Fate of Tobacco Mosaic Virus after Entering the Host Cell

Tobacco leaves were inoculated with tobacco mosaic virus labeled with ³²P or ³⁵S. After various intervals, extracts of the leaves were prepared. In extracts from leaves infected for 5 to 360 min, about 40 to 60% of the virus retained on leaves was recovered in the pellet of the homogenate centrifuged at 12 000 × g. The virus associated with the 12 000 × g pellet was dissociable by treatment with pancreatic RNase, alkali or sodium dodecyl sulfate (SDS). The parental virus extracted by SDS from the pellet at 12 000 × g had a large amount of partially uncoated virus possessing naked RNA. Analysis by density gradient centrifugation suggested that, in addition to partially uncoated virus, some fragmented RNA was also associated with the 12 000 × g pellet. This fragmented RNA seemed to be derived from partially uncoated virus. Density gradient analysis of SDS extracts from the 12 000 × g pellet suggested that some of the virus underwent uncoating at the internal regions of the virus particle.     

Chemical suppression of the symptoms of two virus diseases

Carbendazim applied at the rate of 2 g per plant to the roots of tobacco (Nicotiana tabacum cv. White Burley) plants before infection with tobacco mosaic virus (TMV) caused very considerable reduction in the severity of disease symptoms in systemically infected leaves but did not affect their virus content. Leaves of untreated, infected plants had a greatly reduced chlorophyll content 100 days after infection whereas the chlorophyll content of leaves of infected plants treated with carbendazim was similar to that of normal uninfected leaves. Carbendazim had no effect on the infectivity of TMV in vitro or on the local lesion reaction of N. glutinosa plants when inoculated with TMV. Carbendazim was applied to lettuce cv. Cobham Green at a total rate of o-i g per plant before and after they were infected with beet western yellows virus and the plants were then grown on in the field. At harvest time (50 days after infection) almost all the treated virus-infected plants were of a normal green appearance, whereas the untreated controls were almost all very severely yellowed and unmarketable.     

Biomass Measurement of Methanogens in the Sediments of Tokyo Bay Using Archaeol Lipids

An archaeal ether-linked lipid, archaeol, was determined to be a biomass indicator for methanogens both in the laboratory enriched culture and in marine sediments. The archaeol measurement method described by Ohtsubo et al. in 1993 was modified and applied to marine sediments. We compared the amount of archaeol with the cell number of methanogens or methane concentration in laboratory enriched culture of methanogens from marine sediment. Good correlations were obtained as follows: (Methane, mmol) = 11.2 × (Archaeol, mg): r= .996 or (Cell number) = 1.13 × 10¹¹× (Archaeol, mg): r= .995, respectively. In the sediments of Tokyo Bay, archaeol was measured from approximately 46 to 561 ng/dry g sediment at the entrance to 267 to 4160 ng/dry g sediment at the innermost area. Using the coefficient from the laboratory experiment, these data corresponded to cell numbers of 5.2 × 10⁶ to 4.7 × 10⁸/dry g sediment. These values were 1 or 2 orders of magnitude higher than those obtained by culture methods in previous studies. Although dead or decomposed cells might be detected, archaeol measurement is useful for estimating the biomass of methanogens because of the good correlation between methane concentration and archaeol content in marine environments. In this study, we found a correlation of (Methane, mmol) = 0.012 × (Archaeol, mg): r= .932, n= 17 in marine sediments. Received December 21, 1998; accepted June 16, 1999     

Fate of Tobacco Mosaic Virus after Entering the Host Cell

Diminutive viral RNAs recovered from tobacco leaves inoculated with ³²P-TMV were investigated. At 3.5 hr after inoculation, most of the viral RNA without coat protein revealed two peaks after sucrose density gradient analysis of SDS-extract from 12,000 × g leaf pellet. The first peak appeared between bacterial ribosomal RNA of 16 S and 5 S and the second peak was around 5 S. These two peaks were digestible with RNase and they appeared as early as 5 min after inoculation. These diminutive RNAs seemed to be derived from partially uncoated parental virus by abscission of their naked RNA tails. The active formation of these diminutive RNAs and their early appearance after inoculation seemed to indicate that most of the inoculated TMV received incomplete uncoating.     

Subcellular fractionation of epiphyseal cartillage isolation of matrix vesicles and profiles of enzymes,phospholipids, calcium and phosphate

Epiphyseal cartilage was fractionated into subcellular components by non-enzymatic methods, and analyzed for activity of marker enzymes, for phospholipids, and for calcium and inorganic phosphate. Alkaline phosphatase, a marker enzyme for matrix vesicles and plasma membranes, was concentrated in the 100 000 × g (microsomal) pellet and, upon subsequent frationalism, in the low-density fractions from the sucrose gradient. Mitochondrial and endoplasmic reticular enzymes were localized primarily in the 20 000 × g pellet, lysosomal enzymes predominantly in the supernate from the microsomal pellet. Two phospholipids characteristic of matrix vesicles, sphingomyelin and phosphatidylserine, were enriched in the low-density sucrose fractions; however, unlike matrix vesicles, there was no depletion in phosphatidylcholine or increase in lysophospholipids. Ca and inorganic P were concentrated in the higher-density fractions, the amounts in the lower-density fractions being some- what lower than those seen in matrix vesicles. The alkaline phosphatase-rich, low-density fractions were thus not identical to matrix vesicles isolated by collagenase digestion, but rather appear to be composed primarily of plasma membranes. Enzyme profiles indicate they were relatively free of mitochondrial, endoplasmic reticular and lysosomal contaminants. The data further indicate that significant modification of the phospholipid, electrolyte, and possibly enzyme content of chondrocyte plasma membranes, must occur during blebbing and matrix vesicle formation.     

Purification and Some Properties of an Isolate of Beet Yellows Virus from Ukraine

A purification procedure, which yielded up to 15–30 mg of beet yellows virus (BYV) per 100 g of infected Tetragonia expansa leaves, has been developed. The procedure included sap clarification with Triton X-100, and two cycles of ultracentrifugation through sucrose cushion, which contained PEG-6000 and NaCl. A specific antiserum was prepared, and BYV infection was successfully detected by the double-antibody sandwich (DAS) ELISA in infected sugar beet leaves and roots diluted up to 1 × 10⁵ and 1 × 10⁴, respectively. The virus concentration was demonstrated to decrease in infected sugar beet roots slowly during 7 months, thus allowing successful diagnosis of planting material in winter storage. BYV presence in Myzus persicae aphids was also reliably detectable using the DAS-ELISA. In a competitive DAS-ELISA test, the Ukraine and the British BYV isolates were found serologically indistinguishable.     

Expression of the tobacco mosaic virus movement protein alters starch accumulation inNicotiana benthamiana

Nicotiana benthamiana plants were transformed with the movement protein (MP) gene of tobacco mosaic virus (TMV), usingAgrobacterium-mediated transformation. Plants regenerated from the transformed cells accumulated 30-kDa MP and complemented the activity of TMV MP when infected with chimeric TMVs containing defective MR These transgenic plants displayed stunting, pale-green leaves, and starch accumulations, indicating that TMV MP altered the carbon partitioning for leaves involved in TMV cell-to-cell movement.     

Purification and some properties of strawberry mottle virus

Strawberry mottle virus (SMoV) (three isolates: HJ, 3E and N) were transmitted to Chenopodium quinoa plants by sap inoculation. All three isolates induced very similar symptoms consisting of chlorotic spots and ringspots in inoculated leaves, and vein chlorosis, mottling, and dwarfing of the upper leaves. SMoV isolate HJ was purified from infected C. quinoa by homogenisation with 10 mM phosphate buffer, pH 7.2 containing 5% Triton X-100, followed by differential, sucrose density-gradient and CsCl equilibrium density-gradient centrifugations. A fraction with a buoyant density of 1.42g^- cm^-3 after CsCl density-gradient centrifugation was highly infectious to C. quinoa and contained many isometric virus-like particles c. 37 nm in diameter. These virus-like particles were never found in fractions from uninfected preparations. Electrophoretic analysis of a fraction containing virus-like particles revealed that these particles might have a single coat protein subunit with the apparent molecular mass of 26 K daltons and one nucleic acid of 6.6 kilobases. Double-stranded RNA analysis of isolate HJ-infected or uninfected C. quinoa and Fragaria vesca var. semperflorens seedling line ‘Alpine’ plants showed that both infected plants had two infection-specific dsRNA bands of mol. wts 4.5 and 3.9 × 10⁶.     

Preparation and polypeptide composition of plasma membrane and other subcellular fractions from wild oat (Avena fatua) aleurone

Plasma membranes can be isolated from a variety of plant tissues by first preparing a post-mitochondrial membrane fraction enriched in plasma membranes, by differential centrifugation, and partitioning this on a dextran-polyethylene glycol two-phase system. With wild oat aleurone, however, we observed that differential centrifugation could not be used to produce a microsomal fraction enriched in plasma membrane. Approximately 70% of the plasma membrane in aleurone homogenates was pelleted by sequential centrifugation at 100 g× 10 min and 1000 g× 10 min. The remainder sedimented at 112 000 g× 1 h. All the material that was pelletable by centrifugation was, therefore, subjected to dextran-polyethylene glycol two-phase partitioning. The plasma membrane marker enzymes glucan synthase II (GSII, EC 2. 4. 1. 34) and UDP-glucose:sterol glucosyltransferase (SGT, EC 2. 4. 1.) were enriched in the upper phase, whereas cytochrome c oxidase activity (EC 1. 9. 3. 1), a mitochondrial marker enzyme, was depleted. The presence of endoplasmic reticulum (ER) and protein body membranes in the phase system was assessed by probing western blots, of SDS-PAGE separated proteins, with polyclonal antiserum either to binding protein (BiP, an ER marker) or to tonoplast intrinsic protein (TIP, a protein body membrane marker). BiP and TIP were present in the lower phase, but were not detected in the upper phase. In addition, the polypeptide patterns of material in the upper and lower phases were very different. These observations suggested that high purity aleurone plasma membrane had been isolated. Although the procedure for isolating plasma membranes was applicable to both aleurone protoplasts and layers, the polypeptide patterns of plasma membranes prepared from these sources were very different. The major protein components of wild oat aleurone were 7 S and 12 S storage globulins. These proteins were present in the lower phase, but not in the plasma membrane enriched upper phase, after aqueous two-phase partitioning. Differential centrifugation studies showed that it was necessary to homogenise aleurone in a buffer of pH 6. 0 or less if a soluble protein fraction, essentially devoid of storage globulins, was to be obtained. The use of these fractionation techniques is discussed in relation to photoaffinity labelling of gibberellin (GA)-binding proteins in aleurone.     

Lactones Produced through Thermal Oxidation of Higher Fatty Acids

Inhibitors of plant virus infection with systemic effects were found in the culture filtrates of Basidiomycetes such as Fomes fomentarius and Schizophyllum commune. These inhibitors were widely distributed in Agaricales and Polyporales. The inhibitors designated as BAS (Basidiomycete Antiviral Substance) were highly active against the mechanical transmission of tobacco mosaic virus (TMV). No toxic effect was observed on the host plants. BAS-F, a polysaccharide produced by F. fomentarius, almost completely inhibited infection, when BAS-F at 2 μg/ml was applied to the same surface of leaves of Xanthi-nc tobacco 24 h before TMV inoculation to the upper surface of the leaves, and 500/0 inhibition was shown when BAS-F at 10 μg/ml was applied to the under surface of leaves. BAS-F also induced systemic resistance to the non-treated leaves when it was applied to only one leaf of the plant. BAS-F also had similar effects against the infection of TMV on bell pepper and tomato plants.     

Induced Resistance in Hypersensitive Tobacco Against Tobacco Mosaic Virus by Injection of Intercellular Fluid from Tobacco Plants with Systemic Acquired Resistance

Acquired resistance in hypersensitive tobacco plants against tobacco mosaic virus (TMV) was induced by components occurring in the intercellular fluid (IV) obtained from virus-infected plants or by plant cell wall components. Induced resistance could be transmitted through seed to the progeny. Lesion size and number were reduced significantly when the progeny was tested by TMV-inoculation. IV was extracted from the upper uninoculated leaves of four times TMV-inoculated Nicotiana tabacum cv. ‘Xanthi’ nc plants. Injection of IV from induction-inoculated plants (SAR-IV) into leaves of healthy plants followed by TMV-infection reduced lesion size significantly. A concentration of 5 × 10^?7 g SAR-IV/ml was still active. IV from healthy plants was inactive. The IV's were partly purified by gel filtration on a Sephadex G-50 column. Some fractions were active in inducing resistance as expressed in reduction of lesion size. Fractions of control-IV were inactive. It is still unknown whether the active substances in SAR-IV are in fact cell wall fragments acting as regulatory molecules in disease resistance.     

Gonadotropin and prostaglandins binding sites in rough endoplasmic reticulum and Golgi fractions of bovine corpora lutea.

Highly purified rough endoplasmic reticulum and three subfractions of golgi were prepared from 105,000g pellet of the homogenate by centrifugation in floatation and sedimentation discontinuous sucrose gradients. Highly purified plasma membranes were also prepared from 9,000g pellet of the same homogenates for assessment under the same experimental conditions. Although 5′-nucleotidase, a marker for plasma membranes, was markedly enriched in plasma membranes, very little or none of this enzyme activity was found in other fractions. Very little or no NADH cytochrome c reductase activity, a marker for rough endoplasmic reticulum, was found in fractions other than rough endoplasmic reticulum. Galactosyl transferase, a marker for golgi, was found and enriched in all the fractions; however, enrichment in golgi fractions was higher than in other fractions. Very little or no lysosomal marker activity, i.e., acid phosphatase, was found in rough endoplasmic reticulum or golgi fractions as compared to lysosomes. These marker enzyme data suggested that rough endoplasmic reticulum and golgi fractions were relatively pure with little or no cross contamination with other organelles. The [¹²⁵I]human choriogonadotropin ([¹²⁵I]hCG), [³H]prostaglandin (PG)E₁, and [³H]PGF_2a specifically bound to rough endoplasmic reticulum and golgi fractions in addition to plasma membranes. The enrichments of binding in the former two fractions, in some cases, were as high as plasma membranes itself. The specific binding of some of the ligands was found to be partially latent in rough endoplasmic reticulum and golgi fractions but not in plasma membranes. Marker enzyme data, ratio between bindings and marker enzyme activities (an index of organelle contamination), and partial latency of binding suggest that rough endoplasmic reticulum and golgi fractions intrinsically contain gonadotropin and PGs binding sites.     

Purification and properties of cassava green mottle, a previously undescribed virus from the Solomon Islands

A sap-transmissible virus obtained from cassava with a green mottle disease occurring at Choiseul, Solomon Islands, was transmitted to 30 species in 12 plant families and was readily seed-borne in Nicotiana clevelandii. In cassava plants infected by inoculation with sap, the first leaves to be infected systemically developed a mottle with some necrosis whereas leaves produced subsequently were symptomless but contained the virus. Most other species developed chlorotic or necrotic local lesions and systemic mottle or necrosis. This was followed, in several species, by production of small symptomless virus-containing leaves. The virus was cultured in N. clevelandii; Chenopodium quinoa was used for local-lesion assays. Leaf extracts from infected N. clevelandii were infective after dilution to 10–⁵ but usually not at 10–⁶, after heating for 10 min at 60°C but not at 65°C, and after storage at 20°C for at least 12 days. The virus has isometric particles of 26 nm diameter which sediment as three components, all containing a protein of mol. wt c. 53000. The two fastest sedimenting components respectively contain single-stranded RNA of mol. wt, estimated after glyoxylation, c. 2.9 × 10⁶ and 2.3 × 10⁶. Both RNA species are needed for infection of plants. In tests with antiserum prepared to purified virus particles, the virus was detected in cassava and N. clevelandii by gel-diffusion precipitin tests, by immunosorbent electron microscopy and by ELISA. Despite its similarity to nepoviruses, the virus did not react with antisera to 18 members of the group. It was named cassava green mottle virus and is considered to be a previously undescribed nepovirus.     

Characterization of sub-genomic double-stranded RNAs from virus-infected plants

Double-stranded RNAs (sub-RFs) smaller than the double-stranded RNAs (RFs) corresponding to genomic RNAs of tobacco mosaic (TMV) and cowpea chlorotic mottle (CCMV) viruses were isolated from infected plants and characterized. Seven of the 12 sub-RFs of TMV that were found ranging in size from 3.00 – 0.42 × 10⁶ daltons corresponded to twice the size of the 7 sub-genomic mRNAs reported by Goelet and Karn (8). Six sub-RFs of CCMV were found ranging from 0.98 – 0.39 × 10⁶ daltons with the most abundant species corresponding to twice the size of RNA 4. The kinetics of incorporation of ³H-uridine into sub-RFs were different from that into RFs. Incorporation into sub-RFs was slow and linear whereas that into RF turned over rapidly.     

Fine Structure of Healthy and TMV-Infected Tobacco Cells Grown in Tissue Culture

Three types of tobacco (Nicotiana tabacum cv. Havana 38) callus: 1) healthy stem callus, 2) TMV-infected stem callus, 3) TMV-infected leaf callus; and leaves differentiated from healthy stem callus, and from TMV-infected leaf callus were compared for fine structure. In addition, the fine structure was observed of plastids in cells of leaves differentiated from callus isolated from stem sections of TMV-infected hybrid tobacco plants (N. tabacum cv. Havana 38 ×N. glutinosa) grown under high temperature. The cytoplasmic organelles in tissue cultured cells were similar to those in cells of greenhouse-grown tobacco plants. Except for plastids, TMV infection did not noticeably affect morphologically other cellular organelles in tissue culture cells. In TMV-infected leaf callus, numerous small bodies were seen in plastid-like bodies, while vesicle-like structures were observed in the stroma of plastids in leaves differentiated from callus of hybrid tobacco inoculated with TMV. Morphological variations of mitochondria, such as swelling and vacuolization of the inner matrix, occurred frequently in TMV-infected leaf callus. Needle-like crystalline inclusions or looped inclusions composed of many fine, long filaments were considered TMV particles orientated parallel to each other. The TMV particles were detected in the cytoplasm of tissue culture cells.     

A culture unit system for the study of responses of mycorrhizal and non-mycorrhizal seedling to treatments

The time-dependence of Mn accumulation was confirmed in potato foliage (Solanum tuberosum. L.cv. Norland) grown in solution culture. Older leaves grown at 0.61 mM Mn had substantially higher Mn concentrations than younger leaves and stem samples. Levels of Mn in older leaves increased steadily from 4000 µg g^–1 at one week to 8–10,000 µg g^–1 at 6 weeks, but were relatively constant in the emerging leaves. Even foliage grown at low Mn levels (0.01 mM Mn) had 4 fold gradients in Mn concentration from younger (40 µg g^–1) to older leaves (180 µg g^–1).At 0.61 mM Mn, concentrations of 3–4000 µg g^–1 in the youngest fully-developed leaves did not bring about any decline in yield, and levels of up to 5000 µg g^–1 occurred in individual potato leaves before Mn toxicity symptoms were observed. Potato foliage grown at the high Mn had similar leaf numbers, but showed an increased stem length and smaller leaves than foliage grown at 0.01 mM Mn. In particular, the leaf area of the middle and lower leaf fractions were affected by the high Mn level.The ability of rapidly growing plants to withstand high concentrations of Mn is discussed in relation to the pattern of dry matter and Mn accumulation shown by potato foliage.     

Effect of chitosan on tobacco mosaic virus（TMV） accumulation,hydrolase activity,and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus（TMV） in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV（2 μg/mL） and chitosan（1 mg/mL） were lower in the early period of infection（3 days after inoculation）, by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases（proteases, RNases） in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid（PTA）-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles（18 nm in diameter, 300 nm long）, these suspensions contained abnormal（swollen, “thin” and “short”） virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that “thin” virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.     

Quantitative and qualitative study of the bacterial flora of farmed freshwater prawn (Macrobrachium rosenbergii) larvae

Quantitative and qualitative studies of the bacterial flora of farmed freshwater prawn (Macrobrachium rosenbergii) larvae in Saudi Arabia were performed, and isolates identified where possible. Physico‐chemical characteristics, bacterial counts, and the nature of the bacterial flora of larvae rearing tank water, sediment, tank wall surfaces, larval surface, supplied water, and feed were investigated. Bacterial counts ranged from 2.1 ± 1.3 × 10⁵ to 2.2 ± 0.8 × 10⁷ colony forming units (CFU) ml^?1 in tank water; 4.4 ± 0.9 × 10⁷ to 8.3 ± 1.7 ×10⁹ CFU g^?1 in tank sediment; 8.6 ± 1.0 × 10² to 9.8 ±0.7 × 10⁴ CFU cm^?2 on the tank wall surface; 1.3 ± 1.1 × 10⁴ to 7.7 ± 1.6 × 10⁶ CFU per larva surface, 7.9 ± 1.2 × 10⁵ to 5.0 ± 1.5 × 10⁷ CFU g^?1 in washed larval tissue slurries, 9.1 ± 0.7 × 10³ CFU ml^?1 in supplied water, and 2.4 ± 1.9 ×10¹⁰ CFU g^?1 in mixed feed. Fourteen bacterial genera were identified, including Chryseomonas sp., Vibrio spp., Cellulomonas sp., Aeromonas hydrophila, and Pasteurella sp. The tank water and sediment had similar bacteria to those on the prawn larvae. Chryseomonas sp., Cellulomonas sp. and Vibrio sp. were the most dominant species (prevalence >10%) in tank water; Chryseomonas sp., Pseudomonas alcaligenes and Shewanella putrefaciens in the sediment; Ps. alcaligenes and Cellulomonas sp. on the tank wall surface; Chryseomonas sp., and Cellulomonas sp. on the larval surface; and Chryseomonas sp., Vibrio vulnificus, Sh. putrefaciens and V. alginolyticus in the washed larval tissue slurries (prevalence 10%). Pseudomonas alcaligenes, Moraxella sp., Serratia liquefaciens, Gordona sp. and Burkholderia glumae were absent in larvae but identified in the culture water, tank sediment, and tank wall surface. Pseudomonas sp., Chryseomonas sp., Pasteurella sp. and V. alginolyticus were the prevalent bacteria (>12%) in supplied water. The feed contained V. alginolyticus, A. hydrophila and Cellulomonas sp. as the dominant bacteria (>13%). In the culture water and larvae samples, 83% of the feed and supplied water bacteria were identified.     

Host range and some properties of potato mop-top virus

Potato mop-top virus (PMTV) was transmitted by inoculation of sap to twenty-six species in the Solanaceae or Chenopodiaceae and to Tetragonia expansa; species in eleven other plant families were not infected. The virus was cultured in inoculated leaves of Nicotiana tabacum cv. Xanthi-nc or in N. debneyi. Diagnostic local lesions were produced in Chenopodium amaranticolor. In winter, ten solanaceous species were slowly invaded systemically but the first leaves infected were those immediately above inoculated leaves. When transmitted to Arran Pilot potato by the vector Spongospora subterranea, PMTV induced all the main types of shoot and tuber symptoms found in naturally infected plants. Isolates of PMTV from different sources differed considerably in virulence. PMTV-containing tobacco sap lost infectivity when heated for 10 min at 80 °C, diluted to 10^-4, or stored at 20 °C for 14 weeks. Infectivity was partially stabilized by 0·02% sodium azide. When sap was centrifuged for 10 min at 8000 g, infectivity was mainly in the sediment. Infective sap contained straight rod-shaped particles about 20 nm wide, with lengths up to 900 nm and crossbands at intervals of 2·5 nm. Many of the particles were aggregated side-to-side, and the ends of most seemed damaged. The slight infectivity of phenol-treated leaf extracts was abolished by pancreatic ribonuclease. The present cryptogram of PMTV is R/*:*/*:E/E:S/Fu.     

Soluble and bound forms of intracellular acid carboxypeptidase inAspergillus saitoi

The enzymological, physical, and immunological properties of soluble and bound forms of intracellular acid carboxypeptidase isolated from fresh mycelia ofAspergillus saitoi are reported. In the broken mycelia, about 60% of the total activity was found in the 2,000×g precipitate, with most of the remainder in the 100,000×g supernantant. The highly purified enzymes, I_a and I_b, from the 100,000×g supernatant were found to be homogeneous by such criteria as disc gel electrophoresis at pH 9.4 The bound enzyme, II, was solubilized from the 2,000×g precipitate by self-digestion at pH 6.4 and was highly purified by chromotography. The two forms of intracellular enzymes, the soluble enzymes (I_a and I_b) from the 100,00×g supernatant and the solubilized enzyme (II) from the 2,000×g precipitate, were closely related to, but not completely identical with, the extracellular acid carboxypeptidase.