Oscar Touster

Alterations in tissue levels of uridine diphosphate glucose dehydrogenase, uridine diphosphate glucuronic acid pyrophosphatase and glucuronyl transferase induced by substances influencing the production of ascorbic acid

Chemical agents known to enhance the production of l-ascorbic acid in animals were studied in vivo to determine their effects on tissue levels of three enzymes which may be concerned with the biosynthesis of the vitamin. The agents were found to have differential effects on the enzymes tested. Barbital and Chloretone, but not other enhancing agents, increase the activity of UDPG dehydrogenase in liver extracts. This alternation in activity is prevented by ethionine and by hypophysectomy. The guinea-pig enzyme was similarly affected by the two drugs, although ascorbic acid is not produced. Carcinogenic hydrocarbons and aminopyrine increase the activity of glucuronyl transferase in liver. The hepatic level of UDP-glucuronic acid pyrophosphatase, on the other hand, does not respond appreciably to the enhancing agents, although ethionine and barbital administered together elevate this enzyme in liver without stimulating ascorbic acid excretion. In vitro, barbiturates have an inhibitory action on the pyrophosphatase. The alterations in enzyme activities are discussed in relation to the possibility that ascorbic acid is synthesized via a pathway involving glucuronyl transferase rather than UDP-glucuronic acid pyrophosphatase.

[8] Solubilization and polyacrylamide gel electrophoresis of me mbrane enzymes with detergents

Publisher Summary Polyacrylamide gel electrophoresis is an excellent and widely employed method for the separation and identification of soluble enzymes, but membrane proteins, including enzymes, present difficulties because of their presumed lipoprotein character. Large lipoprotein molecules or aggregates are unlikely to enter gels composed of 5–10% acrylamide that is the best compromise between firmness and large pore size, or, if they enter, fail to resolve satisfactorily. Consequently, it is necessary to solubilize or disaggregate the membrane proteins prior to electrophoresis. Disaggregation or solubilization has most often been accomplished by the method of Takayama or modifications in which membranes are treated with phenol, acetic acid, and urea, subsequent electrophoresis being accomplished in gels containing 5 M urea and 35 % acetic acid.

The similar effects of swainsonine and locoweed on tissue glycosidases and oligosaccharides of the pig indicate that the alkaloid is the principal toxin responsible for the induction of locoism

A neurological condition resembling that observed in hereditary mannosidosis occurs in animals ingesting spotted locoweed and plants of the genus Swainsona. Swainsonine has been isolated from these plants and has been suggested to be the primary causative agent in inducing the pathological condition. This alkaloid has also been found to increase tissue acid alpha-D-mannosidase levels in rats while lowering liver Golgi mannosidase II levels. In the present study, the effects of locoweed and swainsonine were directly compared for the first time, with the pig as experimental animal. Both increased most lysosomal acid glycosidase activities in most tissues, decreased liver Golgi mannosidase II levels, increased plasma hydrolase levels, and greatly increased tissue oligosaccharide, especially Man5GlcNAc2 and Man4GlcNAc2. These results indicate that swainsonine is the agent in locoweed responsible for the enzymatic and oligosaccharide changes. The behavior of the animals was also similarly affected by swainsonine and locoweed.

The solubilization and gel electrophoresis of membrane enzymes by use of detergents

Abstract The solubilization and electrophoresis of membrane proteins with phenol and acetic acid or urea usually have deleterious effects on enzyme activities. A method is reported of solubilizing rat-liver plasma membrane proteins, by the use of the detergents sodium dodecyl sulfate, sodium deoxycholate, and Triton X-100, for subsequent electrophoresis of the enzymes on 7% polyacrylamide gel. An important feature of the method is the incorporation of detergent into the gel and the electrophoresis buffer. By use of this procedure, non-specific esterases, an alkaline phosphatase, and alkaline phosphodiesterase are detectable on the gels. The method is compared with other techniques currently being used, and it would appear that this method will have general usefulness in the study of membrane enzymes.

Marked differences in the swainsonine inhibition of rat liver lysosomal α-d-mannosidase, rat liver golgi mannosidase II, and jack bean α-d-mannosidase

Abstract Swainsonine, a plant toxin, strongly inhibits certain α- d -mannosidases but has no effect on others [ D. R. P. Tulsiani, T. M. Harris, and O. Touster (1982) J. Biol. Chem.257, 7936–7939]. The reversible inhibition of jack bean and lysosomal α- d -mannosidases has previously been suggested to be similar in nature but quite complex. Specific differences in the action of swainsonine on these two enzymes and on Golgi mannosidase II are reported. (a) The inhibition of the jack bean mannosidase, but not rat liver lysosomal α- d -mannosidase or Golgi mannosidase II, is increased by preincubation with the alkaloid. (b) The inhibition of the jack bean and lysosomal enzymes, but not mannosidase II, is competitive at inhibitor concentrations of ⩽0.5μ m . (c) The inhibition of jack bean α-mannosidase is largely irreversible, its very limited reversibility being partially dependent upon the swainsonine concentration used and on the time of preincubation with the inhibitor. On the other hand, the inhibition of lysosomal α-mannosidase is largely reversible, as shown by dilution experiments and by the use of [3H]swainsonine. Golgi mannosidase II shows intermediate reversibility, the results indicating two modes of binding; one rapid and irreversible, the other much slower and reversible.

Production of hybrid glycoproteins and accumulation of oligosaccharides in the brain of sheep and pigs administered swainsonine or locoweed.

Swainsonine and swainsonine-containing plants produce biochemical and neurological changes in several mammalian species. The toxin is a potent inhibitor of liver lysosomal alpha-D-mannosidase and Golgi mannosidase II. The inhibition of the latter enzyme causes the production of abnormal glycoproteins containing hybrid oligosaccharides instead of complex types in a variety of cultured cells. In view of the widespread occurrence and biological importance of N-linked glycoproteins in the central nervous system, we initiated studies to determine the structure of oligosaccharides in glycoproteins prepared from the brain of control, swainsonine-fed, and locoweed-fed animals. The results presented here indicate that the feeding led to alteration in the structure of brain glycoproteins. Over 25% of the glycoproteins which presumably contained complex-type oligosaccharides were modified and now contained hybrid oligosaccharides. The structure of the N-linked oligosaccharide (glycopeptide) was established by (a) studying the binding properties of the glycopeptide to immobilized lectins of known sugar specificity, and (b) comparing the size of the glycopeptide before and after treatment with exo- and endoglycosidases. The production of hybrid oligosaccharides occurred despite the apparent absence of mannosidase II in brain. The relationships of the altered structure of brain glycoproteins, accumulation of mannose-rich oligosaccharides in the brain, and abnormal behavior of the animals administered swainsonine or locoweed are discussed.

Swainsonine, a potent mannosidase inhibitor, elevates rat liver and brain lysosomal α-d-mannosidase, decreases Golgi α-d-mannosidase II, and increases the plasma levels of several acid hydrolases

Swainsonine, a toxic plant alkaloid reported to be the agent that induces in animals a neurological condition very similar to the hereditary lysosomal storage disease mannosidosis, and to inhibit the formation of complex glycoproteins of the asparagine-linked class, was recently shown [D. R. P. Tulsiani, T. M. Harris, and O. Touster, (1982) J. Biol. Chem.257, 7936–7939] to be a highly potent and specific inhibitor of Golgi mannosidase II in addition to being a strong inhibitor of lysosomal mannosidase. In the present study the effect of administered swainsonine on tissue enzyme levels was investigated. The activity of Golgi mannosidase II was markedly decreased (22% of control) without changes occurring in the activities of several other Golgi enzymes. However, the effects of swainsonine on lysosomal enzymes was unexpected. In liver, acid mannosidase increased markedly, instead of decreasing as would be expected from a compound reported to induce a mannosidosis-like condition. Similarly, the principal change in brain was a substantial increase in lysosomal mannosidase levels. In plasma, most lysosomal enzymes increased. These results indicate that the pathological effects of swainsonine are not solely attributable to its being an inhibitor of lysosomal α-d-mannosidase and are probably a consequence of abnormal processing of glycoproteins.

Biochemical characterization of the lysosomes of ehrlich ascites tumor cells

Abstract 1. 1. The presence of lysosome-like particles in Ehrlich ascites tumor cells has been demonstrated by the use of biochemical methods. The intracellular distribution pattern of 4 lysosomal marker enzymes (deoxyribonuclease II, aryl sulfatase, acid phosphotase, and β-galactosidase) and the demonstration of the latency of these enzymes were the two major criteria applied to the identification of the organelles in the particulate fraction obtained by differential centrifugation. Studies on the physical properties of these lysosomes revealed an increased stability as compared to similar particles of rat liver and kidney. The tumor cell lysosomes are further distinguished from those of liver, for example, by having a lower density than mitochondria, the liver lysosomes being denser than mitochondria. 2. 2. A partial purification of deoxyribonuclease II from Ehrlich ascites cells was undertaken because of the contradictory reports in the literature concerning the presence of the enzyme in these cells. The results show that the enzyme is indeed present and displays the characteristics of the deoxyribonuclease II found in normal tissues.

Changes in plasma hydrolase activities in hereditary and streptozotocin-induced diabetes.

Abstract Reports of elevated plasma levels of acid hydrolases in diabetic patients prompted us to investigate these enzymes in genetically diabetic (db/db) mice and in streptozotocintreated rats and mice. The homozygous (db/db) mice showed decreased, rather than increased, levels of plasma hydrolases as compared to their heterozygous (db/+) nondiabetic controls. Similarly, mice (Swiss and db/+) with streptozotocin-induced diabetes showed lowered plasma hydrolase activities. On the other hand, drug-induced diabetes in rats was accompanied by increased hydrolase levels, the increase being reversed by insulin treatment. In investigating the underlying mechanism of the changes observed in rats, leukocytes and blood platelets were ruled out as sources of the additional plasma lysosomal enzymes, and evidence was obtained suggesting that the diabetic animal was normal in regard to the ability to remove lysosomal glycosidase from blood. However, perfusion of diabetic liver resulted in release of more acid hydrolase activity than did perfusion of normal liver, and perfusion with glucagon stimulated enzyme release. These results suggest that liver is a possible source of the added enzyme found in streptozotocin-treated rat plasma.

Some aspects of the cellular biochemistry of lysosomal and related glycosidases

Glycosidases are particular objects of contemporary research chiefly for three reasons: a) interest in lysosomal processes and lysosomal diseases, b) interest in understanding the basis for the occurrence of abnormal glycoproteins and glycolipids on the surface of tumor cells, and c) the value of purified glycosidases in structural studies on glycoproteins and glycolipids. In this essay we shall focus on questions concerning the characteristics and origin of these enzymes and on methodological questions relevant to current investigations in mammalian systems. Rather than reviewing the extensive and diverse literature, we shall cite only those reports which are relevant to this effort to appraise our understanding of certain aspects of the cellular biochemistry of glycosidases. Lysosomes are now recognized as being involved in a considerable number of important biological processes. The capacity of lysosomes to degrade extensively proteins 1, mucopolysaccharides 2, glycoproteins 2, 3, glycolipids, and nucleic acids 4 is now well established. Although, with one exception, a myeloperoxidase in one class of lysosomes in polymorphonuclear leukocytes 5, all of the enzymes that have been conclusively shown to be lysosomal are hydrolases, the degradative action of these enzymes of course does not limit their functions to what may be termed purely digestive actions. In addition to