Spheroplasts, preparation

Capsaicin.—It is known that phenylalanine acts as the source of the vanillylamine moiety of capsaicin (132) in Capsicum annuum and that valine (136) is used for the construction of the acyl part.2 It has been noted107 that whilst radioactive valine (136) was incorporated into capsaicin (132) and its dihydro-derivative (133), L-[U-14C]leucine [as (137)] labelled the capsaicin analogues (134) and (135) in Capsicum fruits both amino-acid precursors were very considerably better incorporated into capsaicinoid metabolites by using spheroplasts prepared from the placentas of Capsicum fruits. or-Ketoisovaleric acid (138) and cr-ketoisocaproic acid... 

Table VI. Changes in CMC-saccharifying Activities of Pseudomonas Cellulase Components During Incubation with Broken Spheroplast Preparation...

As shown in Table VI, only slight changes in the relative CMCase activity were found in the mixtures of the cellulase components A and C with any of the broken spheroplast preparations, while a considerable increase in the activity was noticed in the mixtures with the component B. The enhancement in the mixtures treated with those SI and S3 was particularly high, which were both the broken spheroplasts from the cultures of an exo-type cellulase synthesis the cellulase activity was enhanced five times that of the original. S2 preparation from the culture of an endo-type cellulase synthesis was much lower in the activating... 

Spheroplasts prepared according to ( ) were shocked and illuminated for 2 hours, according to (51,54). 

More recently, we have obtained electron micrographs suggesting penetration of tubular pili through the outer membrane (Fig. 7). These observations suggest that the true anchoring site is at the plasma membrane. Plasma membrane anchoring of pili is also supported by the fact that the pili remained associated with spontaneously lysed cells, as well as to spheroplast preparations. 

Approximately 1 p% of wild-type or mutant pCLI plasmid DNA is used for transformation of yeast cells. Spheroplasts of Saccharomyces cerevisiae strain GRF 18023,24 are prepared following the protocol of Burgers and Percival25 with some modification. High copy number transformants are directly selected on minimal medium plates lacking leucine and containing 8% glucose (to repress lysozyme expression) and 1 M sorbitol as an osmotic stabilizer. 

The spheroplasts are resuspended in 2 ml of STC by very gentle stirring with a 1-ml sterile pipette tip. The competent cells should be used immediately at best or may be stored for up to 5 hr. The above preparation should be sufficient for 20 transformations. 

The NAD-dependent 7a-HSDH was found to be associated with both cytoplasmic and cell membrane fractions during the preparation of spheroplasts of B. fragilis [20]. Moreover, the activity of this enzyme in B. fragilis increased 3-5-fold as the cultures entered stationary growth phase. However, the mechanism of regulation of enzyme synthesis is not yet known. 

Intracellular enzymes are prepared by plasmoptysis or sonic oscillation of spheroplasts or shocked cells. 

Since the occurrence of cellulase components A and B in pairs at first suggests the possible mutual conversion between them, it was investigated if such possibility would be realized by some cytoplasmic materials. For this purpose, plasmoptysized spheroplasts were prepared from cells grown on 0.5% sophorose (SI) or 0.5% cellobiose culture (S2) and those on culture under controlling supply of cellobiose (S3). (Although, as already stated, spheroplasts retained about 20% of the cell-... 

Novobiocin also causes chaining in Streptococcus faecium [23], although this effect is not produced in all cocci [13]. The induction of spheroplasts in Escherichia coli by novobiocin has also been reported [24] and, in fact, the antibiotic has been recommended for preparing spheroplasts in Gramnegative bacteria [25]. In contrast, however, it has been shown by various workers [26-28] that novobiocin does not induce spheroplasts in Serratia marcescens or in various strains of E. coli and [28] that it may even prevent spheroplast induction caused by benzylpenicillin in hypertonic medium (Figure 2.1). 

The lysis of cell wall and the preparation of spheroplast of Mucor mucedo, which belongs to the same Mucorales as Rhizopus, by a purified chitosanase from Bacillus R-4 has been described. ... 

Yeast strain differences and/or differences in culture medium result in a range of efficiencies of spheroplasting with the enzyme preparations we use. Pretreatment has allowed us to employ the conditions described below with different strain backgrounds grown in different media. Because we have made changes to our previously reported method for preparing yeast nuclei (Aris and Blobel, 1991), which serves as the starting material for the isolation of nucleoli, we present the entire method for nuclei here. 

Note It may be better to use lyticase (Boehringer Mannheim), because it is a cleaner preparation, but spheroplasting conditions will need to be worked out for this enzyme. 

---