corresponding values of bovine chymotrypsin A. It also showed a lower level of kininase activity. Camel chymotrypsin was more susceptible than its bovine equivalent to inhibition by soybean trypsin inhibitor and aprotinin. It showed the same pH optimium as bovine chymotrypsin A for its esterolytic activity, but was more dependent on CaCl₂ for long-term stability.     

Purification of carnitine acetyltransferase from skeletal muscle of the camel (Camelus dromedarius)

The enzyme carnitine acetyltransferase (CAT) catalyzes the reversible transfer of short-chain acyl groups between coenzyme A and L-carnitine, and hence, plays an important role in the -oxidation of fatty acids. Purification and characterization of CAT from desert animal species may help in explaining the involvement of secondary pathways for energy production in these species. In this paper, we report the purification and partial characterization of CAT from the Arabian camel. CAT was purified from the skeletal muscle of the Arabian camel by ammonium sulfate and acetone fractionation, followed by chromatography on DEAF-Sepharose, agarose-Co A and Superose 12 gel filtration columns. CAT was purified by 2937-fold to a specific activity of 94 Units mg^–1. The purified CAT was a monomer of 59 kDa as judged by native and SDS-PAGE, and showed a pl of 5.2. The enzyme displayed maximum activity with propionyl-Co A. Apparent K_m for acetyl-, propionyl- and butyryl-Co A were 27.7, 17.3 and 29 M respectively, while palmitoyl-Co A was not a substrate.     

Genotypic characterization of Iranian camel (Camelus dromedarius) isolates of Echinoccocus granulosus

In the present study, Echinoccocus granulosus isolates collected from camels (Camelus dromedarius) in eastern Iran were characterized based on the nucleotide sequences of mitochondrial CO1 and NDI genes. The molecular results for camel isolates demonstrated that at least 2 different genotypes are present, i.e., a buffalo genotype (G3) and the camel genotype (G6). Although the sequences of the Iranian camel genotype (G6) are completely homologous to the reference sequence of G6 (M84666) of E. granulosus , a nucleotide mutation (C to T at position 168) was detected in the CO1 sequences of the Iranian G3 isolates (HM626405) when compared with the reference G3 genotype (M84663). The findings of the present study represent the demonstration of the buffalo strain in camels. As previously reported, humans can be infected by this genotype; accordingly, the epidemiological importance of this genotype in the camel population should be considered in further studies.     

Purification and partial characterization of ceruloplasmin receptors from rat liver endothelium

Ceruloplasmin (CP), a circulating glycoprotein, is known for its copper transport. Recently the spectrum of its activity has been increased to include numerous enzymatic functions. CP binds to the liver endothelium and is transported across the cell via a mechanism involving receptor-mediated endocytosis. To isolate CP receptors, we obtained purified preparations of liver endothelium in rats. The membrane was then isolated by ultracentrifugation and solubilized in Triton X-100. Membrane proteins were labeled with 125I and passed through an affinity column in which CP was covalently linked to Sepharose 4B. Most of the radioactivity was eluted with buffer during the first 5 days. When no more radioactivity was eluted with buffer, elution was done either competitively with cold excess CP or 1 M NaCl. By this technique, a sharp single peak of radioactivity was obtained and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. Under both conditions receptors appeared as a single band with Mr of 35,000 containing 3% carbohydrate and an isoelectric point of 5.2.     

Forestomach motility in the camel (Camelus dromedarius)

There is a cyclical pattern of motility in compartments 1 and 2 of the forestomach of the camel which can be categorized into A- and B-contractions. An average motility cycle is composed of 7 A- and 5 B-contractions and lasts 5 min, including a pause of 2.3 min. The glandular sacs within the caudal sac of compartment 1 contract 1.7 sec earlier than the caudal sac. The proximal part of the canal between compartment 2 and 3 contracts 1.2 sec prior to the distal part. Forestomach motility is stimulated by distention of the cranial sac of compartment 1 and inhibited by distention of the tubiform portion of compartment 3.     

Monophyletic origin of domestic bactrian camel (Camelus bactrianus) and its evolutionary relationship with the extant wild camel (Camelus bactrianus ferus)

The evolutionary relationship between the domestic bactrian camel and the extant wild two-humped camel and the factual origin of the domestic bactrian camel remain elusive. We determined the sequence of mitochondrial cytb gene from 21 camel samples, including 18 domestic camels (three Camelus bactrianus xinjiang , three Camelus bactrianus sunite , three Camelus bactrianus alashan , three Camelus bactrianus red , three Camelus bactrianus brown and three Camelus bactrianus normal ) and three wild camels ( Camelus bactrianus ferus ). Our phylogenetic analyses revealed that the extant wild two-humped camel may not share a common ancestor with the domestic bactrian camel and they are not the same subspecies at least in their maternal origins. Molecular clock analysis based on complete mitochondrial genome sequences indicated that the sub-speciation of the two lineages had begun in the early Pleistocene, about 0.7 million years ago. According to the archaeological dating of the earliest known two-humped camel domestication (5000–6000 years ago), we could conclude that the extant wild camel is a separate lineage but not the direct progenitor of the domestic bactrian camel. Further phylogenetic analysis suggested that the bactrian camel appeared monophyletic in evolutionary origin and that the domestic bactrian camel could originate from a single wild population. The data presented here show how conservation strategies should be implemented to protect the critically endangered wild camel, as it is the last extant form of the wild tribe Camelina.     

Semen-induced ovulation in the bactrian camel (Camelus bactrianus)

Bactrian camels (63 female female, 8 male male) were used in the breeding season to determine the factors that will induce ovulation. After insemination of semen samples into the vagina, the ovaries were checked for ovulation by rectal palpation. The results indicated that ovulation was induced by the seminal plasma, but not by the spermatozoa, and the incidence of ovulation after insemination was 87%. Most of the females (66%) had ovulated by 36 h after insemination and the rest by 48 h, as after natural service. The least amount of semen required to elicit ovulation was about 1.0 ml. Intramuscular injections of LH, hCG and LHRH also caused ovulation, even in females that had not ovulated in response to insemination.     

Purification and comparison of phosphoenolpyruvate carboxykinase from the liver and kidney of the Arabian camel (Camelus dromedarius)

1. Phosphoenolpyruvate carboxykinase was partially purified from camel liver and kidney by ammonium sulphate fractionation, gel filtration and ion-exchange chromatography. 2. The specific activity of the purified preparation from liver was 39.2 mumol/min per mg protein. 3. When isolated from the kidney the specific activity of the enzyme was very much higher 155.5 mumol/min per mg protein. 4. The enzyme from the two sources were similar in their pH optimum which was approx. 7.2 and their relative stability to thermal inactivation at 60 degrees C. 5. The mol. wt of the enzyme from both organs was estimated at 80,000 +/- 5000.     

Purification and characterization of luteinizing hormone from the dromedary (Camelus dromedarius)

Luteinizing hormone (LH) has been purified from 150 dromedary pituitaries and its partial physicochemical, biological and immunological characterization has been achieved. Purification of the hormone was monitored by a porcine LH radioreceptor assay (RRA). In this system, the final camLH preparation exhibited an activity 0.6-fold that of highly purified porcine LH. The acid half-dissociation of camLH at equilibrium was observed at pH 4.2. A homologous camLH RRA was developed using the testicular plasma membrane fraction from prepubertal camels and radioiodinated, highly-purified camLH. Pituitary and chorionic gonadotropins (CG) from several mammalian species were compared to camLH in this system. The equine gonadotropins eLH and eCG were shown to be 6 times less potent in the camel RRA than in the porcine RRA, whereas the LH from other species exhibited similar activities in both systems. This particularity of camel LH receptors offers a new tool for the study of structural features of gonatropin interactions with their receptors.     

Clinical and anatomical studies of the camel (Camelus dromedarius) genitalia

Clinical and anatomical studies of the genitalia of 294 camel (197 males and 97 females) were carried out between May 1984 and October 1985. Preslaughter (clinical) examination was followed by detailed post-mortem (anatomical) examination and dissection of genitalia at the abattoir. Measurements and weights of the various segments of genitalia were made to establish the baseline data for breeding soundness evaluation in the dromedary in Northern Nigeria.     

Control of luteolysis in the one-humped camel (Camelus dromedarius)

Blood plasma concentrations of 13,14-dihydro-15-keto PGF2 alpha (PGFM) were measured in groups of mature non-pregnant and pregnant camels to study PGF2 alpha release patterns around the time of luteolysis and the timing of the signal for pregnancy recognition. Injection of each of four camels with 10 and 50 mg of PGF2 alpha showed clearly that five times the dose of exogenous hormone produced five times the amount of PGFM in peripheral plasma, thereby indicating that, as in other animal species, PGFM is the principal metabolite of PGF2 alpha in the camel. Serial sampling of three non-pregnant camels on each of days 8, 10 and 12, and three pregnant camels on day 10, after ovulation for 8 h showed a significant (P < 0.05) rise in mean plasma PGFM concentrations only on day 10 in the non-pregnant, but not the pregnant, animals. A single intravenous injection of 20, 50 or 100 iu oxytocin given to three groups of three non-pregnant camels on day 10 after ovulation did not increase their basal serum PGFM concentrations. However, daily treatment of six non-pregnant camels between days 6 and 15 (n = 3) or 20 (n = 3) after ovulation with 1-2 g of the prostaglandin synthetase inhibitor, meclofenamic acid, inhibited PGF2 alpha release and thereby resulted in continued progesterone secretion throughout the period of meclofenamic acid administration. These results showed that, as in other large domestic animal species, release of PGF2 alpha from, presumably, the endometrium controls luteolysis in the dromedary camel. Furthermore, reduction in the amount of PGF2 alpha released is associated with luteal maintenance and the embryonic signal for maternal recognition of pregnancy must be transmitted before day 10 after ovulation if luteostasis is to be achieved. However, the results also indicate that, in contrast to ruminants, the release of endometrial PGF2 alpha in the non-pregnant camel may not be controlled by the release of oxytocin.     

Purification and partial characterisation of camel milk xanthine oxidoreductase

Xanthine oxidoreductase (XOR) was purified in the presence of dithiothrietol from camel milk with yields of up to 22.2mg/l that were comparable to those obtained from bovine and human milk sources. On SDS-PAGE, the freshly purified camel milk XOR had a protein flavin (A280/A450) ratio of 5.3 +/- 0.4 and appeared homogenous with a single major band of approximately Mr 145.3 KDa. Surprisingly, in all the batches (n = 8) purified camel milk XOR showed no detectable activity towards xanthine or NADH. The molybdenum content of camel XOR was comparable to human and goat milk enzymes. After resulphuration, camel milk XOR gave a specific activity of 1.1 nmol/min/mg and 13.0 nmol/min/mg enzyme towards pterin (fluorimetric assay) and xanthine (spectrophotometric assay) respectively. This activity was markedly lower than that of human, bovine and goat enzymes obtained under the same conditions. These findings suggest that the molybdo-form of camel enzyme is totally under desulpho inactive form. It is possible that camel neonates are equipped with an enzymic system that reactivates XOR in their gut and consequently generates antibacterial reactive oxygen species.     

Comparison of virokine from camel pseudocowpoxvirus (PCPV) with Interleukin 10 of the Dromedary camel (Camelus dromedarius)

Cellular interleukin-10 (IL-10) gene from the peripheral blood mononuclear cells of the healthy Dromedary camel (Camelus dromedarius) and viral IL-10 (vIL-10) from the skin scabs of the Dromedary camels infected with contagious ecthyma (a parapoxviral infection in the camels) were amplified by polymerase chain reaction, cloned and characterized. Sequence analysis revealed that the open reading frame (ORF) of dromedarian camel IL-10 is 537 bp in length, encoding 178 amino acid polypeptide while open reading frame of vIL-10 from camel is 561 bp, encoding 187 amino acid polypeptide. The Dromedary camel IL-10 exhibited 62.6% and 68.5% sequence identity at the nucleotide and amino acid level, respectively, with vIL-10 from camel. Sequence analysis also revealed that the Dromedary camel IL-10 shared 99.4% and 98.3% identity at the nucleotide and amino acid level, respectively, with the Bactrian camel (Camelus bactrianus). But vIL-10 from camel shared 84.7% and 83.4% sequence identity at the nucleotide and amino acid level, respectively, with vIL-10 from reindeer (Rangifer tarandus), which is a ruminant species belonging to the order Artiodactyla. The present study was conducted to evaluate the evolutionary origin of the camel parapoxvirus with parapoxviruses of cattle and sheep and the resultant sequence analysis revealed that camel parapoxvirus is closely related to cattle parapoxvirus than sheep parapoxvirus (Orf virus).     

Levels of glycosylated haemoglobin in the Arabian camel (Camelus dromedarius)

Proportions of glycosylated haemoglobin (Hb) were determined in 10 Arabian camels (Camelus dromedarius) and were compared with normal controls (n = 59) and diabetic patients (n = 47) using the thiobarbituric acid (TBA) method. The level of glycosylated haemoglobin (5.5%) in camels is significantly different from that of normal healthy humans (4.9%) (P less than 0.001). Whereas the glucose levels were comparable, this difference in percentages of glycosylated haemoglobin may be explained by the difference in survival time between human and camel red blood cells.     

The purification, characterization and regulatory properties of liver phosphofructokinase in the Arabian one-humped camel (Camelus dromedarius)

1. Phosphofructokinase from camel liver was purified to homogeneity more than 3600-fold, and the yield of the preparation was 46%. 2.The sodium dodecyl sulphate-treated purified enzyme migrated as a single band in 10% polyacrylamide gel. 3. The enzyme is a tetramer, with a monomer Mr 90,000. 4. The regulatory properties of the purified enzyme from camel liver were studied at pH 7.0. 5. The enzyme displayed cooperativity with respect to fructose 6-phosphate and was inhibited by high concentrations of ATP. 6. The enzyme was also inhibited by citrate, phosphocreatine and 2,3-bisphosphoglycerate. 7. On the other hand, ADP, AMP, glucose 1,6-bisphosphate and fructose 2,6-bisphosphate were all found to be strong activators for camel liver phosphofructokinase.     

Anatomical subdivisions of the stomach of the Bactrian camel (Camelus bactrianus)

Twenty stomachs of Bactrian camels (Camelus bactrianus) were studied by gross dissection. Based on the configuration of the stomach and the structure of the mucous membrane, the stomach was divided into three ventricles that differ from the arrangement described for ox and sheep. The first and second ventricles of the proventriculus of camel form one stomach rather than two different stomachs. These ventricles of the proventriculus do not correspond to the rumen and reticulum of ox and sheep. The third ventricle appears to be the abomasum. One part of the abomasum has reticular mucosal folds that indicate it is not the reticulum. A second part of the abomasum has longitudinal mucosal folds suggesting it is not the omasum. Three glandular sac areas associated with the preventriculus and abomasum are also described.