SHORT COMMUNICATIONS I 5 ~

BBA 23476

A rnethod for separation and estimation of chondroitin sulphate isomers by electrophoresis on cellulose acetate paper

The separation of chondroitin sulphate B from chondroitin sulphate isomers A and C has been reported by TRUNDLE AND ZVIANN 1 and HARUKI AND I ( IRK 2, using paper electrophoresis, and by BERENSON AND DALFERES ~ using fibreglaSs chromatog- raphy.

The method reported in this communication is more rapid and gives a clearer separation than any previously reported.

The method is basically that of HARUKI AND KIRK ~ but uses cellulose acetate paper (Oxoid) instead of paper as supporting medium for electrophoresis in zinc acetate. Quanti tat ive estimation of the various fractions may be carried out.

The cellulose acetate strips were soaked in o.I M zinc acetate, blotted dry ~and 2o-#1 samples containing 2o-3o #g mucopolysaccharide were applied as a line at the origin. The samples were run in o.I M zinc acetate using a potential of o.25 mA/cm for 3 h at room temperature. The cellulose acetate strips were then transferred directly to 1% alcian blue in 2 % acetic acid for 5 rain followed by washing in tap water for 15 min. The strips were dried between filter paper at room temperature and then cleared in liquid paraPSn. The position and relative densities of the stained muco- polysaccharides were recorded with a Joyce-Loebl scanning densitometer using maximum slit width, an Ilford 2o 4 red filter and a recording arm ratio of I :IO., Corresponding electrophoresis strips were run in veronal acetate (pH 8.4) buffer for I h at 0.25 mA/h; under these conditions chondroitin sulphate isomers do not separate.

The samples of chondroitin sulphate were obtained from cow aorta by digestior~ with proteolytic enzymes and precipitation with alcohol after the method of KIRK AND DYRBYE ~. The mixture of mucopolysaccharides was dialysed and the constituent mucopolysaccharides separated using DEAE-Sephadex and eluting with NaC1 solution of increasing ionic strengths ~. Chondroitin sulphates were obtained in the fraction eluted with 1.25 M NaC1 in o.oi M HC1.

The total mucopolysaccharide content of the eluates from the Sephadex column was obtained using the carbazole method of Dlsc~I~ ~, as modified by BITTER AND' MI:I~ ~. Relative concentrations of chondroitin sulphate B to chondroitin sulphates A

~-- . #!AtC~

~ ~ Origin Distance from origin(cm)

Fig. I. A densi tometer trace of alcian blue stained chondroit in sulphate isomers obtained f rom cow aor ta subjected to electrophoresis on cellulose acetate paper. The upper trace shows a single peak of chondroi t in sulphates A, 13 and C run in veronM buffer. The lower trace shows chondroit in sulphate 13 separated f rom chondroi t in sulphates A and C after elecfcrophoresis in zinc acetate.

Biochim. Biophys. Acta, ~77 (z969) ~57-~58.

~5~ SHORT COMMUNICATIOX S

and C were ob ta ined from the dens i tomete r readings b y measuremen t of the area under the carves (Fig. z).

i n order to ob t a in reproducib le resul ts i t was found necessary to change tt.~e zinc ace ta te solut ion in the etectropimresis t a n k af ter each run as the mo la r i t y of the solut ion al ters dur ing a run due to depos i t ion of zinc on the ca thode.

To check t h a t the m e t h o d was measur ing chondro i t in su lpha te B, a sample o~! chondro i t in su lpha te ob ta ined from the Sephadex column was incuba ted wi th testicw, lar hya lu ron idase s and dialysed, then e lec t rophores is was carr ied out on a sample in zinc ace ta t e as above. 0 n l y one line was ob ta ined on s ta in ing the e lectrophoresis s t r ips . This resul t confirms the presence of chondro i t in su lpha te B as chondro i t in su lpha tes A and C and hya lu ron ic acid are d iges ted b y tes t icn ta r hyaluronidase*~

The au tho r t h a n k s Mr. D. R o a ~ s for technica l assistance.

Department of Biochemistry, University of Sheffield, She~eld (Great Britain)

R~ E. So PROem

I D. S. TRUNDLE AND G. V. ~ANX, Biochim. Bi@hys. Acta, IO~ (~965) 127. 2 F. HAR~JKI AND J. E. KIRK, Biochim. Bi@ltys. Acta, ~36 (~967) 39x-393. 3 G. S. BER~NSON AND E. R. DALFZRES, Biochim. Biophys. Acta, 58 (~962) 34. 4 J- E. KIRK aND M. DYR~VX, J. Gerontol., ~2 (I957) 23. 5 IV[. SCH~ID% Biochim. Bio~hys. Acta, 63 (~962) 346. 6 Z. DlSCUE, J. Biol. Chem., I67 (1947) I89. 7 T. Bl~C~ga aND H. M. MUIR, Anal. Biochem., 4 (1962) 33 o. 8 K. MEYER, E. DAVIDSON, A. LINKER AND P. HOFFMAN, Biochim. Bi@hys. Acta, 2I (~956) 506.

Rece ived October 3~st, I968

Biochim..~?iophys. Acta, z77 (~969) I57-~58

~BA 23482

The relationship between milk accumulation and enzyme activities in the

involuting rat mammary gland

L a c t a t i o n in the ra t is a ccompan ied b y an increase in the ac t i v i t y of a large n u m b e r of m a m m a r y g land enzymes which subsequen t ly decline r ap id ly af te r weaningl,K I t is known tha t hormones of the an te r io r p i t u i t a r y p l ay an lmpor tanz rote in the in i t i a t ion and ma in t enance of l ac t a t i on (see ref. 3 and hypophysecvomy of the l ac t a t ing r a t leads to a r ap id decline in mi lk p roduc t ion and an a lmos t equa l ly r a p i d decline in the ac t iv i t ies of a n u m b e r of enzymes l,~. The ac t iv i t ies of different enzymes decrease to different ex t en t s so t h a t wi th in 2 4 h of opera t ion a charac- t e r i s t i ca l ly changed p a t t e r n of enzyme a c t i v i t y has been establ ished. These changes af te r h y p o p h y s e c t o m y are r ep roduced a lmos t exac t ly if the l i t te r is r emoved from the l ac t a t i ng ra t and the g lands a l lowed to become engorged wi th milk and it is a ques t ion of some in te res t whe ther th is common response is due ~o s imi lar changes in the levels of c i rcula t ing hormones or whe the r changes wi th in the engorged g land itself in some w a y reproduce the condi t ions following h y p o p h y s e c t o m y .

~och~m. ~io~]zys. At#a, ~77 (~968) ~58-16o