s¢ 23040 SKORT COMMUNICATIONS

Starch-gel electrophoresis of ~-casein

411

Electrophoresis of whole casein in urea-containing starch gels 1 has been shown to be a powerful tool for the identification of its numerous components, except the fraction called K-casein. With the latter extremely diffuse and blurred zones are observed 1, 3. Despite the fact that during free electrophoresis K-casein has nearly the same mobility as the as-casein 3, the mobility of the blurred K-casein zone in the starch gel is much lower than that of as-casein. I t is probable that molecular sieving by the starch gel is responsible for this effect 4. Indeed, it is known that of all the casein components K-casein has the strongest tendency to aggregate3, 5. Moreover, preliminary sedi- mentat ion studies in this laboratory showed these aggregates to consist of very asymmetrical particles, not unlike those observed with /~-casein 6. SWAISGOOD AND BRUNNER 5 found that even in a medium containing 7 M urea, K-casein does not dis- aggregate completely. Only after the reduction of the disulphide bonds by 2-mercapto- ethanol, does the molecular weight decrease to a value of about 2o000. This led us to the idea of investigating the influence of 2-mercaptoethanol on the starch-gel pat tern of K-casein. As will be demonstrated below, the addition of 2-mercaptoethanol

Fig. i . Effect of add i t ion of 2 -mercap toe thano l on t he s ta rch-gel e lectrophoresis p a t t e r n of K-casein. a, whole casein, p ro te in concen t r a t ion 1.5%, 0.022 M 2 -mercap toe thano l ; b, K-casein, p ro te in concen t r a t ion 2%, o.o22 M 2 -mercap toe thano l ; c, t h e s ame as b b u t w i t h o u t 2 - m e r c a p t o e t h a n o l

addi t ion.

Biochim. Biophys. Acta, 90 (1964) 411-414

412 S H O R T C O M M U N I C A T I O N S

leads to a very satisfactory resolution of the diffuse K-casein zone into a large number of well defined bands.

Whole casein of bulk milk and of the milk of individual cows was prepared by iso-electric precipitation. From this K-casein was isolated by the procedure of McKENZlE AND WAKE 7. The method of WAKE AND BALDWIN 1 of preparing starch gels was modified as follows. 2-Mercaptoethanol was added to the cooling gel at 5 °0 with careful mixing to give a final concentration of 0.022 M. As a consequence of the weakening action of 2-mercaptoethanol on the gel strength the starch concen- tration had to be increased from 11. 4 to 12.2 %. The casein samples applied to the gel contained 1.5-3 % protein, 2 % starch and likewise 7 M urea and 0.022 M 2-mer- captoethanol in 0.076 M Tris-citrate buffer (pH 8.6). Electrophoresis was carried out at room temperature.

The results of the addition of 2-mercaptoethanol on the starch-gel electrophoretic pattern of K-casein of bulk milk are shown in Fig. I. A number of sharp bands has appeared in the previously blurred region of K-casein. The relative mobilities of these bands were measured according to WAKE AND BALDWIN I and are collected in Table I, together with the relative mobilities of some other known casein components. It is seen that some values are slightly altered after the addition of 2-mercaptoethanol. By chromatographic purification likewise with the addition of 2-mercaptoethanol, to be described in a future paper, we isolated a fraction, mainly consisting of the bands 0.52 and 0.60. This fraction flocculated readily with the milk-clotting enzyme rennin in the absence of Ca 2+, which is characteristic for K-casein 3.

Whole caseins of individual Friesian cows were then investigated by the same technique. From Fig. 2 it is evident that differences exist in the numbers and mobili- ties of the K-casein bands of the individual cows. It is probable that the different K-bands represent genetic variants of the same protein, as was observed with the other casein components s-l°. This hypothesis is supported by the fact that the casein samples c-g in Fig. 2 originate from 5 daughters of the same sire. Apparently these

T A B L E I

RELATIVE MOBILITIES OF eASY.IN COMPONENTS IN UREA-STARCH-GEL ELECTROPHORESIS

H. 4 % starch 12.2 % starch Component without 2-mercaptoethanol with 2-memaptoethanol

? o . 1 8 0 . 2 4

K - - 0 . 5 2 K - - 0 . 6 0

? - - 0 . 6 6

? - - 0 . 7 2

? - - 0 . 7 4 ? - - 0 . 7 8 ? - - o . 8 1

? - - 0 . 8 4

f l - B 0 . 7 3 0 . 9 o f l - A 0 . 8 0 0 . 9 2

? 0 . 8 6 0 . 9 5 R e f . b a n d 1 I .OO i .oo

? i . 0 4 1 . 0 4

~s -C 1 . 0 7 i . 0 7 0¢s-g I . IO I . I O

o~s-A 1 . 1 5 1 . 1 5

B i o c h i m . B i o p h y s . A c t a , 9 o ( 1 9 6 4 ) 4 1 1 - 4 1 4

S H O R T C O M M U N I C A T I O N S 413

Fig . 2. Electrophoresis of whole casein samples (protein concentration 3 % ) i n urea-starch gels containing 0 . 0 3 0 M 2 - m e r c a p t o e t h a n o l . a , reference sample of casein of pooled m i l k ; b - h , caseins

of individual Friesian cows.

T A B L E I I

DISTRIBUTION OF THE K-CAS]~IN BANDS 0 .52 AND 0 .60 AMONG 48 ERIESIAN AND 22 iRIS[ cows

Band number Breed

0.52 0.52 + 0.60 0.60

Friesian 2 21 25 M R I J 8 IO 4

5 cows are homozygous for the K-casein represented by band o.6o. On the other hand Cow h is seen to be homozygous for K-casein 0.52, whereas Cow b seems to represent the heterozygous species.

In orientating experiments we investigated a larger number of individual cows of two major Dutch breeds, the so-called Friesian and MRIJ cows. Distribution numbers of the bands 0.52 and 0.60 are collected in Table II.

Netherlands Institute for Dairy Research, Ede (The Netherlands)

D. G. SCHMIDT

B i o c h i m . B i o p h y s . Ac ta , 9 0 (1964) 4 1 1 - 4 1 4

414 SHORT COMMUNICATIONS

1 R. G. WAKE AND R. L. BALDWIN, Biochim. Biophys. Acta, 47 (1961) 225. 2 j . M. NEELIN, D. ROSE AND H. TESSlI~R, J. Dairy Sci., 45 (1962) 153.

T. A. J. PAYENS, Biochim. Biophys. At/a, 46 (1961) 441. 4 0 . SMITHIES, Arch. Biochem. Biophys., Suppl. I (1962) 125. 5 H. E. SWAISGOOD AND J. R. BRUNNER, Biochem. Biophys. Res. Commun., 12 (1963) 148. e T. A. J. PAYENS AND B. W. VAN MARKWlJK, Biochim. Biophys. Acta, 71 (1963) 517 . v H. A. McK~NzlE AND R. G. WAKE, Biochim. Biophys. Acta, 47 (1961) 240. s M. P. THOMPSON, C. A. KIDDY, L. PEPPER AND C. A. ZlTTLE, Nature, 195 (1962) iooi . 9 R. ASCHAFFENBURG, Nature, 192 (1961) 431.

l0 R. ASCHAFFENBURG, J. Dairy Res., 3 ° (1963) 251.

Received April I4th, 1964 Biochim. Biophys. Ac/a, 90 (1964) 411-414

sc 23 o43 The metabolism of 9(10)-hydroxystearic acid by the

cellular slime mold, Dictyostelium discoideurn

The enzymatic mechanism of direct desaturation of long-chain fat ty acids is being actively investigated in several laboratories z. Requirements for NADPH and molecu- lar 0 2 have been clearly shown 2, and in at least one instance Fe ~+ and FAD are also necessary a. The fa t ty acid substrate must be present as the CoA thioester 2.

These cofactor requirements have suggested an oxygenase mechanism for de- saturation, with participation of a hydroxylated intermediate 2, but direct evidence for this postulation has not yet been found*, s. Despite these negative results, the possible existence of hydroxy acid intermediates has not been excluded, since such compounds might occur only as enzyme-bound forms 5.

Growing amebae of the cellular slime mold, Dictyostelium discoideum desaturate palmitate and stearate directly to palmitoleate and oleate, respectively 6. Suspensions of resting cells of D. discoideum are capable of desaturating as much as 2.5 mg (IO/~moles) of palmitate per g (wet wt.) of cells in 5 h (F. DAVlDOFF, unpublished observations). The analysis of isotope incorporation experiments with D. discoideum is facilitated by the fact that palmitate and stearate are not significantly degraded to acetate. Acids of chain length Clo or less are degraded to acetate which is then incorporated into long-chain fat ty acids mainly by elongation of medium chain-length fat ty acids. There appears to be very little synthesis de novo of fa t ty chains 6. For these reasons, the growing culture might be a sensitive system in which to detect the direct conversion of hydroxy acids to unsaturated acids, if this conversion does in fact occur. The results to be presented in this paper indicate, however, that 9(10)- hydroxystearate is not converted directly to oleate, but is degraded to acetate, behaving in this respect like fa t ty acids of chain length Cj0 or less.

9(Io)-Hydroxy[I-z4C~stearate was synthesized from [I-14C]oleic acid by hydro- boration 7. The product was purified by preparative gas-liquid chromatography, and had the same retention time on QF-I and SE-3o liquid phases as authentic methyl- 9(Io)-hydroxystearate.

Vegetative cells of D. discoideum, aggregateless stain Agg 204, were grown in submersion culture on lipid-extracted Escherichia coli as described elsewhere s. Radio- active 9(Io)-hydroxystearic acid was added as the albumin complex to the growing culture. After 24 h, the cells were harvested, washed by centrifugation, and the lipids

Biochim. Biophys. Acta, 90 (1964) 414-416