香菇和侧耳属间原生质体的电融合研究

从培养在液体培养基中的香菇、美味侧耳和平菇的单核菌丝用酶法分离了原生质体。施加0.5MHz、500PV/cm的正弦波和μs、6000PV/cm方形脉冲的电场诱导下使其电融合。电融合后的融合子和原生质体在固体培养基上植板培养成菌落。在显微镜下检查融合子菌株菌丝的锁状联合选出从融合子长成的菌株。香菇和美味侧耳的融合菌株产生频率为61.53%,香菇和平菇的融合菌株产生频率为32.58%。根据融合菌株与亲本的拮抗作用和他们的过氧化物同工酶和酯酶同工酶的电泳酶谱与其亲本酶谱的不同,证实这些融合菌株是从融合的异核体生长成的。同时讨论了电融合方法和结果。     

Characterization of subunit structural alterations which occur during purification of nitrate reductase from Escherichia coli

Nitrate reductase from Escherichia coli, purified to homogeneity after release from membranes by deoxycholate treatment, was composed of two subunits of 155,000 (α) and 58,000 (β) daltons and contained no cytochrome b₁. Analysis of fractions at different stages of purification by gel electrophoresis and immunoprecipitation revealed that during the early steps of the purification cytochrome b₁ dissociated from the enzyme and the β subunit was altered in size as determined by sodium dodecyl sulfate-gel electrophoresis. Analysis of the peptide patterns obtained by partial proteolysis of isolated α and β subunits established that these subunits are composed of distinct sequences and ruled out a precursor-product relationship between the two subunits. The β subunit was altered during the purification by loss of a 2000-dalton fragment, apparently from its carboxyl terminus. The protease inhibitor tosyllysine chloromethylketone protected nitrate reductase from more extensive degradation by endogenous proteases during the purification but did not prevent the removal of the 2000-dalton fragment. This carboxyl terminal fragment was part of a 15,000-dalton sequence which was removed by trypsin and which was required for the self-associating character of the unmodified enzyme monomers. From the structural changes which occurred during the purification procedure, it is proposed that the carboxyl terminal segment of the β subunit is involved in the binding of nitrate reductase to cytochrome b₁ and its association with the membrane.     

Sequence relationship of glycosylated and unglycosylated gag polyproteins of Moloney murine leukemia virus.

Both glycosylated and unglycosylated polyproteins coded by the gag gene are produced in cells infected with Moloney murine leukemia virus. GpP80gag is a glycosylated precursor of a larger gag glycoprotein exported to the cell surface, whereas Pr65gag is an unglycosylated precursor of the virion internal structural proteins. GpP80gag contains not only carbohydrate, but also additional polypeptide sequences not found in Pr65gag. In the experiment reported here, we localized the differences between GpP80gag and Pr65gag with respect to the domains of the individual gag proteins. This was done by comparison of partial proteolytic cleavage fragments from Pr65gag, from GpP80gag, and from the unglycosylated form of GpP80gag (P75gag) which had been immunoprecipitated by antisera specific for gag proteins p30, p15, and p10. We conclude that the additional polypeptide sequences in GpP80gag are located at or very near the amino terminus of the polyprotein. The carbohydrate in GpP80gag is attached to polypeptide sequences held in common between GpP80gag and Pr65gag.     

Bubble flow in the downflow section of an airlift tower

Characterization of the two-phase flow in the downflow section of the airlift tower is necessary for accurate modeling of the airlift tower. A Split-cylinder airlift tower was investigated for superficial gas velocities ranging from 0.0683 to 0.3315 m/sec for an air–water system. Statistical cross-covariance techniques were used to yield velocities, void fractions, and flow rates corresponding to upward and downward components of bubble flow in the downflow section of the airlift tower. From these results the fraction of incoming air entrained in the downflow section was determined as a function of superficial gas velocity and position.     

Comparison of adipose glycerol kinase of hyperglycemic obese mice and lean litter-mates

Summary Glycerol kinase activity found in the epididymal adipose tissue of lean litter-mates of hyperglycemic obese mice exhibits two distinct Km values. The Km(s) obtained graphically by a Hoftsee plot are 40 and 637 m. The glycerol kinase of obese mice showed 29 times more total activity per fat pad or 9 times more activity per mg of protein as compared to that of the lean controls. The increased glycerol kinase activity found in the obese mice predominantly exhibited low Km value. Increase in the activity with the high Km value was minimal. The apparent molecular weight of adipose glycerol kinase is approximately 60,000–65,000. A higher activity of glycerol kinase in the epididymal adipose tissue of the obese mice (ob/ob) has been reported by Treble and Mayer ¹. The significance of this increased enzyme in obesity² has been investigated by many investigators^3–6, using either whole epididymal fat pad or isolated fat cells as the source of glycerol kinase. However, little is known regarding the properties of this enzyme, and even the characterization of the liver glycerol kinase is not yet complete^{7, 8}.We have recently shown that adipose glycerol kinase of Sprague-Dawly rat and of Swiss mice exhibit two Km values, and the apparent molecular weight is approximately 55,000–60,000⁹. The present study was undertaken in order to establish whether the enzyme in ob/+ mice also exhibits two Km values and if so, whether the activity associated with both Km(s) is increased. We found that the specific conditions for enzyme activity determination and the basis on which the activity is expressed are quantitatively important in clarifying the difference in glycerol kinase activity of ob/ob and ob/+ mice. The increased activity in ob/ob mice is predominantly the low Km enzyme, and the apparent molecular weight is approximately 60,000–65,000.Supported in part by the Juvenile Diabetes Foundation. We thank Mr. CHARLES ROSENTHAL for technical assistance.     

Nodule infection by bean yellow mosaic virus in Phaseolus vulgaris.

Infection of root nodules of beans, Phaseolus vulgaris L., by bean yellow mosaic virus (BYMV) and the effect of the disease on the specific activity of the nodule are reported. Infectivity and serological microprecipitin assays with two sources of BYMV antiserum demonstrated that nodules from bean plants whose leaves had been inoculated with BYMV contain BYMV antigen. The disease reduced the fresh weights of tops, roots, and root nodules and induced premature nodule decay and/or nodule drop. The disease also reduced leghemoglobin content, on a plant weight basis, and N2 fixation rate, on an individual plant basis, as measured by the acetylene reduction assay. The increased leghemoglobin content per gram-nodule in BYMV-infected nodules relative to healthy nodules might be associated with multiplication of the virus in the nodule and/or unknown cellular effects derived from the BYMV-Rhizobium interaction.     

Effects of temperature on diphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart. Aspects of the kinetics, stability, and quarternary structure of the enzyme.

A temperature-dependent conformational change of the active DPN-linked isocitrate dehydrogenase was observed. When initial reaction kinetic data were examined between 35 and 5 degrees, the Hill number (n) varied from 2 at higher to n approaching unity at lower temperatures, with an inflection point at 17 degrees. The presence of manganous isocitrate in the incubation media shifted the transition temperature for enzyme inactivation by 5,5'-dithiobis(2-nitrobenzoate) from 8-16 degrees. These temperature-dependent transitions were paralleled by progressive changes in sedimentation velocities from s20, w of 10.4 at 25 degrees to 7.3 at 10 degrees as measured by active band centrifugation. The linear Arrhenius plot for apparent V max and the constancy of S0.5 for the substrate manganous isocitrate between 35 and 5 degrees suggest that this temperature-dependent conformational change may not be solely related to manganous isocitrate. Further indications of equilibria between different species of enzyme in solution and effects of substrates and cofactors on conformation came from studies of specific activity of enzyme diluted into buffers at 3 and 25 degrees. Dilution to concentrations between 10 and 25 mum enzyme resulted in relatively rapid protein concentration-dependent inactivation which could be prevented and fully reversed by manganous isocitrate. No further substantial inactivation was found subsequent to this phase at 25 degrees. Lowering the temperature of the dilution buffer to 3 degrees favored formation of enzyme species exhibiting a further time and pH-dependent loss of activity which became independent of protein concentration below 7 mum enzyme. The rate of cold inactivation was reduced by raising the ionic strength of the buffer and its progress could be arrested by manganous isocitrate; however, the substrate did not restore the original activity.