Isolation of chitin synthetase from Saccharomyces cerevisiae. Purification of an enzyme by entrapment in the reaction product

Chitin synthetase, in the zymogen form, was extracted with digitonin from a particulate fraction from Saccharomyces cerevisiae and converted into active form by treatment with immobilized trypsin. When the activated enzyme was incubated with UDP-GlcNAc and other components of an assay mixture, a chitin precipitate formed, trapping a large portion of the synthetase. The enzyme was easily extracted frm the chitin gel with a recovery of approximately 50% and an enrichment of approximately 100-fold. Further purification was obtained by repeating the chitin step. After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the purified synthetase showed a major band corresponding to Mr 63,000, a weaker band at Mr 74,000, and some other minor bands. Under nondenaturing conditions, an Mr of 570,000 was calculated for the enzyme from Stokes radius and sedimentation coefficient determinations. After electrophoresis in a nondenaturing gel and incubation with the components of the standard assay, chitin was formed and precipitated in the gel, yielding an opaque band. Soluble oligosaccharides were not precursors for insoluble chitin, suggesting that synthesis of chitin chains takes place by a processive mechanism. N-Acetylglucosamine stimulated the purified synthetase only slightly and did not participate as a primer in the reaction. The same chain length, somewhat more than 100 units of GlcNAc, was determined in samples of chitin that had been synthesized either in vivo, or with a membrane preparation or with purified synthetase. These results suggest that chitin synthetase itself is capable both of initiating chitin chains without a primer and of determining their length.     

Immobilization of enzymes in porous supports: Effects of support-enzyme solution contacting

Proteins have been immobilized in porous support particles held in a fixed-bed reactor through which protein solution is continuously circulated. Changing the recirculation flow rate alters the observed immobilization kinetics and the maximum enzyme loading which can be achieved for glucose oxidase and glucoamylase on carbodiimide-treated activated carbon and for glucoamylase immobilized on CNBr-Sepharose 4B. Direct microscopic examination of FITC-labelled protein in sectioned Sepharose particles and indirect activity-loading studies with activated carbon-enzyme conjugates all indicate that immobilized enzyme is increasingly localized near the outer surface of the support particles at larger recirculation flow rates. Restricted diffusion of enzymes may be implicated in this phenomenon. These contacting effects may be significant considerations in the scaleup of processes for protein impregnation in porous supports, since apparent activity and stability of the final preparation depend on internal protein distribution.     

Light saturation curves and quantum yields in reaction centers from photosynthetic bacteria.

Reaction centers isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26 mutant were irradiated with laser pulses of variable energy and the amount of photooxidation of the primary electron donor bacteriochlorophyll was measured. The resultant light saturation curve fits an exponential function and not a hyperbolic or hyperbolic tangent function. Analysis using either a Poisson statistical model or a simple kinetic model predicts an exponential light saturation curve in the limit where the light pulse is long relative to any transient intermediate states. The absolute quantum yield of photochemistry was found to be 0.98, utilizing the entire light saturation curve. Distortions from the simple exponential light saturation behavior are predicted when very short laser pulses are used.     

Structural characterization of virion proteins and genomic RNA of human parainfluenza virus 3

The virion proteins and genomic RNA of human parainfluenza virus 3 have been characterized. The virion contains seven major and two minor proteins. Three proteins of 195 X 10(3) molecular weight (195K), 87K, and 67K are associated with the nucleocapsid of the virion and have been designated L, P, and NP, respectively. Three proteins can be labeled with [14C]glucosamine and have molecular weights of 69K, 60K, and 46K. We have designated these proteins as HN, F0, and F1, respectively. HN protein has interchain disulfide bonds, but does not participate in disulfide bonding to form homomultimeric forms. F1 appears to be derived from a complex, F1,2, that has an electrophoretic mobility similar to that of F0 under nonreducing conditions. A protein of 35K is associated with the envelope components of the virion and aggregates under low-salt conditions; this protein has been designated M. The genome of human parainfluenza virus 3 is a linear RNA molecule with a molecular weight of approximately 4.6 X 10(6).     

Structure of defective DNA molecules in Epstein-Barr virus preparations from P3HR-1 cells.

Epstein-Barr virus (EBV), isolated from P3HR-1 cells, induces early antigen and viral capsid antigen upon infection of human B-lymphoblasts. The strong early antigen- and viral capsid antigen-inducing activity is only observed in P3HR-1 virus preparations harboring particles with defective genomes, suggesting that this biological activity is directly associated with the defective DNA population. After infection of EBV genome-carrying Raji or EBV genome-negative BJAB cells, defective genomes of P3HR-1 EBV DNA are replicated in excess, depending on the multiplicity of infecting EBV particles. Hybridization of the DNA from such infected cells with 32P-labeled EBV DNA after HindIII cleavage reveals six hypermolar fragments. Mapping of these fragments shows that they form one defective genome unit containing four nonadjacent regions (alpha, beta, gamma, and delta) of the nondefective P3HR-1 EBV DNA. Two of the segments (alpha and beta) contain ca. 17 and 13 megadaltons, respectively, from the terminal regions of the P3HR-1 genome, whereas the two smaller segments (gamma and delta) contain ca. 3.7 and 3.0 megadaltons, respectively, originating from the central portion of the genome. In the defective molecule, the regions gamma and delta are present in the opposite orientation compared with nondefective P3HR-1 EBV DNA. Tandem concatemers are formed by fusion of the alpha and beta regions. Our model suggests that tandem concatemers of three defective genome units can be packaged into virions in P3HR-1 cells.     

Characterization of newly isolated monoclonal antibodies against MHC of a Japanese wild mouse

We have already developed nine B10.MOL congenic strains carrying H-2 haplotypes derived from Japanese wild mice, Mus musculus molossinus, with the C57BL/10 genetic background. To obtain monoclonal antibodies against the H-2 antigen of the Japanese wild mouse, we carried out cell fusion using spleen cells from the animal immunized with one of the B10.MOL strains, B10.MOL-SGR (H-2 ^wm7). As a result, 19 hybridomas producing monoclonal antibodies were produced. Analysis with the intro-H-2 recombinants derived from B10.MOL-SGR indicated that 8 of them reacted with the class I and II with the class II molecule. The class I antibodies were tested for their cross -reactivities on wild mice and on the panels of standard inbred and B10.MOL strains. Most of the antibodies reacted with both the Japanese wild mice and the other subspecies, including standard inbred, while two antibodies highly specific for the donor H-2K region reacted with only three wild-derived mice, two M. m. molossinus from Anj o and Shizuoka, Japan, and one M. m. domesticus from Pigeon, Canada. In addition, all of the other four antibodies reactive with the K antigen of B10.MOL-SGR also reacted with the same three wild mice. The wild mice belonging to different subspecies might share very similar H-2K antigenic determinants in spite of their genetic and geographical remoteness.     

Effect of gamma-ray irradiation on alcohol production from corn

Cracked corn was irradiated with gamma rays at 0-100 Mrad and the effects of the irradiation on sugar yield, susceptibility to enzymatic hydrolysis of starch, yeast growth, and alcohol production were studied. Gamma irradiation at 50 Mrad or greater produced a considerable amount of reducing sugar but little glucose. At lower dosages, gamma irradiation significantly increased the susceptibility of corn starch to enzymatic hydrolysis, but dosages of 50 Mrad or greater decomposed the starch molecules as indicated by the reduction in iodine uptake. About 12.5% reducing sugar was produced by amylase treatment of uncooked, irradiated corn. This amount exceeded the level of sugar produced from cooked (gelatinized) corn by the same enzyme treatment. The yeast numbers in submerged cultivation were lower on a corn substrate that was irradiated at 50 Mrad or greater compared to that on an unirradiated control. About the same level of alcohol was produced on uncooked, irradiated (10(5)-10(6) rad) corn as from cooked (121 degrees C for 30 min) corn. Therefore, the conventional cooking process for gelatinization of starch prior to its saccharification can be eliminated by irradiation. Irradiation also eliminated the necessity of sterilization of the medium and reduced the viscosity of high levels of substrate in the fermentation broth.     

Immobilization of glucoamylase and glucose oxidase in activated carbon: effects of particle size and immobilization conditions on enzyme activity and effectiveness

Glucoamylase and glucose oxidase have been immobilized on carbodiimide-treated activated carbon particles of various sizes. Loading data indicate nonuniform distribution of immobilized enzyme within the porous support particles. Catalysts with different enzyme loading and overall activities have been prepared by varying enzyme concentration in the immobilizing solution. Analysis of these results by a new method based entirely upon experimentally observable catalyst properties indicates that intrinsic catalytic activity is reduced by immobilization of both enzymes. Immobilized glucoamylase intrinsic activity decreases with increasing enzyme loading, and similar behavior is suggested by immobilized glucose oxidase data analysis. The overall activity data interpretation method should prove useful in other immobilized enzyme characterization research, especially in situations where the intraparticle distribution of immobilized enzyme is nonuniform and unknown.     

Alterations in the Structure of the Oligosaccharide of Vesicular Stomatitis Virus G Protein by Swainsonine

Swainsonine, an inhibitor of glycoprotein processing, inhibits the formation of the normal oligosaccharide chain of the G protein of vesicular stomatitis virus. Thus, when vesicular stomatitis virus was grown in baby hamster kidney cells in the presence of swainsonine (15 to 500 ng/ml) and labeled with [2-(3)H]mannose, the oligosaccharide portion of the G protein was completely susceptible to the action of endoglucosaminidase H. However, the normal viral glycoprotein is not susceptible to this enzyme. Various enzymatic treatments and methylation studies of the mannose-labeled oligosaccharides suggest that swainsonine causes the formation of a hybrid-type oligosaccharide having an oligomannosyl core (Man(5)GlcNAc(2)-Asn) characteristic of neutral oligosaccharides plus the branch structure (NeuNAc-Gal-GlcNAc) characteristic of the complex oligosaccharides. A structure for this hybrid oligosaccharide is proposed. Swainsonine had no effect on the incorporation of [(14)C]leucine into viral proteins, nor did it change the number of PFU produced in these cultures. It did, however, slightly decrease the incorporation of [(3)H]glucosamine and increase the incorporation of [(3)H]mannose. Vesicular stomatitis virus raised in the presence of swainsonine bound much more tightly to columns of concanavalin A-Sepharose than did control virus. Swainsonine had to be added within the first 4 or 5 h of virus infection to be effective. Thus, when 100 ng of the alkaloid per ml was added at any time within the first 3 h of infection, essentially all of the glycoprotein was susceptible to digestion by endoglucosaminidase H. However, when swainsonine was added 4 h after the start of infection, 30% of the glycopeptides became resistant to endoglucosaminidase H; at 5 h, 70% were resistant. The effect of swainsonine was reversible since removal of the alkaloid allowed the cells to form the normal complex glycoproteins. However, the time of removal was critical in terms of oligosaccharide structure.