Harry Beevers

The glyoxylate cycle as a stage in the conversion of fat to carbohydrate in castor beans

Abstract 1. 1. Cell-free supernatant fractions of castor beans catalysed the following reactions: 2. (a) the aldol fission of isocitrate, to produce equal amounts of glyoxylate and succinate; 3. (b) the activation of acetate in the presence of CoA and ATP; 4. (c) the condensation of acetyl CoA with oxaloacetate, to form citrate, and with glyoxylate, to form malate; 5. (d) the overall formation of malate from acetyl CoA and either citrate or isocitrate. 6. 2. These reactions of the glyoxylate cycle, when coupled with the reactions leading from fat to acetyl CoA, and from malate to carbohydrate, provide a route for the net conversion of fat to carbohydrate.

Metabolic production of sucrose from fat.

T HE production of sucrose from reserve fat occurs on a large scale in some germinating seeds. For example, an ungerminated castor bean contains some 260 mgm. of fat (70 per cent of its dry weight) and 15 mgm. of total carbohydratesl. During germination at 25° C. in the dark over a period of 8 days the total fat content of the seedling falls to about 50 mgm. while the carbohydrate rises to 230 mgm., and this is in spite of the fact that sugars are being used extensively in the growth and respiration of the seedling proper. Thus there is a production of more than 1 gm. of sugar (principally sucrose) for each gram of fat consumed. This interconversion is strictly confined to the endosperm tissue (respiratory quotient (RQ) about 0·35); sucrose moves into the growing seedling where it supports carbohydrate respiration (RQ = 1·0). In considering the pathway of this interconversion, we should note first that glycerol is converted quantitatively into sucrose in the endosperm and quite efficiently in the cotyledons also 2 • The labelling pattern in the hexose produced from glycerol-1,314C shows that the intact 3-carbon unit is incorporated, presumably after conversion to triose 2 • It should be clear, however, that the synthesis of sucrose from the glycerol moiety of the fat could account for only a ;;mall fraction of the conversion actually observed. Thus the problem resolves into the synthesis of sucrose from long-chain fatty acids. The metabolism of fatty acids in plant tissues has been intensively investigated by Stumpf et al. 3 • Although enzymic mechanisms inducing <X-oxidation engaged earlier attention, the metabolism of specifically labelled acids by tissue-slices pointed to the operation of a [3-oxidation pathway in vivo, and such a sequence has indeed been shown to occur in mitochondria from peanuts and other tissues when these were suitably fortified with co-factors. Formidable problems remain in elucidating the details of utilization of the unsaturated acids, but it seems safe to conclude that the breakdown results in the production of acetyl coenzyme A (CoA)3• Certainly the efficiency of the overall conversion of fat to carbohydrate is such that the loss of carbon as carbon dioxide during the production of 2-C units must be very small indeed. Now it has been established that acetate is utilized readily by slices of endosperm tissue•. In a 9-hr. experiment in which specifically labelled acetates were provided, it was found that at least 33 per cent of the added carboxyl carbon and 68 per cent of the added methyl carbon were converted to sucrose; the amounts converted to carbon dioxide were 46 per cent and 10 per cent, respectively. This net synthesis of sugar from acetate, with little diversion to other products, posed a problem analogous to that presented by those microbial cells which can grow on acetate as a sole source of carbon. The elucidation of the glyoxylate cycle5, by which a net synthesis of a 4-C acid such as malate can be achieved from two acetate units, offered a basis for its solution. An examination of castor bean tissues showed that the requisite enzymes, and particularly those newly described from micro-organisms, malate synthetase and isocitratase, were indeed present. A proposal was made, therefore, that the pathway of synthesis involved the production of a dicarboxylic acid by the glyoxylate cycle followed by conversion of malate into hexose•. Since this time we have carried out extensive experiments on tissues of fatty seedlings and extracts which have confirmed this suggestion and firmly establish the pathway of sucrose synthesis from acetate in such material. This evidence will now be summarized.

Transpiration, a prerequisite for long-distance transport of minerals in plants?

The major “benefit” alleged to accrue from transpiration (the evaporative loss of water from plant surfaces) is that it is essential for the long-distance transport of mineral ions, but the possible interrelation between these two processes has rarely been tested. Transpiration was experimentally dissociated from mineral supply by growing sunflowers (Helianthus anuus) in hydroculture and providing mineral nutrients only during the nights. These plants grew as well as a control group that received nutrients only during the day and transpired 12–15 times more water during the exposure period. It thus appears that convective water transport in the xylem, brought about by root pressure and the resultant guttation, “growth water,” and Münchs phloem counterflow is in itself sufficient for long-distance mineral supply and that transpiration is not required for this function.

The occurrence and assay of isocitrate lyase in algae

Abstract A modified method of isocitrate lyase assay is described which, coupled with the phenylhydrazone assay of keto acids, allows an accurate and definitive measurement of the enzyme activity. This eliminates the ambiguity associated with the use of only one assay of a nonspecific nature, such as the semicarbazone or phenylhydrazone methods. The detection of isocitrate lyase is described in acetate-grown cultures of Chlorella vulgaris, Chlamydomonas dysosmos, Chlorogonium elongatum, Gonium quadratum, Polytomella agillis , and Polytomella caeca . Its presence is very strongly indicated in Euglena gracilis , grown on acetate or ethanol, but cannot be conclusively established, due to complicating reactions which rapidly utilize glyoxylate. With the exception of the Polytomella species, all these organisms could be grown autotrophically, and under these conditions did not produce isocitrate lyase.

The glyoxylate cycle in Polytomella caeca

Abstract 1. 1. The two enzymes unique to the glyoxylate cycle, isocitrate lyase and malate synthase, have been shown to be present in Polytomella caeca . 2. 2. Malate synthase is virtually confined to the particulate fraction. Isocitrate lyase is predominantly cytoplasmic, but is present in small amounts in the particles. 3. 3. Tracer experiments showed malate to be the most highly labeled compound at short times after feeding acetate-2-C 14 . Radioactivity in malate, as a percentage of the total incorporated, was at its maximum before succinate reached its maximum, thus suggesting that malate did not arise primarily from succinate and therefore supporting the suggestion that the glyoxylate cycle was operating. An unexpectedly low level of labeling occurred in citric acid.

Trans-aconitate in plant tissues

Abstract Measurements of 14 C (from acetate- 14 C) in cis - and trans -aconitates show that 95 per cent of the total aconitate is present in the trans form in corn tissues. Trans -aconitate-1,5,6- 14 C was metabolized by corn and pea root sections but not by carrot discs. It is suggested that aconitate in corn tissues is out of equilibrium with other TCA cycle acids because it accumulates preponderantly in the trans form in the tissue.

Purification and properties of malate synthetase from castor beans

Abstract Malate synthetase has been purified some 300-fold from crude extracts of germinating castor-bean seedlings. The enzyme was completely inactive in the absence of divalent cations and was maximally activated by Mg++. The Km for acetyl coenzyme A was 6.7 · 10−5M and that for glyoxylate was 8.5 · 10−5M. None of a variety of aldehydes substituted for glyoxylate. No evidence was obtained that the enzyme could catalyze the breakdown of malate.

Lipoxidase and the oxygen absorption of homogenates from corn seedlings

Abstract Homogenates from 2 1 4 - day corn seedlings absorbed oxygen most rapidly at pH 5.0 and a second smaller maximum occurred at pH 7.2. The homogenates could be separated into two fractions, one of which contained lipoxidase and the other contained substrates for lipoxidase. Several lines of evidence led to the conclusion that the lipoxidase-substrate system accounts for the major portion of the oxygen absorption of the homogenate at pH 5.0 but at pH 7.2, other enzymes systems must contribute to the oxygen absorption.

[49] Proteins and phospholipids of glyoxysomal membranes from castor bean

Publisher Summary This chapter describes a method for obtaining proteins and phospholipids of glyoxysomal membranes from castor bean. For an accurate analysis of protein and phospholipid components of membranes of an organelle, the first requirement is that the organelle fraction should be uncontaminated with other cell constituents. In the particular case of glyoxysomes from castor bean endosperm, this requirement is close to being realized, because this plant material yields a remarkably good separation of major organelles in a single linear sucrose density gradient. On such a gradient the glyoxysomes are found as a clear separate protein band at equilibrium density 1.25 g/nil and they are virtually uncontaminated by other organelles or membranes, as revealed by the measurements of marker enzymes and the uniformity of structures in electron micrographs. The glyoxysomes are seen in such pictures as spherical organelles surrounded by a single unit membrane. There are no membranes within the amorphous matrix. The second requirement for an accounting of membrane constituents is that, the matrix components are completely removed.

Oxidation of N-acetylindoxyl by an enzyme from plants☆

Abstract Some properties of an enzyme system from plants which is capable of oxidizing N-acetylindoxyl have been described. The final product is tentatively identified as acetylisatic acid, and since the same product is obtained when the enzyme is allowed to act on N-acetylisatin (which can be derived by chemical oxidation of N-acetylindoxyl) this is supposed to be the primary product when N-acetylindoxyl is oxidized by the enzyme.