.5~. ]g . , Heir 3, 1967 Kurze OriginMmitteilungen 7t

DAN: Z. Naturforsch. 14b, 234 (1959). - - [6] OOAWA, T., F. OeATX U. X. SnlSATA: Biochim. Biophys. Acta 112, 223 (1966). - - [6'] K~Eumz, W., u. P. W z B ~ : Z. Naturwiss. 53, l i (1966). - - [7] WESE~, P.: Z. Naturforsch. 18b, 1105 (t963). - - [8] ORNSTEIN, L.: Ann. N.Y. Acad. Sci. 121, 321 (t964).

P r e c i p i t a t i o n o f R i b o s o m e s f r o m E. c o l i B b y P o l y m y x i n B M. TXUB~R*

Max v. Pe t t enkofe r - Ins t i tu t ff~r Hygiene und Medizinische Mikrobiologie der Universitgt , IVliinchen

Po lymyxin t3, a cyclic, basic oligopeptide antibiotic, binds to some exten t to the small particle fraction of gramnegat ive and -positive bacteria during its bactericidal action [1]. Like other polyamines po lymyxin B is able to precipitate ribo- nucleic acid [2]. The following communica t ion describes the precipitat ion of r ibosomes from E. colt t3 by po lymyxin t3.

Po lymyxin t3 sulfate was pu t - /00% ~ - -

e0 i

$o

#o

$o

0 $0 40 GlO I00 200 ~00

Fig. i. Precipitation of riboso- mes from E. coli B and of yeast RNA by polymyxin B.Abscissa (log.): ~g polymyxin ]3 sulfate per mi. Ordinate: O. D.~, of supernatant (%). - - ~ RNA 100 ~glml; b Ribosomes 200 p.g/ ml; c Ribosomes 40 ~g/ml

chased f rom Pfizer G. m. b. H., Karlsrube, (bactericidal acti- v i ty 766t uni ts per rag). Pre- para t ion of ribosomes, assay of r ibonuclease I and sucrose densi ty gradient centr ifugat ion were performed as cited else- where [3]. Precipi ta t ion of ri- bosomes by po lymyxin B was de termined in 0-05 M Tris-HCl buffer pH 7.5 af ter 2hrs at 40 C and I0 rain centr ifugat ion at f0000 g by measur ing the optical densi ty of the superna- t au t at 260 m~x. As shown in Fig. I, r ibosomes were precipitated by poly- myxin t3 wi th similar pa t t e rns as h ighpolymer yeast IRNA. Full precipi tat ion at a given a m o u n t of r ibosomes was de- pendent on the a m o u n t oi poly- myx in B present in the reaction

mixture. I f the content of r ibosomes in the mixture was diminished, less po lymyxin B was necessary for their preci- pi tat ion. The observed precipitat ion of r ibosomes was preven- ted by 0.01 M l~{[gCI 2. Tile washed r ibosome-polymyxin pellet was soluble in 1.0 M NaC1. The la tent ribonuclease I of ribo- somes was precipitated together wi th the r ibosomes (100%) thereby remaining fully active against external yeast RNA. As detected b y zone centrifugation, in t0-r 70 S sedimenting particles were formed f rom 50 S r ibosomes in the presence of am oun t s of po lymyxin t3 not sufficient to precipitate ribosomes. These da ta suggest t h a t po lymyxin B acts like other polyamines or basic antibiotics (e. g. spermine or s t rep tomycin [~]): format ion of electrostatic bonds between its 5 free y-aminogroups of diamino butyr ic acid residues [5] and the negative charged phospha te groups of r ibosomal RNA at suitable p H and ionic force. The binding of po lymyxin B to yeast RNA was directly seen by thinlayer ch roma tography of acid hydrolysa tes of po lymyxin precipitated RNA. Only the 4 amino acids occurring in the po lymyxin B molecule (leucine, phenylalanine, threonine and diamino butyr ic acid [6]) were found, a t levels indicating a RNA-po lymyx in ]3 sulfate ratio of abou t I :f (weight:weight) . This ratio is very similar to the ratio reported previously [2] and agrees wi th the precipi tat ion pa t t e rn shown in Fig. t. Note added i~e proo]: While this manusc r ip t was in preparat ion, a paper was published describing the precipitat ion of ribo- somes f rom E. colt B by the po lymyxins 18, B1, B2, D 1, E 1 and E 2 [6]. The authors got results identical with those reported here.

Eingegangen am 1t. Oktober t966

* Present address: Department of Molecular Biology, Albert Ein- stein College of l\iedicine, New York 6t, N. Y. [1] NEWTON, B. A.: Bacteriol. Rev. 2o, 14 (1956). - - [2] LAr'TER- RAD~, C., and ~'[. MACH~BOmUF: Ann. Inst. Pasteur 78, 753 ( I 9 5 0 ) . - [8] T ~ u ~ , ~{.: Arch. Mikrohiol. ss, 3I ('I966). - - [4] P ~ T ~ A X ~ , M. L. : The physics1 and chemical properties of ribosomes. Amster- dam-London-New York: Elsevier PubI. Co. 1964. - - [5] Suzu~I, T., et al.: J. Biochem. s6, 335 (I964). - - [6] NAKAJ~MA, K., and J. Ka- WAS~ATA: Biken J. 9, 45 (t966).

M e l a n o p h o r e n - s t i m u l i e r e n d e W i r k s a m k e i t y o n A m i n o s ~ i u r e n

MANFRED GERSCH und ANN]tLI]~SE ]~ALTIN

Zoologisches In s t i t u t der Universitgt, Jena

Nach den vorliegenden ]3efunden kann das Pentapept id His- Phe-Arg-Try-Gly als sog. , t i e n n s e q u e n z " des MSI-I angesehen werden [1- -3] , wei Ies noch ffir dessen Wirknngscharak te r be- s t immend ist. Es wurde daher bier untersucht , ob und ge- gebenenfalls in welcher Weise einzelne Aminosgmren allein schon eine Wirkung auf die Melanophoren yon Amphibien auszui~ben vermOgen. Zur ~]berprCtfung gelangten in in-vitro- Testen an hypophyseek tomier ten R a n a escuZe~ta und in in- vivo-Versuchen mit Hautstt~ckchen die im a-MSH enthal tenen Aminosgmren in der L-Xonfiguration, teilweise diese auch in der D- und DL-Konfiguration. Der in-vitro-Test erwies sich als bedentend empfindlicher.

'11 I ,l'i L o / i f . i . i i l l 1 ~ r ' 5er ~ M~t i

5

in-v im- 7Dst

"'"L' ' "" i I i , , ~ . I i

His i Phe I Try i I Zy s I Pro I I/a/

q

3

2

' I I ' I o I i , , tr I 5er ' >~ell GIu

i n -v i i t o - Test

HIll i l I I , r r I i Ii li , i I , , i li , ~i

/ / is ' Pke ' X r f ' ' 5% ' Zgs ' Pro ' Va/

Fig. 1. Melanophoreu-Index (Ordinate) der verschiedenen Amino- s ~ u r e n . - - - - L - F o r m ; . . . . . D-Form; . . . . . DL-Form

Die Ergebnisse zeigt Fig. 2. Von den t3 im ~-MSH vorkom- menden Aminos~uren war L-Histidin die einzige, die sowohl in vivo als auch in vitro Expans ion der Melanophoren hervor- rief. AuBer dem maximalen Effekt dutch L-Histidin erwiesen sich in vitro v o n d e r Xennsequenz des Pentapepi ids n u t noch T r y t o p h a n (L-Try und DL-Try) und L-Arginin schwach wirksam. Bemerkenswer t ist weiterhin die deutliche Wirksam- keit yon L- und ~-Methionin, worauf schon frfihere Ergeb- nisse h indea ten [4]. t ieine der un te rsuchten Aminos~uren verursachte dagen umgekehr t eine l{onzentrat ion dispergier- ter Melaningranula.

Bet der Beurtei lung der Ergebnisse ist zwar nicht zu ilber- sehen, dab in allen FXllen die erforderliche ]s der wirksamen TestlOsungen (0, 5 %) unverb~ltnismXBig hoch liegt, und auBerdem tier erreichte Expans ionsgrad un te r dem dutch MSH erzielten zuri~ckbleibt. Andererseits sprechen eden doch nu t bes t immte Aminos~uren an. Obgleich diesen gewiB keine alleinige Spezifit~t zukommt, mug angenommen werden, dab sie wesentlich fflr die Wirkungsweise des Gesamtmolekfils mi tbes t immend stud. Demgegent~ber scheinen Phenylalanin und Glycin ffw diese Funkt ion der Kennsequenz entbehrlich zu seth. Bemerkenswer t ist weiterhin die relativ geringe ZahI wirksamer AminosNuren des MSH-Molekfils im Vergleich zu Ergebnissen am Uterus der Ra t te [5].

Eingegangen am 21. Oktober 1966

[1] LI, CH. H. : The Biochemical Aspects of hormone action, S. 4--659 (ed. by A. B. EISENSTEIN). Boston: Little, Brown & Co. 1964. - - [2] SC~{WYZER, R.: Ergeb. PhysioL biol. Chem. u. exptI. PharmakoI. 53, ~--4t (~963). [3] Sc}{wvzEJL R., u. C~. H. LI: Nature 182, 1669-- 1670 (1958). - - [4] Lo, T. B . , J .S . DIxoNu. CH.H. LI:Bioehem. et Biophys. Acta 53, 584--586 (196I). - - [5] GERSCH, M., ST. STOCK- LOSOWA U. K. RICHTER: Zool. Jahrb., Abt. Allg. Zool. u. Physiol. 73 (1967) (ira Druek).

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