A procedure for purifying jack bean urease for clinical use

Urease with a purity meeting the requirements of analytical use was purified from jack bean meal through steps consisting of 20% acetone extraction, heat treatment, acid precipitation, and lyophilization. For extraction of urease, one part of bean meal was mixed with 5 parts of 20% acetone containing 1 mM EDTA and 1 mM 2-mercaptoethanol, and stirred at 20 degrees C for 5 min. Milky substances in the extract were removed by heat treatment. Urease in the clear yellow supernatant was precipitated by adjusting the pH of the solution to 5.4 with citric acid. The acid precipitated urease was neutralized by dissolving in 0.015 M phosphate buffer, pH 8.5 (final pH 6.8 to 7.0) and then lyophilized. By this procedure, the purity of the enzyme was increase 14.7 fold, the recovery of activity was 63%, and the yield was 6.75 g from 1 kg of bean seeds. The specific activity of the preparation was 411 units/mg protein (240 units/mg solid), and the free ammonia content was less than 0.01 microgram per unit. Some other proteins were present in the urease preparation as examined by gel filtration and gradient polyacrylamide gel electrophoresis. The molecular weight of the enzyme estimated by gel filtration was 480,000. However, two urease activity bands with molecular weight of 230,000 and 480,000 were observed in the polyacrylamide gel electrophoregram. From the result of determination of blood urea nitrogen (BUN), this simple purification procedure could be used for practical preparation of urease from jack bean meal for clinical analysis.     

The subunit structure of jack-bean urease

1. Urease of specific activity 160-180 Sumner units/g. (Sumner, 1951) was purified from jack-bean meal. The preparation was pure on the basis of polyacryl-amide-gel electrophoresis and N-terminal studies. 2. By using both the 1-fluoro-2,4-dinitrobenzene method and the phenyl isothiocyanate method a single N-terminal methionine residue was found. 3. A single C-terminal sequence -Tyr-Leu-Phe was found by studies with carboxypeptidase A, carboxypeptidase B and hydrazinolysis. 4. N-Bromosuccinimide cleavage showed that five unique tryptophan sequences were present: Trp-Ala, Trp-Glu, Trp-Gly, Trp-Met and Trp-Arg. 5. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate showed that urease had a subunit molecular weight of 76000. 6. The yield of N- and C-terminal amino acids, the number of tryptic peptides and tryptophan sequences and the above polyacrylamide-gel electrophoretic measurement all suggest that urease contains a single structural subunit of molecular weight 75000.     

Analysis of volatile fractions of Schisandra chinensis (Turcz.) Baill. using GC-MS and chemometric resolution

The two-dimensional data obtained from GC-MS has been used qualitatively and quantitatively to determine the components of the volatile fractions of Schisandra chinensis obtained by six different extraction methods. Sub-window factor analysis (SFA) was employed to confirm the identities of components determined in different samples. With the help of SFA, and other chemometric techniques, peak purity in the chromatograms was determined, and overlapping peaks were resolved to yield a pure chromatographic profile and mass spectrum for each component. It is demonstrated that the accuracy of qualitative and quantitative analysis may be greatly enhanced using chemometric resolution methods, such methods being particularly valuable with respect to the analysis of complex samples such as traditional Chinese medicines. It is further demonstrated that different extraction methods give rise to volatile fractions of S. chinensis which differ qualitatively and quantitatively in their composition.     

Scaled down method for the reproducible recovery to high purity of human serum albumin from low volume blood samples

A basic need for a protein-based dosimeter is a purified protein. In this communication we present an isolation protocol and an HPLC-based assay which allows one to determine the purity of the isolated albumin. A total of 168 human blood samples were collected from workers of a benzene processing plant and from nearby countryside at Kohtla-Järve, Estonia. Albumin was isolated from plasma by sequential precipitation and the purity was determined by HPLC. The amount of albumin present in plasma varied between the individuals, being 147 ± 26 mg/5 ml (n=168), which is about 59% of plasma albumin. However, the isolated albumin was highly pure (100.9 ± 8.2%, n=5). All albumin samples analyzed demonstrate two peaks in HPLC analysis. The two peaks detected were collected and subjected to MS analysis, which demonstrates a difference of 120 mass units between the two albumin products isolated. We have developed an assay, which is easy to carry out and is not too labor intense. The HPLC analysis can be applied to confirm the purity of the isolated albumin as well as to confirm the quantity of the albumin in samples.     

Purification of catecholase from Solanum melangena (brinjal)

Catecholase was purified from cortex of Solanum melangena (brinjal) on natural affiant, lignin. The elution profile showed seven peaks with the 6th peak having 4616-fold purity. The 6th pure fraction loaded on PAGE showed two protein bands on staining with Coomassie brilliant blue, one at the point of application and other near the dye front. These bands exhibited catecholase activity, when stained with 4-methyl catechol and proline, Basic fuschin and ethidium bromide showed positive tests, indicating that catecholase is a ribonucleoglycoprotein.     

Effect of Actinomycin D on Virus-induced Ribonucleic Acid Polymerase Formation in Foot-and-Mouth Disease Virus-Infected Baby Hamster Kidney Cells

Actinomycin D, at a concentration that inhibits cellular ribonucleic acid (RNA) synthesis, inhibited the production of foot-and-mouth disease virus-induced RNA polymerase in baby hamster kidney cells. Inhibition was proportional to exposure time and reached 85% when actinomycin D was added 90 min before infection. Polymerase production was inhibited to the same extent in growth and minimal media, and the kinetics of its appearance were slightly different than in untreated cells. Enzyme preparations from actinomycin-treated cells having one-third to one-tenth the activity of untreated samples gave products with RNA profiles similar to those of controls. The 37S viral peak, 20S ribonuclease-resistant peak, and 26 to 28S peaks were present in all cases. Actinomycin D did not consistently inhibit virus production in either medium. Insulin did not prevent the actinomycin induced inhibition of polymerase and virus production from occurring.     

Jack been urease (EC 3.5.1.5). II. The relationship between nickel, enzymatic activity, and the "abnormal" ultraviolet spectrum. The nickel content of jack beans

At low pH, EDTA promotes the loss of the tightly bound nickel ions from jack bean urease. The specific activity of soluble enzyme after partial EDTA-promoted inactivation is a linear function of the nickel content. The results are consistent with the presence of 2.0 nickel ions per 97 000-dalton subunit in pure urease. The time scale for loss of enzymatic activity and nickel under these conditions is similar to that for loss of the "abnormal" tail absorption in the ultraviolet and visible absorption spectrum of urease (including the shoulder at approximately 420 nm). This indicates that nickel in urease is essential for enzymatic activity and establishes that the metal ions are in part responsible for the tail absorption in the ultraviolet spectrum of urease. After partial inactivation in the presence of EDTA either at low pH or in 2.5 M guanidinium chloride at neutral pH, urease did not regain activity in the presence of Ni2+. As yet apourease has not been produced reversibly. Jack bean seeds grown hydroponically without added nickel were low in both urease activity and nickel (10 and 6%, respectively, of parent seeds). Several other metal ions were readily available. This result suggests that metal ions other than nickel cannot substitute for nickel in the formation of normally active urease.     

Purification of the Proteinaceous α-Amylase Inhibitor from Phaseolus Vulgaris by Affinity Chromatography with Immobilized Enzyme or Antibody

A protein inhibiting salivary and pancreatic a-amylase of mammalian origin is contained in dry seeds of beans (Phaseolus vulgaris). Starting from a crude bean extract, the amylase inhibitor may be purified about 30fold in one step to apparent homogeneity by chromatography on matrix-bound salivary amylase. Compared with protein obtained by a conventional purification procedure and in similar yield, the amylase inhibitor obtained by affinity chromatography had the same specific activity (4.5 (akat inhibitor units/mg protein). A one step purification from crude extracts to homogenous inhibitor with the same specific activity was achieved by immuno-affinity chromatography on immobilized rabbit antibody raised against pure amylase inhibitor. The yield was 60 % that of a conventional purification. Criteria of purity of the inhibitor protein were thin-layer electrofocussing and immuno-electrophoresis.     

Characterization of human placental alkaline phosphatase by activity and protein assays, capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Placental alkaline phosphatase (PLAP) that had been isolated from human placenta was further purified using subsequent ion-exchange chromatography (IEC), affinity chromatography (AC) and centrifugal membrane concentration (CMC). During the process, the PLAP samples from the different stages of purification were characterized regarding purity and activity. This was accomplished by combining Lowry analysis, enzymatic activity assay, capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE) and matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS). The sample obtained after IEC had a rather low specific activity (6.8 U/mg) and appeared to contain several major contaminants, among which was human serum albumin (HSA). AC followed by CMC yielded PLAP with a specific activity of 128 U/mg. The purity and identity of the protein was indicated by MALDI-TOF-MS yielding a spectrum with one major peak at m/z 58 101. Interestingly, CZE of the pure PLAP revealed a cluster of peaks, which probably reflects the presence of various glycoforms and/or oligomers. The same analytical approach was used to characterize commercially available PLAP. This sample showed a moderate specific activity (15 U/mg) and appeared to be highly impure containing various other proteins.     

磁化水浸种对西瓜种子萌发及幼苗生理的影响

用不同强度的磁化水(0．05T、0．08T、0．10T、0．15T)浸种西瓜种子研究磁化水对西瓜种子萌发及幼苗生理的影响,结果表明：与对照相比,各种强度磁化水浸种都能提高种子的发芽率、发芽势、发芽指数和幼苗的抗坏血酸含量,其中0．10T磁化水处理后西瓜种子发芽率、发芽势提高幅度最大;对抗坏血酸含量的影响以0．08T处理最明显;经过不同强度磁化水处理后幼苗中的过氧化物酶同工酶酶、酯酶同工酶活性得到不同强度的增强,不同品种表现不同。实验结果说明磁化水处理后能明显提高西瓜种子的萌发能力,并能提高幼苗的各种生理活性。     

The Interrelationship of Canavanine and Urease in Seeds of the Lotoideae

Seeds of 29 species of canavanine-synthesizing legumes wereassayed for their urease and canavanine production. All of theexamined species possess detectable urease activity. In general,the leguininous seeds richest in urease also had the most canavanine. The urease content of the jack bean seed, Canavalia ensiformis(L.) DC., is formidable and disproportionally greater than thequantity of stored canavanine. The massive urease content ofthe seed cannot be rationalized by the magnitude of the canavaninepool. Analysis of eight species of Mucuna demonstrated that canavanineis not stored in the seeds of these plants. Mucuna species donot appear to be unique in having seeds that do not concurrentlyproduce urease and canavanine.     

Renaturation, purification and characterization of streptokinase expressed as inclusion body in recombinant E. coli

Recombinant protein purification is facilitated using high expression systems which produce larger quantities of streptokinase protein as inclusion bodies. As the accumulation of active streptokinase is toxic to the host cells, we have optimized the conditions to achieve large amounts of streptokinase in the form of inclusion bodies. The solubility and yield of pure protein are highly dependent on various combinations of chemical additives, ionic and non-ionic detergents and salts, with solubilizing agents followed by refolding of denatured protein into active form. As the extraction of the purified streptokinase from inclusion bodies requires denaturation and a subsequent refolding step, careful balancing steps were needed to develop under different controlled conditions. Here the purified fragments of refolded proteins were screened to select the conditions that yield the active streptokinase having native conformation. The maximum specific activity of the purified streptokinase was achieved by these methods. The refolded recombinant streptokinase was analyzed by RP-HPLC showing a purity of 99%. Size exclusion chromatography profile shows that there are minimal aggregates in the active streptokinase protein and the percentage of renaturation is around 99%.     

幽门螺旋杆菌重组尿素酶B亚基的纯化及其免疫学性质的研究

目的:幽门螺旋杆菌(Hp)尿素酶是Hp重要的定制因子和致病因子,Hp尿素酶活性位点位于Hp尿素酶B亚基(UreB),研发基于UreB的Hp疫苗是一种很有前景的防治Hp感染的策略。方法:主要利用基因克隆技术从幽门螺旋杆菌标准菌株SS1(Hp SS1)获得Hp尿素酶B亚基基因,并构建含有重组Hp尿素酶B亚基(rUreB)基因的重组表达载体pET-rUreB及其重组菌株;重组菌株经蛋白表达和优化后,利用Ni-NTP镍离子亲和层析和DEAE Sepharose FF阴离子交换层析纯化重组尿素酶B亚基(rUreB),并进一步通过腹腔注射免疫BALB/c小鼠,研究rUreB的免疫学性质。结果:通过基因克隆技术成功获得了Hp尿素酶B亚基基因,并成功构建了重组表达载体pET-rUreB及其重组菌株BL21(DE3)/pET-rUreB,经蛋白表达优化及纯化,可获得高纯度(96.5%)的重组蛋白rUreB。重组蛋白rUreB辅以弗氏佐剂腹腔注射免疫BALB/c小鼠,经间接ELISA鉴定小鼠能够产生针对天然Hp尿素酶和UreB的高滴度特异性抗体,且能够显著性抑制Hp尿素酶的活性。结论:重组Hp尿素酶B亚基能够在大肠杆菌表达系统中获得较高水平的表达,具有较高的免疫学特异性,其抗体能够有效抑制Hp尿素酶活性。为研究基于尿素酶的防治Hp感染的Hp疫苗奠定了一定的实验基础。     

Induction of urease by urea analogues in Evernia prunastri thallus

Urease activity in Evernia prunastri (L.) Ach. thallus is induced by incubation of lichen samples on 20 m M N,N-dimethylformamide and 20 m M N-formylurea or 40 m M thiourea although, in these two last cases, activity subsequently decreases again. The induction of enzyme activity is repressed by including 40 μ M cycloheximide in the medium. Filtration through Sepharose 6B of cell-free extracts from thalli incubated on 20 m M N,N-dimethylformamide shows a main peak of urease activity which has a molecular weight of about 560000 dalton. However, those extracts from thalli floated on 20 m M N-formylurea and 40 m M thiourea show several peaks of similar enzyme activity, which have molecular weights of about 1 100000, 670000, 260000 and 140000 dalton and 1 100000, 670000 and 140000 dalton respectively.
A time-course of urease activity could be related to the accumulation of lichen phenols in the thallus for samples incubated on N,N-dimethylformamide and thiourea.     

Isolation, Purification and Partial Characterisation of Urease from Seeds of Water Melon (Citrullus vulgaris)

Urease from seeds of water melon was purified to apparent homogeniety upto a sp act of 3750 units/mg protein with 31% recovery. Enzyme showed single protein band on native PAGE by urease specific staining. The mol wt of the enzyme was 4,70,000 and the preparation was free from bound nucleotides (A₂₈₀/A₂₆₀=1.14). The enzyme exhibited maximum activity in 50 mM Tris-acetate buffer (pH 8.5). The Km for urease was 8 mM. The enzyme was not inhibited by 25 mM of EDTA in 50 mM Tris-acetate buffer (pH 8.0 and 8.5).     

Purification of human erythrocyte glucose 6-phosphate dehydrogenase and glutathione reductase enzymes using 2',5'-ADP Sepharose 4B affinity column material in single chromatographic step

The enzymes of glucose 6-phosphate dehydrogenase and glutathione reductase were purified from human erythrocytes in one chromatographic step consisting of the use of the commercially available resin 2',5'-ADP Sepharose 4B by using different washing buffers. Ammonium sulfate (30-70%) precipitation was performed on the hemolysate before applying to the affinity column. Using this procedure, G6PG, having the specific activity of 22.9 EU/mg proteins, was purified with a yield of 43% and 9150-fold; GR, having the specific activity of 20.7 EU/mg proteins, was purified with a yield of 26% and 8600-fold. The purity of the enzymes was checked on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and each purified enzyme showed a single band on the gel. This procedure has advantages of preventing of enzyme denaturation, short experimental duration, and use of less chemical materials for purification of the enzymes.     

Purification of urease from Ureaplasma urealyticum

We have purified urease from the Mollicutes, Ureaplasma urealyticum, using high performance liquid chromatography methods and DEAE-Sephadex chromatography. While only small amounts of material could be utilized in these methods, urease was purified at least 180-fold, yield a major band on SDS-PAGE of 66,000 daltons, a minor band of 64,000 daltons, and several faint bands of lower molecular mass. These results suggest that the 380,000 dalton intact urease is a pentamer or hexamer of these two larger subunits. The highly purified urease from DEAE-Sephadex retained full activity for at least 20 days at 4 degrees C in sodium phosphate buffer (pH 7.2) with 1% bovine serum albumin. The estimated specific activity of the DEAE peak fractions, 180 IU/micrograms, is at least 90-fold greater than that of jack bean urease.     

Effects of temperature on the specific dynamic action of the southern catfish, Silurus meridionalis

Specific dynamic action (SDA), the energy expended on all physiological processes that is associated with meal digestion and assimilation, is strongly affected by temperature. We assessed the effects of temperature on the postprandial metabolic response and calculated SDA of the southern catfish, Silurus meridionalis. The fish was fed with experimental diets at a meal size of 4% body mass, and by using an 8-chamber, continuous-flow respirometer the oxygen consumption rate was determined at a 2 h interval until the postprandial oxygen consumption rate returning to the preprandial level, at four different temperatures. The energy expended on SDA (SDA(E)) were 2.71, 3.07, 3.16, and 3.62 kJ, the SDA(coefficients) (energy expended on SDA quantified as a percentage of the digestible energy content of the meal) were 7.70, 9.44, 10.36, and 11.12%, and the peak metabolic rates (R(peak)) of SDA were 3.48, 4.31, 5.96, and 7.30 mg O2 h(-1), at 17.5, 22.5, 27.5, and 32.5 degrees C respectively. The relationships between those parameters and temperature were: SDA(E)=1.74+0.0559T (n=26, r(2)=0.676), SDA(coefficient)=4.10+0.223T (n=26, r(2)=0.726), and R(peak)=-1.34+0.264T (n=26, r(2)=0.896). The SDA durations showed a slow-fast-slow tendency of decrease with increasing temperature, and were 88.00, 85.71, 67.71, and 66.50 h at 17.5, 22.5, 27.5 and 32.5 degrees C respectively. Two separate peaks appeared during the SDA response at 17.5 degrees C, and it might be due to a rapid startup of the mechanical process with a lag of the biochemical process, which suggested that the peaks of "mechanical component" and "biochemical component" of SDA might be separated when temperature was low enough.     

Effects of temperature on the specific dynamic action of the southern catfish, Silurus meridionalis.

Specific dynamic action (SDA), the energy expended on all physiological processes that is associated with meal digestion and assimilation, is strongly affected by temperature. We assessed the effects of temperature on the postprandial metabolic response and calculated SDA of the southern catfish, Silurus meridionalis. The fish was fed with experimental diets at a meal size of 4% body mass, and by using an 8-chamber, continuous-flow respirometer the oxygen consumption rate was determined at a 2 h interval until the postprandial oxygen consumption rate returning to the preprandial level, at four different temperatures. The energy expended on SDA (SDA(E)) were 2.71, 3.07, 3.16, and 3.62 kJ, the SDA(coefficients) (energy expended on SDA quantified as a percentage of the digestible energy content of the meal) were 7.70, 9.44, 10.36, and 11.12%, and the peak metabolic rates (R(peak)) of SDA were 3.48, 4.31, 5.96, and 7.30 mg O2 h(-1), at 17.5, 22.5, 27.5, and 32.5 degrees C respectively. The relationships between those parameters and temperature were: SDA(E)=1.74+0.0559T (n=26, r(2)=0.676), SDA(coefficient)=4.10+0.223T (n=26, r(2)=0.726), and R(peak)=-1.34+0.264T (n=26, r(2)=0.896). The SDA durations showed a slow-fast-slow tendency of decrease with increasing temperature, and were 88.00, 85.71, 67.71, and 66.50 h at 17.5, 22.5, 27.5 and 32.5 degrees C respectively. Two separate peaks appeared during the SDA response at 17.5 degrees C, and it might be due to a rapid startup of the mechanical process with a lag of the biochemical process, which suggested that the peaks of "mechanical component" and "biochemical component" of SDA might be separated when temperature was low enough.     

Human renal renin. Complete purification and characterization

Complete purification of human renin from noncancerous, autopsied kidneys is reported. A 480,000-fold purification was achieved to yield renin with a specific activity of 950 Goldblatt units/mg. This preparation satisfied multiple criteria of purity as tested by polyacrylamide gel electrophoresis, isoelectric focusing, specific activity, analytical ultracentrifugation, and immunodouble diffusion. The molecular weight of the pure enzyme determined by sedimentation equilibrium is 40,000. The apparent molecular weight estimated by gel filtration is 41,000. The enzyme has an isoelectric point of pH 5.7. Human renin shows an affinity for concanavalin A, suggesting the presence of carbohydrates. These properties and the amino acid composition of human renin are different from those of renin obtained from other mammalian species. Human renin antibodies prepared with the pure enzyme preparation showed negligible cross-reactivity with renin from other mammalian species. The activity with homologous human renin substrate has a pH optimum of 6, whereas with substrates from other mammalian species the optima were in higher or lower pH ranges.