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p-Hydroxy-DL-phenylglycin 16 , farblose Nadeln aus n-Propanol/Wasser 3 : 1 , Schmp. 215 —216 °C, Zers., 46 mg ( = 55% d. Theorie). 3.5-Dihydroxy-DL-phenylglycin, farblose Nadeln aus n-Propanol/Wasser 3 : 1 , Sdimp. 232 — 234 °C, Zers., 23 mg ( = 25,1% d. Theorie). Elementaranalyse: C 8 H 9 N0 4 Gef. C 52,24 H 5,11 Ber. C 52,45 H 4,92 N 7,89, N 7,65. Herrn Prof. Dr. K. MOTHES danken wir für ständig anregende Diskussionen. Acid Carboxypeptidases: Their Occurrence in Plants, Intracellular Distribution and Possible Function H. ZUBER Department of Molecular Biology and PH. MATILE Department of General Botany Swiss Federal Institute of Technology Zurich, Switzerland (Z. Naturforsch. 23 b, 663—665 [1968] ; eingegangen am 17. November 1967) Saure Carhoxypeptidase, welche Phenylalanin von CbO-Leu-Phe-OH bei pH 5,3 abspaltet, ist ein in höheren Angiospermen weit verbreitetes Enzym. Es scheint dagegen in primitiven Angiospermen (Fagus und Magnolia) sowie in Gymnospermen und Kryptogamen (Ausnahme: Equisetum) zu fehlen. Bei Zitrone ist die Aktivität saurer Carboxypeptidase von Natur und Alter der extrahierten Gewebe abhängig (Exocarp und Endocarp der Früchte, Blätter). In Zellen der Wurzelspitzen von Maiskeimlingen ist das Enzym in meristematischen Vakuolen lokalisiert. Funktionell ist die saure Carboxypeptidase als lysosomales Enzym, welches am intrazellulären Proteinabbau teilnimmt, anzusprechen. 1 has purified and characterized a carboxypeptidase extracted from the exocarp of citrus fruit. A study of the occurrence of similar enzymes in various organisms of the plant kingdom has now yielded evidence that carboxypeptidases with a pH optimum around 5 are common constituents of many angiospermous plants (Table 1 ) . It is unlikely that the carboxypeptidases which occur in different plant species are identical with respect to structure, activity and specificity; rather would it seem that they belong to a family of enzymes, subsequently termed acid carboxypeptidases, which hydrolyze typical substrates at a pH of 5.3. With two exceptions all of the angiospermous plants tested so far contain acid carboxypeptidase activity: from the point of view of phylogenetic classification, it should be noted that Fagus and Magnolia, which lade this enzyme, belong to the primitive orders of Fagales and Polycarpicae. CarboxypeptiZUBER 1 H. ZUBER, Nature [London] 201, 613 [1964]. 2 F . FELIX and J . LABOUESSE-MERCOUROFF, Biochim. b i o p h y s i c a Acta [Amsterdam] 21, 303 [1956]; F.FELIX and N.BRUILLET 122, 127 [1966]; 139, 382 [1967], K . MORIHARA, T.OKA, and H . TSUZUKI dase is also absent in extracts from needles of the gymnosperm Picea. Of some Cryptogams tested the horse-tail Equisetum contains acid carboxypeptidase; considering the phylogenetic situation of the Equisetales, it appears that acid carboxypeptidases may have originated independently at the evolutionary levels of the Pteridophyta (class of Equisetales which has not passed through further development) and of the Angiosperms. Carboxypeptidase activity, finally, is present in the thermophilic fungi Talaromyces duponti (Ascomycetes) Humicula lanuginosa (Deuteromycetes); these enzymes, however, belong to a different family of alkaline carboxypeptidases since they are optimally active at pH 8 — 9. Similar enzymes have recently been isolated from yeast and Streptomyces Fradiae 2 . The total carboxypeptidase activity which is extractable from a certain mass of plant material seems to be dependent both on the species of plant and on the kind of tissue used. It appears from the data listed in Table 1 that a very high yield was obtained by extracting cauliflower buds, a tissue which consists almost exclusively of metabolically very active meristematic cells. Unauthenticated Download Date | 6/18/17 2:37 PM Plant Tissue Yield Units per 100 g of fresh weight C r values Acid carboxypeptidase Artichoke (Cynara scolymus) Tomato (Solanum lycopersicum) Orange (Citrus aurantiacum) Pea (Pisum sativum) Cauliflower (Brassica oleracea) Spinach (Spinacia oleracea) Nettle (Urtica dioica) Corn (Zea mais) Beech (Fagus silvatica) Magnolia spec. Spruce (Picea excelsa) Horse-tail (Equisetum arvense) Fern (Athyrium filix-femia) Talaromyces du ponti Bracts Fruit Fruit, Exocarp Seedling Flowerbuds Leaf Leaf Seedling Leaf Leaf Needle Shoot Sporophyll Mycelium 1 619 150 600 400 11 692 4 761 300 2 090 0 0 0 114 0 0 0.020 0.023 0.040 0.0004 0.042 0.016 0.005 0.002 0 0 0 0.012 0 0 Alkaline carboxypeptidase Talaromyces du ponti Mycelium 17 300 0.143 Table 1. Occurrence of carboxypeptidases in plants. The tissues were extracted with 2.3% NaCl as described previously Precipitation of the protein with (NH 4 ) 2 S0 4 (70% saturation) yielded a crude enzyme product which was dialyzed against water and lyophilized. The determination of the carboxypeptidase activity present in 1 mg of crude product was based on the hydrolysis of CbO-Leucyl-phenyl- alanine-OH (2 x 1 0 ~ 2 M substrate in 0.1 M Na-citrate-buffer pH 5.3; production of phenylalanine in 10, 30, 60 and 120 min of incubation at 37 °C). Q-values (proteolytic coefficient) represent the quotient of kx (velocity constant of first order reaction) and E (concentration of protein in mg protein-N). (1 u n i t = C t x /ug protein-N; 15% N.) In lemon the exocarp of very young fruits contains twice as much carboxypeptidase than the same tissue of mature fruits (Table 2). Thus the carboxypeptidase content seems to be related to the metabolic activity of cells. In citrus not only the fruit but experiments had shown that approximately onethird of the total activity present in an extract from corn seedlings (grown under sterile conditions at 28 °C for 40 hrs) is sedimentable. An even higher proportion of the enzyme is structurally bound in extracts from root tips (approx. 5 mm long), prepared by grinding the tissue in the presence of sand and a medium containing 0.5 M sorbitol, 0.05 M tris-HCl-buffer pH 7.6 and 1 mM EDTA. Differential centrifugation of the low-speed supernatant of a root tip extract yielded a mitochondrial (15 min 20,000 g ) , a microsomal (30 min 150,000 g) and a soluble fraction. Carboxypeptidase activity is present in all of these subcellular fractions; thus, the enzyme appears to be both soluble and structurally bound (Table 3 ) . The highest specific activity is present in the mitochondrial fraction which, according to a previous study 3 contains particles carrying a considerable number of lysosomal enzymes. Several types of lysosomes can be distinguished; one of them is identical with small vacuoles which, in contrast to large vacuoles, are not destroyed when the tissue is ground. These vacuoles are not sedimentable in media which contain even low concentrations of Tissue Size of fruit Exocarp Endocarp Exocarp Exocarp 2— 4 cm 2— 4 cm 6 cm 10 — 15 cm Leaf Leaf Age 1 year 2 years Colour of exocarp dark-green — yellowish-green yellow Yield Units C1-values per 100 g 3 419 709 2 787 1 606 0.066 0.066 0.100 0.093 8 666 12 329 0.130 0.093 Table 2. Carboxypeptidase activities in various tissues of lemon. Experimental conditions are the same as indicated in Table 1. also the leaf contains the enzyme: in contrast to the above findings, more activity is present in extracts from old leaves. Of all the tissues tested so far the crude enzyme product from old lemon leaves contained the highest carboxypeptidase activity. In order to elucidate the functional significance of the acid carboxypeptidase its intracellular distribution was studied in a suitable object. Preliminary 3 PH. MATILE, Planta 79, 181 [1968]. Unauthenticated Download Date | 6/18/17 2:37 PM Fraction Specific activity Cell-free extract Mitochondrial fraction Microsomal fraction Soluble fraction Light lysosomes (Meristematic vacuoles) Heavy lysosomes 2.221 1.415 0.641 1.751 11.55 6.10 Table 3. Carboxypeptidase activity in subcellular fractions from root tips of corn seedlings. The activities are expressed as /u.M phenylalanine produced in 60 min per mg of protein. surcrose; the other types of lysosomes, however, sediment ealisy in 0.4 M sucrose. Hence, two classes of lysosomes can be obtained from a mitochondrial fraction which is overlayered on a discontinuous gradient with steps of 0.4 and 1.17 M sucrose; upon centrifugation (2 hrs at 39,000 rpm in a Spinco SW 39 rotor) the mitochondria form a pellet, whilst heavy and light lysosomes are trapped at the surface of 1.17 and 0.4 M sucrose, respectively. The data in Table 3 show that carboxypeptidase is present in both types of lysosomes. The highest specific acti vity is contained in the light lysosomes — that is, in meristematic vacuoles. According to early observations of cytologists4 the large vacuole of expanded (differentiated) plant cells originates from tiny vacuoles of meristematic cells. It may therefore be assumed that the enzyme acivity found in the soluble cell fraction is localized in vacuoles large enough to be disrupted during homogenization of the cells. This assumption is sup ported by the finding that the lysosomal enzymes 4 A. G 5 u i l l i e r m o n d , G . M a n g e n o t , and L. P l a n t e f o l , Traite de Cytologie vegetale, Le Frangois, Paris 1933. P h . M a t i l e and A. W ie m k e n , Arch. Mikrobiol. 56, 148 [1967]. present in preparations of isolated vacuoles of yeast cells are completely soluble if the cells are submitted to a procedure of homogenization which causes the rupture of these delicate organelles5. Functionally, the acid carboxypeptidases seem to be involved in the intracellular digestion of protein, a process which, together with protein synthesis, is responsible for the phenomenon of protein turnover. From the localization of this and many other lysosomal enzymes in meristematic vacuoles 6 it must be con cluded that this organelle represents the locus of intracellular digestion. The acid carboxypeptidases of higher plant cells seem to represent lysosomal enzymes analogous to what were formarly termed the cathepsins 7 of ani mal cells. In contrast to their alkaline counterparts (intestinal carboxypeptidase A and B, fungal carb oxypeptidase) acid carboxypeptidases seem not to be metalloenzymes (no inhibition with E D T A ). They seem to function intracellularly whereas the neutral or alkaline carboxypeptidases rather re present secretion products with an extracellular function. Acid and alkaline carboxypeptidases have different active sites, but since they catalyze the same reaction they are analogous enzymes. The authors are indebted to D r. P. D o h rn (director of the Zoological Station, Naples, Italy) for kindly permitting them to use his laboratory facilities, to the Swiss National Science Foundation (projects no. 3426 and 4393) for financial support, and to CIBA Ltd., Basel, for supplying the strains of fungi employed in this present study. 6 P h . M a t i l e , Z. Naturforschg. 21b, 871 [1966]. 7 J. S . F r u t o n , G . W. I r v i n g , and istry 141, 773 [1941] ; L. M . 237, 1082 [1962]. M. B G J. biol. Chem and R. S h e r m a n ergm ann, reenbaum Unauthenticated Download Date | 6/18/17 2:37 PM