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Biomedical subjects

M Sarwar

Publications and source records attributed to M Sarwar.

At least 19 recordsLinked to original sources
PubMed2006

Effect of different non-chloride sodium sources on the performance of heat-stressed broiler chickens.

1. One hundred and eighty 1-d-old broiler chicks were used to evaluate the effect upon broiler performance during severely hot summer months of three different sodium salts: sodium bicarbonate (NaHCO3), sodium carbonate (Na2CO3) and sodium sulphate (Na2SO4), in starter and finisher diets having an identical electrolyte balance (DEB) of 250 mEq/kg. 2. The non-chloride sodium salts were added to contribute the same amount of sodium and were substituted at the expense of builder's sand in the basal diets containing common salt (NaCl) as Na and Cl source. 3. Each diet was fed to three experimental units having 15 chicks each until 42 d of age. Severe heat-stress conditions, maintained in the rearing room, were indicated by high average weekly room temperature (minimum 29.3 degrees C; maximum 38.0 degrees C). 4. Diets containing sodium salts gave better body weight gain, feed intake and feed to gain ratio than the control diet. Sodium salts also enhanced water intake as well as water to feed intake ratio. This effect was more pronounced in broilers fed NaHCO3 supplement (with NaCl in the basal diets). 5. The increased water intake resulted in lower body temperature in heat-stressed birds fed NaHCO3 supplemented diet than in birds fed other sodium salts. A lower mortality rate was noted with NaHCO3 (15.15%), Na2CO3 (13.64%) and Na2SO4 (15.15%) supplements than with the control (33.33%) treatment. 6. Better carcase and parts yield were observed in sodium supplemented broilers. Sodium salts reduced the alkalotic pH and enhanced the blood sodium content, which ultimately improved the blood electrolyte balance and overall performance of heat-stressed broilers. 7. Supplementing broiler diets with sodium salts improved the live performance of heat-stressed broilers and better productive performance was noted with NaHCO3 than other sodium supplements.

Animals↗
PubMed2006

The influence of exogenous multienzyme preparation and graded levels of digestible lysine in sunflower meal-based diets on the performance of young broiler chicks two weeks posthatching.

The study was conducted to investigate the effect of adding multienzyme preparation (Rovabio Excel AP, Adisseo Asia Pacific Pte Ltd., Singapore) at 2 inclusion levels of sunflower meal (SFM; 20 and 30% of the diets) with 3 levels of digestible Lys (0.8, 0.9, and 1.0%) with and without enzyme in a 2 x 2 x 3 factorial arrangement. Each diet was offered to 4 replicates of 51 one-day-old straight-run Hubbard broiler chicks (n = 2448) in a practical vegetable-based mash diet having 2,750 kcal of ME/kg and 19% CP during 2 wk posthatching (1 to 14 d of age). Feed formulation was based on digestible amino acids, which were calculated from the CP and DM contents of each ingredient using AminoDat 2 (Degussa Corp., Allendale, NJ). The enzyme used in this study was authenticated by the supplier to have minimum level of endo-1,4-beta xylanase (22,000 visco units/g) and endo-1,3(4)-beta glucanase (2,000 AGL units/g) and was added at the rate of 50 mg/kg of finished diet. No significant effect of enzyme or level of SFM was observed on BW gain (BWG), feed intake, or mortality during the experimental period. The BWG and feed:gain for birds fed on 30% SFM with enzyme were comparable to those fed on 20% SFM without enzyme during 1 to 14 d of age. However, enzyme at 20% SFM depressed the BWG (P <or=0.006) and feed conversion ratio (P <or=0.027). A quadratic (P <or=0.036) response of digestible Lys was noted for BWG, whereas it was linear (P <or=0.001) for feed:gain during 1 to 7 and 1 to 14 d. A level of 1.0% digestible Lys was observed best for BWG and feed:gain. For BWG, 0.8 and 0.9% digestible Lys was comparable when it was used at 30% SFM, along with enzyme. In conclusion, enzyme supplementation during 2 wk posthatching has no remarkable effect when used in SFM-based diets. Moreover, digestible Lys may be lowered to 0.8% during the first week but not less than 1.0% during the second week post-hatching.

Aging↗
PubMed2006

Chemical treatments to reduce antinutritional factors in salseed (Shorea robusta) meal: effect on nutrient digestibility in colostomized hens and intact broilers.

This study investigated the effects of chemical treatments of salseed meal (SSM) on nutrient digestibility and digestive enzymes in colostomized hens and intact broilers. Finely ground SSM was treated (820 mL/ kg of SSM DM) with distilled water (pH 5.3), 0.67 M acetic acid (pH 2.4), or 0.67 M sodium hydrogen carbonate (pH 8.2), and incubated for 12 h at 37 degrees C. Five isonitrogenous and isocaloric diets each for colostomized hens (25.6 g of N/kg of DM and 12.5 MJ/kg of DM) and for broilers (33.6 g of N/kg of DM and 12.3 MJ/kg of DM) were formulated. For each group, 1 of these diets was wheat-based (control) whereas the other 4 were SSM-based diets (untreated SSM or SSM treated with water, acetic acid, and sodium bicarbonate). Inclusion of SSM in diets markedly reduced apparent protein and starch digestibility in hens and broilers. Chemical treatments of SSM improved the protein and starch digestibilities in colostomized hens and broilers. Treatment of SSM with alkali reduced the pancreatic hypertrophy in broilers that was observed when SSM was included in the diet. Inclusion of SSM in broiler diets did not affect pancreatic trypsinogen activity; however, chymotrypsinogen and alpha-amylase activities were depressed with its inclusion. Activities of these enzymes were improved by all chemical treatments of SSM. Dietary inclusion of SSM in broiler diets depressed the activities of trypsin, chymotrypsin, and alpha-amylase in the jejunal digesta. Alkali treatment proved to be the most effective in reducing the adverse effects of SSM upon trypsin and chymotrypsin activities. The hens receiving SSM in their diets produced eggs with discolored yolks (dirty greenish-yellow). In conclusion, nutrient digestibility, and pancreatic and intestinal enzymes were markedly depressed with the inclusion of SSM in the diets of the fowl. Bicarbonate was the most effective treatment to improve nutrient digestibility and mitigate, to some extent, the poor digestion of SSM.

Animal Feed↗
PubMed2005

Effect and interactions of dietary sodium and chloride on broiler starter performance (hatching to twenty-eight days of age) under subtropical summer conditions.

One-day-old Starbro male broiler chicks (n = 360) were used to determine the effect of increasing levels of Na+ and Cl- above the NRC (1994) recommendations for growing broilers diets (hatching to 28 d) in extremely hot weather. The average maximum and minimum temperatures recorded were 39 and 32 degrees C, respectively. An average relative humidity was 58.2% during the experimental period. Three levels of dietary Na+ (0.20, 0.25, and 0.30%) and Cl- (0.30, 0.40, and 0.50%) were used in 3 x 3 factorial arrangement while maintaining a dietary electrolyte balance (DEB) of 250 mEq/kg. Higher weight gain (P < 0.002) and maximum water consumption (P < 0.05) were observed for birds fed diets containing 0.25 and 0.30% Na+, respectively. Litter moisture was significantly higher (P < 0.05) for birds fed diets containing 0.25% Na+ and 0.40 and 0.50% Cl-. High dietary Na+ (0.30%) tended to increase breast yield (P < 0.003) and decreased abdominal fat (P < 0.001). There was no effect of Na+ on blood pH or serum HCO3-. Diets containing 0.40% Cl- increased the dressing percentage (P < 0.001) and leg yield (P < 0.001) and decreased serum HCO3- (P < 0.001). There was no effect of dietary Cl- on blood pH, feed intake, weight gain, feed:gain, water intake, water:feed intake, or mortality. Significant dietary effect of Na+ x Cl- was noted only for litter moisture (P < 0.001), dressing percentage (P < 0.05), breast (P < 0.05) and leg (P < 0.001) yields, abdominal fat (P < 0.002), and serum HCO3- (P < 0.001). Birds fed diet containing 0.25% Na+ and 0.30% Cl- performed as well as those fed other diets when the cyclic temperature ranged from 32 to 39 degrees C.

Aging↗
PubMed2003

Human porphobilinogen deaminase mutations in the investigation of the mechanism of dipyrromethane cofactor assembly and tetrapyrrole formation.

Porphobilinogen deaminase mutants that cause acute intermittent porphyria have been investigated as recombinant proteins expressed in Escherichia coli, yielding important insight into the mechanism of dipyrromethane cofactor assembly and tetrapyrrole chain polymerization. A mutation that affects a key catalytic residue, D99G, results in an inactive holo -protein that exists as a complex with two substrate molecules covalently bound to the dipyrromethane cofactor arising from the reaction between the apo -protein and pre-uroporphyrinogen. The R149Q mutant is also devoid of catalytic activity but the mutant protein is unable to assemble the dipyrromethane cofactor from pre-uroporphyrinogen and persists as an unstable, heat-labile apo -protein. The mutant, R173Q, has very low activity and, like R149Q, also exhibits largely as an apo -protein. The inability to reconstitute either R149Q or R173Q with exogenous pre-uroporphyrinogen confirms the importance of these two arginine residues for dipyrromethane cofactor assembly. In contrast, the mutant R167Q exists as a holo -enzyme but the catalytic cycle is severely compromised, leading to the accumulation of stable enzyme-substrate intermediates from the catalytic cycle.

Amino Acid Substitution↗
PubMed2002

5-Aminolaevulinic acid dehydratase: metals, mutants and mechanism.

5-Aminolaevulinic acid dehydratase catalyses the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid. The studies described highlight the importance of a bivalent metal ion and two active-site lysine residues for the functioning of 5-aminolaevulinic acid dehydratase. Dehydratases fall into two main categories: zinc-dependent enzymes and magnesium-dependent enzymes. Mutations that introduced zinc-binding ligands into a magnesium-dependent enzyme conferred an absolute requirement for zinc. Mutagenesis of lysine residues 247 and 195 in the Escherichia coli enzyme lead to dramatic effects on enzyme activity, with lysine 247 being absolutely essential. Mutation of either lysine 247 or 195 to cysteine, and treatment of the mutant enzyme with 2-bromethylamine, resulted in the recovery of substantial enzyme activity. The effects of the site-directed alkylating inhibitor, 5-chlorolaevulinic acid, and 4,7-dioxosebacic acid, a putative intermediate analogue, were investigated by X-ray crystallography. These inhibitors reacted with both active-site lysine residues. The role of these two lysine residues in the enzyme mechanism is discussed.

Amino Acid Sequence↗
PubMed1999

X-ray structure of 5-aminolevulinic acid dehydratase from Escherichia coli complexed with the inhibitor levulinic acid at 2.0 A resolution.

5-Aminolevulinic acid dehydratase (ALAD), an early enzyme of the tetrapyrrole biosynthesis pathway, catalyzes the dimerization of 5-aminolevulinic acid to form the pyrrole, porphobilinogen. ALAD from Escherichia coli is shown to form a homo-octameric structure with 422 symmetry in which each subunit adopts the TIM barrel fold with a 30-residue N-terminal arm. Pairs of monomers associate with their arms wrapped around each other. Four of these dimers interact, principally via their arm regions, to form octamers in which each active site is located on the surface. The active site contains two lysine residues (195 and 247), one of which (Lys 247) forms a Schiff base link with the bound substrate analogue, levulinic acid. Of the two substrate binding sites (referred to as A and P), our analysis defines the residues forming the P-site, which is where the first ALA molecule to associate with the enzyme binds. The carboxyl group of the levulinic acid moiety forms hydrogen bonds with the side chains of Ser 273 and Tyr 312. In proximity to the levulinic acid is a zinc binding site formed by three cysteines (Cys 120, 122, and 130) and a solvent molecule. We infer that the second substrate binding site (or A-site) is located between the triple-cysteine zinc site and the bound levulinic acid moiety. Two invariant arginine residues in a loop covering the active site (Arg 205 and Arg 216) appear to be appropriately placed to bind the carboxylate of the A-site substrate. Another metal binding site, close to the active site flap, in which a putative zinc ion is coordinated by a carboxyl and five solvent molecules may account for the activating properties of magnesium ions.

Binding Sites↗
PubMed1999

Metabolic studies of [18F-alpha-methyl]tyrosine in mice bearing colorectal carcinoma LS-180.

Brain and tumor uptake of [18F-alpha-methyl]tyrosine (18F-AMT) and the incorporation into each of four fractions (lipid, RNA, DNA and protein) were investigated in mice bearing LS180 colorectal carcinoma. Homogenized tissues were analyzed by the fractionation method into an acid-soluble fraction (ASF) and an acid-precipitable fraction (APF). The APF was further investigated to assess the incorporation of 18F-AMT into each fraction. Incorporation into four fractions of brain and tumor at 60 min post-injection was 20 and 12%, respectively; 10% of the activity was incorporated to lipid in brain and 5% in tumor. There was 5, 2 and 2% incorporation with RNA, DNA and protein, respectively. Metabolites in ASF were analyzed by high-performance liquid chromatography and thin-layer chromatography. There was only one radioactive peak, which corresponded to 18F-AMT. The incorporation of 18F-AMT into lipid was twice that of 18F-AMT in tumor. The uptake of 18F-AMT in tissues was rapid and accomplished before 30 min, and then slowly diffused in blood. These results implied that 18F-AMT was metabolized to protein to only a small extent and trapped as intact 18F-AMT in cells up to 60 min. We conclude that 18F-AMT is a promising tracer for tumor imaging and quantification of the transport rate using two-compartment models.

Animals↗
PubMed1999

Biodistribution studies of the 186Re complex of 3-amino-1-hydroxypropylidene-1,1-bisphosphonic acid in mice.

Preliminary studies of 186Re-labelled 3-amino-1-hydroxypropylidene-1, 1-bisphosphonic acid (APD) were performed to determine its potential for bone pain palliation, and as a treatment for increased bone resorption. The synthesis of 186Re-APD was carried out by reduction of 186Re-perrhenate in the presence of SnCl2. The APD kit, comprising 2.5 mg of APD, 2.5 mg of gentisic acid and 1 mg of Sn++ as SnCl2 2H2O, was prepared in-house. The APD was labelled with 186Re and injected intravenously into normal mice. Mice were subsequently sacrificed at 1, 3, 24, 48, 72, 168 and 240 h post-injection. The greatest accumulation of 186Re-labelled APD was found in bone, resulting in bone-to-blood ratios of 25, 35, 65, 100, 151, 181 and 189, respectively. 186Re-APD showed high uptake in bone, and relatively low uptake in soft tissue, suggesting that 186Re-APD is a potential agent for bone therapy.

Animals↗
PubMed1998

99Tcm-labelled chimeric human/mouse anti-granulocyte antibody bone marrow scintigraphy: a preliminary clinical study.

Bone marrow scintigraphy using 99Tcm-labelled chimeric anti-granulocyte antibody (anti-NCA-95) was performed in 17 patients with haematological disorders and skeletal metastases. Chimeric anti-NCA-95 antibody (chNCA95 Ab, 0.2 mg) labelled with 444 MBq 99Tcm was administered to obtain bone marrow images 4 h post-injection. One week later, an 111In-chloride bone marrow scan was performed on nine patients with haematological disorders. Lumbar bone marrow-to-background (L/B) and ilium-to-background (I/B) uptake ratios were calculated for each scan. In six patients with suspected skeletal metastases, 99Tcm-HMDP bone scans were performed. No patient had any adverse reaction or any immune reaction over 20 weeks. In the patients with haematological disorders, the L/B and I/B ratios of the 99Tcm-chNCA95 Ab scan were 3.41 +/- 0.90 and 1.23 +/- 0.31, whereas those of the 111In-chloride scan were 1.58 +/- 0.32 and 1.00 +/- 0.32, respectively. In assessing findings of irregular central bone marrow uptake and peripheral expansion of the bone marrow, the 99Tcm-chNCA95 Ab scan was much better than the 111In-chloride scan. In the six patients with suspected skeletal bone metastases, three true-positive and two true-negative results were observed. This preliminary study has revealed that 99Tcm-chNCA95 Ab scanning is safe and useful in the diagnosis of haematological disorders and skeletal metastases.

Adult↗
PubMed1998

Biodistribution studies on L-3-[fluorine-18]fluoro-alpha-methyl tyrosine: a potential tumor-detecting agent.

UNLABELLED: Iodine-123-alpha-methyl tyrosine has proven to be a promising SPECT agent for imaging amino acid uptake in tumors. We developed L-[3-(18)F]-alpha-methyl tyrosine (FMT) for PET studies. The aim of this study was to investigate its potential use as a tumor-detecting agent by using tumor-bearing mice. METHODS: We investigated the biodistribution in normal BALB/C mice and BALB/cA nude mice bearing human rectal cancer cell line (LS180) until 120 min postinjection. FMT tumor uptake at 60 min postinjection in mice with LS180 rectal cancer, RPM11788 B-cell lymphoma and MCF7 mammary cell carcinoma was assessed, and the results were compared with 18F-fluoro-2-deoxy-D-glucose (FDG) tumor uptake. The effect of competitive inhibition of large neutral amino acid transport system using unlabeled L-alanine was also investigated. RESULTS: The amount of FMT in blood fell to 1.05%ID/20 g at 60 min postinjection, whereas that in the pancreas was 15.2%ID/20 g, resulting in a high pancreas-to-blood ratio of 14.5. In other organs, initial uptake peaked at 5 min postinjection and then declined with time. In LS180 tumor-bearing mice, peak FMT uptake in tumor was observed at 60 min postinjection. Tumor-to-blood and tumor-to-muscle ratios ranged from 1.60 to 2.94 and from 2.79 to 3.25 over the 120-min observation period. Tumor uptake of FMT was clearly reduced by inhibition of the amino acid transport system. In mice with LS180 and MCF7 tumors, FMT tumor uptake at 60 min postinjection was significantly higher than FDG tumor uptake, whereas in RPM11788 lymphoma, uptake of FDG was significantly higher than FMT tumor uptake. Tumor-to-blood ratios of FMT in mice with LS180, RPMI1788 and MCF7 tumor at 60 min postinjection were 1.82, 5.88 and 3.56, respectively. CONCLUSION: FMT, like other fluorinated amino acids, may become a promising tumor-detecting agent for PET, assuming that efficient methods of radiosynthesis are developed.

Amino Acids↗
PubMed1997

X-ray structure of 5-aminolaevulinate dehydratase, a hybrid aldolase.

5-Aminolaevulinate dehydratase (ALAD) is a homo-octameric metallo-enzyme that catalyses the formation of porphobilinogen from 5-aminolaevulinic acid. The structure of the yeast enzyme has been solved to 2.3 A resolution, revealing that each subunit adopts a TIM barrel fold with a 39 residue N-terminal arm. Pairs of monomers wrap their arms around each other to form compact dimers and these associate to form a 422 symmetric octamer. All eight active sites are on the surface of the octamer and possess two lysine residues (210 and 263), one of which, Lys 263, forms a Schiff base link to the substrate. The two lysine side chains are close to two zinc binding sites one of which is formed by three cysteine residues (133, 135 and 143) while the other involves Cys 234 and His 142. ALAD has features at its active site that are common to both metallo- and Schiff base-aldolases and therefore represents an intriguing combination of both classes of enzyme. Lead ions, which inhibit ALAD potently, replace the zinc bound to the enzyme's unique triple-cysteine site.

Amino Acid Sequence↗
PubMed1996

Independent thallium-201 accumulation and fluorine-18-fluorodeoxyglucose metabolism in glioma.

UNLABELLED: SPECT with 201TI is an effective procedure for evaluating the malignancy of glioma. Our goal was to investigate the diagnostic relevance of both 201TI SPECT and [18F]fluorodeoxyglucose (FDG) PET and the relation between 201TI uptake and glucose metabolism in glioma using comparative SPECT and PET studies. METHODS: Thallium-201 SPECT and FDG dynamic PET studies were performed in 20 patients with untreated glioma (5 with glioblastoma, 5 with anaplastic glioma, 10 with low-grade glioma). Thallium-201 uptake in the tumor was estimated using the 201TI index, defined as the ratio of 201TI uptake in the tumor to that in the contralateral normal brain on SPECT images obtained 15 min after intravenous injection. We measured regional glucose metabolic parameters, including rate constants and regional cerebral metabolic rate of glucose utilization (rCMRgl), in the tumor. We then compared the regional 201TI index and glucose metabolic parameters with the histologic characteristics, malignancy and computed tomographic/ magnetic resonance imaging findings. In addition, we investigated the correlation between the 201TI index and glucose metabolic parameters. RESULTS: Thallium-201 SPECT showed abnormal 201TI uptake in all patients with glioblastoma and anaplastic glioma. Thallium-201 indices of glioblastoma (202.6 +/- 22.1%) and anaplastic glioma (176.6% +/- 26.6%) were significantly higher than that for low-grade glioma (106.7% +/- 13.8%). The rCMRgl value of glioblastoma (17.6 +/- 3.5 mumole/100 g/min) was also significantly higher than that for low-grade glioma (10.8 +/- 4.5 mumole/100 g/min), although rCMRgl showed a large variability in both high- and low-grade glioma. Rate constants of FDG kinetics had no correlation with histological grade of glioma. Some patients with high-grade glioma, however, showed false-negative results with FDG-PET because of high normal brain uptake of FDG. Conversely, most low-grade glioma could not be localized by 201TI SPECT. There was no correlation between the 201TI index and glucose metabolic parameters. CONCLUSION: Thallium-201 indices and rCMRgl values for glioblastoma were higher than those for low-grade glioma. Thallium-201 uptake in the tumor may be independent of increased glucose transport or metabolism. Thallium-201 SPECT and FDG-PET are complementary in the diagnosis of glioma, although 201TI SPECT is more significantly correlated with the malignancy of glioma.

Brain Neoplasms↗
PubMed1994

CYFRA 21-1 as a tumor marker used in measuring the serum fragment of cytokeratin subunit 19 by immunoradiometric assay.

Serum levels of cytokeratin subunit 19 (CYFRA 21-1) were measured in 42 healthy volunteers, 104 cases of malignant diseases, 30 patients with chronic renal failure and 13 patients with nonmalignant and infectious diseases. The reliability of the method was demonstrated after dilution of serum samples and intra- and inter-assay reproducibility. Serum CYFRA-21-1 concentrations were less than 2.00 ng/ml in all healthy controls and 86% of the malignant cases had high serum CYFRA 21-1 levels. However slightly elevated values of CYFRA 21-1 were observed in most chronic renal failure patients. High correlation was observed between serum CYFRA 21-1 and Tissue Polypeptide Antigen (TPA) values (r = 0.90, n = 10) but not with serum alpha-feto protein (AFP) concentrations. Furthermore, cross binding tests with the CYFRA 21-1 tracer/CYFRA 21-1 antibody-coated beads and CYFRA 21-1 tracer/TPA antibody-coated beads also gave an almost linear graph. These results indicate that CYFRA 21-1 and TPA share similar type of antigens.

Biomarkers, Tumor↗
PubMed1992

Effects of varying forage and concentrate carbohydrates on nutrient digestibilities and milk production by dairy cows.

Five Holstein cows with ruminal cannulas were used in a 5 x 5 Latin square design to determine the effect of replacing forage NDF with soyhull NDF and varying concentrations of nonstructural carbohydrates on nutrient digestion and milk production. Diets in which NDF percentage from forage (corn silage: alfalfa hay, 1:1) was 80 (control), 70, or 60 were formulated by substituting soyhulls for forage; total forage was 43.2, 36.7, and 31.1% of the diets, respectively, but total NDF was 31%. Nonstructural carbohydrates were formulated to be 47 (control), 35, or 25% by substituting soyhulls, roasted soybeans, and Ca soaps for concentrate. Ruminal acetate: propionate ratio decreased linearly when diets lower in forage NDF were fed, but it increased quadratically when dietary nonstructural carbohydrates were reduced. Apparent digestibility of OM increased quadratically, but NDF digestibility and lactation performance were unaffected when diets lower in forage NDF were fed. Digestibility of NDF increased linearly when nonstructural carbohydrates were reduced, perhaps because of greater digestibility of soyhull NDF and smaller negative associative effects. Fat from soybeans and Ca soaps was increased as nonstructural carbohydrates decreased. Added fat probably increased fatty acid digestibility and decreased milk protein percentage. Greater FCM production without correspondingly greater feed intake or BW loss increased feed efficiency as nonstructural carbohydrates decreased. In dairy rations containing soyhulls, 60% of dietary NDF from forage should maintain lactation performance, and decreasing nonstructural carbohydrates to 25 to 35% of feed DM, coupled with adding dietary fat, may decrease negative associative effects and improve efficiency of milk production.

Ammonia↗
PubMed1990

Cloning of aminoglycoside phosphotransferase (APH) gene from antibiotic-producing strain of Bacillus circulans into a high-expression vector, pKK223-3. Purification, properties and location of the enzyme.

The aminoglycoside phosphotransferase gene from a butirosin-producing strain of Bacillus circulans was cloned in a high-expression vector (pKK223-3) to give the recombinant plasmid pMS5. Escherichia coli harbouring the plasmid, E. coli JM103[pMS5], was characterized, and several features of the expression of the phosphotransferase were studied. The phosphotransferase activity was best expressed in a medium lacking glucose, and the highest levels of the enzyme were found between 12 and 24 h of growth. The induction of the phosphotransferase expression with isopropyl beta-D-thiogalactopyranoside (inducer) was found to be undesirable as the overproduction of the enzyme led to the killing of the bacteria. The subcellular location of the phosphotransferase, and also the site in vivo of the phosphorylation of neomycin, was found to be in the cytoplasm. The phosphotransferase was purified to homogeneity in good yield (17 mg of purified protein/3 litres of culture) and was shown to be a monomer of Mr 30,000-32,000. The N-terminal amino acid sequence was in agreement with that predicted from the gene sequence and confirmed the absence of any signal sequence. The regiospecificity of the phosphotransferase reaction was studied by m.s. and by 1H-, 13C- and 31P-n.m.r. using ribostamycin as the substrate, and it was found that the antibiotic was phosphorylated at the 3'-hydroxy group.

Amino Acid Sequence↗