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.     

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).     

表面活性剂和Ca~(++)对大麦根质膜透性的作用

阳离子型表面活性剂(CTMA)以低于CMC的浓度、非离子型表面活性剂(Triton-X-100)以高于CMC的浓度引起大麦离体根K~+及可溶性糖的外流,并有浓度效应。5℃时表面活性剂引起溶质外流,CTMA预处理10 min后,Ca~(++)无抑制作用。Ca~(++)与 Triton-X-100同时处理大麦根促进溶质外流。Mg~(++)、Mn~(++)对CTMA及Triton-X-100引起溶质外流的效应与Ca~(++)的相类似,但不如Ca~(++)有效。     

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.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells IV. Two Different Requirements for Protein Synthesis During Simian Virus 40 Deoxyribonucleic Acid Replication

The replication of simian virus 40 (SV40) deoxyribonucleic acid (DNA) was inhibited by 99% 2 hr after the addition of cycloheximide to SV40-infected primary African green monkey kidney cells. The levels of 25S (replicating) and 21S (mature) SV40 DNA synthesized after cycloheximide treatment were always lower than those observed in an infected untreated control culture. This is consistent with a requirement for a protein(s) or for protein synthesis at the initiation step in SV40 DNA replication. The relative proportion of 25S DNA as compared with 21S viral DNA increased with increasing time after cycloheximide treatment. Removal of cycloheximide from inhibited cultures allowed the recovery of viral DNA synthesis to normal levels within 3 hr. During the recovery period, the ratio of 25S DNA to 21S DNA was 10 times higher than that observed after a 30-min pulse with (3)H-thymidine with an infected untreated control culture. The accumulation of 25S replicating SV40 DNA during cycloheximide inhibition or shortly after its removal is interpreted to mean that a protein(s) or protein synthesis is required to convert the 25S replicating DNA to 21S mature viral DNA. Further evidence of a requirement for protein synthesis in the 25S to 21S conversion was obtained by comparing the rate of this conversion in growing and resting cells. The conversion of 25S DNA to 21S DNA took place at a faster rate in infected growing cells than in infected confluent monolayer cultures. A temperature-sensitive SV40 coat protein mutation (large-plaque SV40) had no effect on the replication of SV40 DNA at the nonpermissive temperature.     

Proteins of Vasicular Stomatitis Virus: I. Polyacrylamide Gel Analysis of Viral Antigens

Infection of L cells with vesicular stomatitis virus results in the release, into the cell-free fluid, of four antigenic components separable by rate zonal centrifugation on sucrose gradients. The largest antigens are the infectious (B) particle and a shorter noninfectious, autointerfering (T) particle. The two small antigens are characterized by sedimentation coefficients of approximately 20S and 6S. Treatment of purified B or T particles with sodium deoxycholate results in the release from the particle of a nucleoprotein core which can be purified on sucrose gradient and which has a sedimentation coefficient characteristic of the virus from which it arose. Utilizing purified antigens labeled with (14)C-amino acids during growth, we examined the protein constituents of each antigen by acrylamide-gel electrophoresis. The proteins of B and T particles are identical, each containing one minor (virus protein 1) and three major (virus proteins 2, 3, and 4) proteins, numbered in order of increasing mobility. Virus protein 3 originates from the nucleoprotein core, whereas proteins 2 and 4 come from the coat. The origin of virus protein 1 is not known. The 20S antigen contains a single protein equivalent to virus protein 3, whereas the 6S antigen shows a single protein which is similar to, but probably distinct from, virus protein 2.     

Effect of Oxygen on Heme and Porphyrin Accumulation from δ-Aminolevulinic Acid by Suspensions of Anaerobically Grown Staphylococcus epidermidis

The effect of various conditions on the accumulation of porphyrins and heme by resting suspensions of anaerobically grown cells of Staphylococcus epidermidis was examined. Anaerobically grown cells contain 10 to 15% of the amount of protoheme found in cells grown aerobically. Resting suspensions of anaerobically grown cells, when incubated aerobically in buffer with delta-aminolevulinic acid and glucose for 60 min, exhibited a fourfold increase in protoheme content. At high levels of delta-aminolevulinic acid, there was also a significant accumulation of porphyrins with the solubility and chromatographic properties of coproporphyrin and uroporphyrin. Protoporphyrin was not accumulated. When oxygen was excluded from the incubation mixture, accumulation of protoheme was prevented, but accumulation of coproporphyrin and total porphyrin was enhanced. Nitrate served as an electon acceptor as indicated by its reduction to nitrite; however, nitrate did not substitute for oxygen in causing the accumulation of protoheme. These results suggested that oxygen is required for one of the late steps of heme synthesis in S. epidermidis, possibly for the conversion of coproporphyrinogen to protoporphyrin. The inability of nitrate to substitute for oxygen suggests a role for molecular oxygen as a substrate rather than as an electron acceptor for heme synthesis.