Partial purification and characterisation of Bfi57I and Bfi89I, restriction endonucleases from different strains of Butyrivibrio fibrisolvens

The Escherichia coli nucleoid-associated DNA-binding proteins HU and IHF are required for numerous biological processes, including phage growth (e.g., lambda, phi 80, Mu and f1) and DNA replication. Here, we show that growth of T4 phage is inhibited both in hupA hupB and himA himD double mutants. The growth profile of triple mutants (hupA hupB himA and hupA hupB himD) suggests that HimD subunits can form homodimers, which are functionally competent for supporting in vivo growth of phage T4.     

Partial purification and characterisation of sugarcane neutral invertase

Sugarcane neutral invertase (SNI) has been partially purified from mature sugarcane stem tissue to remove any potential competing activity. The enzyme is non-glycosylated and exhibits catalytic activity as a monomer, dimer and tetramer, most of the activity elutes as a monomer of native M(r) ca 60 k. The enzyme displays typical hyperbolic saturation kinetics for Suc hydrolysis. It has a K(m) of 9.8 mM for Suc and a pH optimum of 7.2. An Arrhenius plot shows the energy of activation of the enzyme for Suc to be 62.5 kJ mol-1 below 30 degrees and -11.6 kJ mol-1 above 30 degrees. SNI is inhibited by its products, with Fru being a more effective inhibitor than Glc. SNI is significantly inhibited by HgCl2, AgNO3, ZnCl2, CuSO4 and CoCl2 but not by CaCl2, MgCl2 or MnCl2. SNI showed no significant hydrolysis of cellobiose or trehalose.     

Partial purification and kinetic studies of exocellular proteinase from Trichophyton mentagrophytes var. erinacei

The dermatophyte Trichophyton mentagrophytes var. erinacei isolated from a patient with tinea cruris was cultured in peptone-glucose broth from which an exocellular proteinase was obtained. The enzyme was partly purified by Sephadex G-100 gel filtration. Its molecular weight was determined to be 33,000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH was 8.5, the optimal temperature 35 degrees C. The proteolytic activity was specifically increased against casein and inhibited by phenylmethylsulphonyl fluoride. The enzyme was identified as alkaline serine proteinase.     

Studies of nicotinic acetylcholine receptor protein from rat brain. II. Partial purification

The pharmacological specificity of the binding of 125I-labeled alpha-bungarotoxin to a 1% Emulphogene BC-720 extract of a rat brain particulate fraction has been investigated. The extract contains a component which possesses the binding characteristics of a nicotinic acetylcholine receptor protein. The crude soluble acetylcholine receptor protein was purified by affinity chromatography utilizing the alpha-neurotoxin of Naja naja siamensis as ligand and 1.0 M carbamylcholine chloride as eluant. A single, batch-wise, affinity chromatography procedure yields an average purification of 510-fold. When this purified material is treated a second time by affinity chromatography, purification as high as 12600-fold has been obtained. Binding of 125I-labeled alpha-bungarotoxin to this purified acetylcholine receptor protein is saturable with a Kd of 1 - 10(-8) M. Nicotine and acetylcholine iodide at concentrations of 10(-5) M inhibit 125I-labeled toxin-acetylcholine receptor protein complex formation by 41 and 61% respectively. At 10(-4) M, carbamylcholine chloride and (+)-tubocurarine chloride give respectively 52 and 82% inhibition. Eserine sulfate and atropine sulfate have no effect on complex formation at a concentration of 10(-4) M. These data support the isolation of a partially purified nicotinic acetylcholine receptor protein.     

Partial purification and some properties of human liver alkaline phosphatase

1. Alkaline phosphatase (EC 3.1.3.1) from human liver was solubilized from the homogenate using 0.2% Triton X-100 containing 0.2 M lithium 3,5-diiodosalicylate, and the pellet obtained was resolubilized with 20% n-butanol. The procedure resulting in 3842-fold purification included acetone fractionation, ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration, hydroxyapatite gel chromatography and further concanavalin A/Sepharose 4B affinity chromatography. 2. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 8.6, and exhibited one-seventh of the alkaline p-nitrophenylphosphatase activity in the hepatic enzyme preparation contains of the alkaline pyrophosphatase activity. 3. The highly purified enzyme was a sialic-acid containing glycoprotein. 4. Sialidase-treated hepatic enzyme clearly presented the phenomenon of delayed mobility, and the delayed enzyme fraction stained more strongly than that of non-treated hepatic alkaline phosphatase. 5. In order to investigate the role of the carbohydrate region(s) of the hepatic alkaline phosphatase molecule on substrate binding, the effect of sialidase treatment on the rate of substrate inhibition of alkaline phosphatase was studied. In the case of hepatic enzyme without sialidase, substrate inhibition of alkaline phosphatase activity was clearly shown, while in the case of the hepatic enzyme with sialidase, there was hardly any substrate inhibition in the range of 1-8 mM p-nitrophenylphosphate.     

The immunosuppressive action of different fractions of Salmonella typhimurium lipopolysaccharide

A non-loading pneumocalorimetric mode of quantitative assessment of physical performance (PP) has been developed in a series of 25 essentially healthy subjects. Based on the correlation-and-regression analysis a formula for PP was found out to be PP = 59.9 x MCC + 33, were MCC is maximum caloric capacity. PP quantitative assessment was proved to be effective in patients presenting with cardiopulmonary problems.     

Partial purification of embryotrophic factors from human oviductal cells

Embryotrophic factors from human oviductal cells were partially purified by liquid chromatographic methods. The conditioned medium from human oviductal cell culture was fractionated successively by concanavalin A (Con-A) affinity chromatography, ion-exchange chromatography and gel filtration. The presence of the embryotrophic activity in the eluates was determined by the stimulatory effects on the development of mouse embryos in vitro. The fraction that did not bind to the lectin Con-A possessed no embryotrophic activity. Ion-exchange chromatography separated the glycoproteins that bound to Con-A into five fractions. Three of them significantly enhanced blastulation as well as conceptus formation. Gel filtration further separated these embryotrophic fractions into five fractions. Three of them with molecular weights of 154 +/- 1, 164 +/- 0.2 and 207 +/- 0.3 kDa significantly stimulated blastulation of mouse embryos. The results of this study demonstrated that several embryotrophic factors with different biochemical properties contributed to the embryotrophic effect of the human oviductal cell/mouse embryo co-culture system.     

Partial purification and some properties of pyroglutamyl peptidase from Enterococcus faecalis

Pyroglutamyl peptidase was partially purified from Enterococcus faecalis ATCC 19433 by anion-exchange chromatography, gel filtration and salting out after lysis of cell walls with N-acetylmuramidase. Pyroglutamyl peptidase was purified 46-fold with a yield of about 2% based on the total activity of the crude extract. The molecular mass of the bacterial enzyme was estimated to be about 82 kD by gel filtration. The pl of the enzyme was 4.2 and the optimum pH and temperatures for the reaction were 7.2-7.5 and 35-45 degrees C, respectively. The enzyme was relatively stable below 45 degrees C, but almost all the activity was lost after heat-treatment at 55 degrees C for 15 min. The apparent K(m) value for pyroglutamyl-beta-naphthylamide was 0.55 mM. The bacterial enzyme specifically cleaved pyroglutamyl residues from the amino termini of pyroglutamyl compounds, such as Pyr-Asn-Gly, Pyr-His-Gly, Pyr-Ala-Glu, Pyr-Ala, neurotensin, thyrotropin-releasing hormone and bradykinin-potentiator B. However, human IgG and Bence Jones protein, which are high-molecular-mass proteins, were not hydrolysed. Neither derivatives of free amino acids, such as Ala-, Gly-, Pro- and Leu-p-nitroanilide, nor benzoyl-DL-Arg-p-nitroanilide were hydrolysed. The activity was strongly inhibited by thiol-blocking reagents (p-CMB, N-ethylmaleimide, monoiodoacetic acid). In addition, protease inhibitors, such as TLCK and PMSF, reduced the activity by 54 to 73%. These results suggest that the bacterial enzyme is a cysteine protease with sulphydryl residues in its active site and, possibly, histidine or serine residues near the active site.     

Partial purification of an endogenous inhibitor of muscarinic ligand binding

An endogenous inhibitory factor (EIF alpha) to the binding of a muscarinic antagonist, [3H]N-methyl scopolamine ([3H]NMS), has been partially (12-fold) purified from the soluble fraction of the ileal longitudinal muscle of guinea-pigs using a heat-treatment, isoelectric fractionation, and DEAE-column chromatography. The EIF alpha inhibited the [3H]NMS binding to the longitudinal muscle membrane with an IC50 of 53.6 micrograms/ml. This was about 230-fold potent than the non-specific inhibition of [3H]NMS binding by bovine serum albumin (BSA). Zn2+ (0.1 mM) almost completely blocked the inhibitory activity of EIF alpha, whereas such the effect of Zn2+ was not observed in the inhibition by BSA. These results suggest that EIF alpha inhibits the [3H]NMS binding to the muscarinic acetylcholine receptor in a different manner from non-specific interaction with the receptor.     

Liquid chromatographic parameters of groups of herbicidal active substances. I. Urea herbicides

Because of the polarity of herbicidal urea derivatives, liquid chromatography is the most suitable method for their determination. The separation of a number of active substances is described. A versatile applicable ternary solvent mixture is used as the mobile phase. Several columns are tested for their separation performances and their capability to retain active substances and some known degradation products. To fully utilize the sensitivity of the photometric detector the UV spectra of the tested compounds are determined.     

Partial purification and substrate specificity of flavin-containing monooxygenase from rat brain microsomes

Flavin-containing monooxygenase (FMO) was partially purified from rat brain microsomes through two successive chromatographies on columns of DEAE Sepharose and 2',5'-ADP Sepharose. The specific activity, benzydamine N-oxidation of partially purified brain FMO, was 122-fold higher than that of microsomes. A single band of 60 kDa was recognized by Western blotting analysis with anti-rat liver FMO. The Km value of brain FMO for thiourea was 4-fold lower, but that for cysteamine was 10-fold higher than that of liver FMO. The enzymatic activity for n-octylamine was detected in neither brain nor liver FMO. Kinetic analysis for neurotoxins also revealed that Km values of brain FMO for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1,2,3,4-tetrahydroisoquinoline (TIQ) and N-methyl TIQ (NMTIQ) were lower than those of liver FMO. These results indicate that rat brain FMO catalyzes several substrates of liver FMO involving neurotoxins, but it seems likely that the kinetic properties of brain FMO are somewhat different from those of liver FMO.     

Anatomy of motor learning. I. Frontal cortex and attention to action

We used positron emission tomography to study new learning and automatic performance in normal volunteers. Subjects learned sequences of eight finger movements by trial and error. In a previous experiment we showed that the prefrontal cortex was activated during new learning but not during during automatic performance. The aim of the present experiment was to see what areas could be reactivated if the subjects performed the prelearned sequence but were required to pay attention to what they were doing. Scans were carried out under four conditions. In the first the subjects performed a prelearned sequence of eight key presses; this sequence was learned before scanning and was practiced until it had become overlearned, so that the subjects were able to perform it automatically. In the second condition the subjects learned a new sequence during scanning. In a third condition the subjects performed the prelearned sequence, but they were required to attend to what they were doing; they were instructed to think about the next movement. The fourth condition was a baseline condition. As in the earlier study, the dorsal prefrontal cortex and anterior cingulate area 32 were activated during new learning, but not during automatic performance. The left dorsal prefrontal cortex and the right anterior cingulate cortex were reactivated when subjects paid attention to the performance of the prelearned sequence compared with automatic performance of the same task. It is suggested that the critical feature was that the subjects were required to attend to the preparation of their responses. However, the dorsal prefrontal cortex and the anterior cingulate cortex were activated more when the subjects learned a new sequence than they were when subjects simply paid attention to a prelearned sequence. New learning differs from the attention condition in that the subjects generated moves, monitored the outcomes, and remembered the responses that had been successful. All these are nonroutine operations to which the subjects must attend. Further analysis is needed to specify which are the nonroutine operations that require the involvement of the dorsal prefrontal and anterior cingulate cortex.     

Partial purification of the ovarian oviposition-inducing factor and estimation of its chemical nature

The ovarian ruptured follicles larger than 0.2 g. of wet weight were collected from laying hens and an ovarian oviposition-inducing factor (OOIF) was extracted from the follicles. The extract was chromatographed on a column of Sephadex G-75. Material from the active region for inducing premature oviposition was subjected to thioglycollate or trypsin treatment. It was found that oxytocic activity of this material was completely destroyed by thioglycollate buy not by trypsin, when tested on the contractility of the isolated hen's uterus. The OOIF was found to be diffusible through a cellophane bag. It is suggested that the OOIF may be a relatively small molecule have a disulfied bridge and it seems to be a hormone or hormone-like substance resembling the posterior pituitary hormones in the chemical nature.     

Influence of phenobarbital on the immunodepressive action of antitumor substances

In the experiments on mice, immunized by mutton erythrocytes, the authors have studied the effect of phenobarbital, in the doses activating drug-metabolizing hepatic systems, on immunodepressive action of imurane, 6-mercaptopurine and cyclophosphane. It has been found that phenobarbital eliminates the immunodepression and reduces considerably the toxicity of the mentioned cytostatics. Under these conditions imurane and 6-mercaptopurine in certain doses render an immunostimulative instead of immunodepressive effect.