S nutrition affects the pools of S available to developing grains of wheat

Developing grains of rain-fed wheat must obtain S from reserves that the plant accumulated during vegetative growth. The effect of S nutrition on the sources of S that were transported from vegetative tissues during grain development was examined. Wheat was grown in solution culture with 2 mM N and either 200 M S (high-S) or 50 M S (low-S). All nutrients were withdrawn either at booting, ear emergence, anthesis, or 8 d post anthesis. At 13 d post anthesis, high-S plants contained sulphate in roots and leaves, GSH (glutathione) in leaves, and S in leaf proteins. Between harvests, sulphate was lost from roots and GSH was lost from leaves. Sulphate and GSH were not detected in roots or leaves of low-S plants at 13 d post anthesis, and between harvests, S in leaf-proteins decreased During reproductive growth, redistribution of sulphate from roots was likely to be in response to S demand, redistribution of GSH was likely to result from the breakdown of chloroplasts, and in low-S plants, hydrolysis of leaf proteins in response to N demand was the most important source of S for grain development.     

Sulphur nutrition changes the sources of S in vegetative tissues of wheat during generative growth

During generative growth, developing wheat grains require nitrogen and sulphur to synthesize storage proteins. The hypothesis that the S required for grain growth can be derived from vegetative tissues was examined by growing plants in nutrient culture containing either 50 M S (low-S) or 200 M S (high-S) and terminating the nutrient supply at various times during generative growth. After terminating the nutrient supply, high-S plants redistributed soluble S to developing grains from pools in roots and leaves, whereas low-S plants remobilized insoluble S (protein-S) from the leaves to the grains. A model for the cycling of S within mature leaves during generative growth is presented.     

Novel kinetics of mammalian glutathione synthetase: characterization of gamma-glutamyl substrate cooperative binding

Glutathione (GSH) synthetase [L-gamma-glutamyl-L-cysteinyl:glycine ligase (ADP-forming), EC 6.3.2.3] catalyzes the final step in GSH biosynthesis. Mammalian glutathione synthetase is a homodimer with each subunit containing an active site. We report the detailed kinetic data for purified recombinant rat glutathione synthetase. It has the highest specific activity (11 micromol/min/mg) reported for any mammalian glutathione synthetase. The apparent K(m) values for ATP and glycine are 37 and 913 microM, respectively. The Lineweaver-Burk double reciprocal plot for gamma-glutamyl substrate binding revealed a departure from linearity indicating cooperative binding. Quantitative analysis of the kinetic results for gamma-glutamyl substrate binding gives a Hill coefficient (h) of 0. 576, which shows the negative cooperativity. Neither ATP, the other substrate involved in forming the enzyme-bound gamma-glutamyl phosphate intermediate, nor glycine, which attacks this intermediate to form GSH, exhibit any cooperativity. The cooperative binding of gamma-glutamyl substrate is not affected by ATP concentration. Thus, mammalian glutathione synthetase is an allosteric enzyme.     

Demand-Driven Control of Root ATP Sulfurylase Activity and SO42- Uptake in Intact Canola (The Role of Phloem-Translocated Glutathione)

The activity of ATP sulfurylase extracted from roots of intact canola (Brassica napus L. cv Drakkar) increased after withdrawal of the S source from the nutrient solution and declined after refeeding SO42- to S-starved plants. The rate of SO42- uptake by the roots was similarly influenced. Identical responses were obtained in SO42- -fed roots when one-half of the root system was starved for S. The internal levels of SO42- and glutathione (GSH) declined after S starvation of the whole root system, but only GSH concentration declined in +S roots of plants from split root experiments. The concentration of GSH in phloem exudates decreased upon transfer of plants to S-free solution. Supplying GSH or cysteine to roots, either exogenously or internally via phloem sap, inhibited both ATP sulfurylase activity and SO42- uptake. Buthionine sulfoximine, an inhibitor of GSH synthesis, reversed the inhibitory effect of cysteine on ATP sulfurylase. It is hypothesized that GSH is responsible for mediating the responses to S availability. ATP sulfurylase activity and the SO42- uptake rate are regulated by similar demand-driven processes that involve the translocation of a phloem-transported message (possibly GSH) to the roots that provides information concerning the nutritional status of the leaves.     

Analysis of glutathione-enhanced differentiation by microfilariae of Onchocerca lienalis (Filarioidea: Onchocercidae) in vitro

Reduced glutathione (GSH), but not its oxidized form (GSSG), stimulated development of Onchocerca lienalis microfilariae to the late first-larval stage in vitro. The degree and frequency of development was dose-related with a peak of activity at 15 mM, a concentration that is similar to known intracellular levels of GSH. To determine the mode(s) of action of this multifunctional compound, other reducing agents (L-cysteine, dithiothreitol), cysteine delivery agents (N-acetyl-L-cysteine, L-thiazolidine-4-carboxylic acid, L-2-oxothiazolidine-4-carboxylic acid), cysteine analogues (S-methyl-L-cysteine, D-glucose-L-cysteine, cysteine ethyl ester), free-component amino acids of GSH (glutamic acid, cysteine, and glycine), a specific metabolic inhibitor of gamma-glutamyl synthetase (buthionine sulfoximine), and an inhibitor of gamma-glutamyl transpeptidase (gamma-glutamyl glutamic acid) were also tested at concentrations of 0.01-50 mM in this system. N-acetyl-L-cysteine at 1-5 mM and D-glucose-L-cysteine at 2.5-10 mM significantly enhanced development. In contrast to those worms maintained in GSH-supplemented medium, microfilariae exposed to GSH for only the first 24 hr showed no enhancement by day 7 in culture. Neither buthionine sulfoximine nor gamma-glutamyl glutamic acid at 0.01-35 mM inhibited the effects of 15 mM GSH or 1 mM N-acetyl-L-cysteine. Results indicate that GSH or other cysteine analogues possessing a free sulfhydryl group must be present in the extranematodal environment to support microfilarial differentiation in vitro.     

Glutathione-mediated transport across intestinal brush-border membranes

Glutathione transport was studied in brush-border membrane vesicles of rabbit small intestine in which gamma-glutamyl transpeptidase (EC 2.3.2.2) had been inactivated by a specific affinity-labeling reagent, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT125). Transport of intact [glycine-2-3H]GSH occurred into an osmotically active intravesicular space of AT125-treated membranes. The 0.1 M NaSCN gradient (Na+ inside greater than Na+ outside) in the transport medium could be replaced with KSCN or NaCl without affecting transport activity. The initial rate of GSH transport followed Michaelis-Menten saturation kinetics (Km = 17 microM). The results suggest that, in these membranes, there was an Na+-independent mediated transport for intact GSH with marked specificity and affinity. In fact glycine, glutamic acid and cysteine did not decrease GSH uptake, as was also true for glycylglycine and glycylglycylglycine; only gamma-glutamylcysteinylglycyl ester, a derivative of GSH, partially inhibited GSH transport.     

Insights into the effects of alpha-synuclein expression and proteasome inhibition on glutathione metabolism through a dynamic in silico model of Parkinson's disease: validation by cell culture data

Dopaminergic neurodegeneration during Parkinson disease (PD) involves several pathways including proteasome inhibition, alpha-synuclein (alpha-syn) aggregation, mitochondrial dysfunction, and glutathione (GSH) depletion. We have utilized a systems biology approach and built a dynamic model to understand and link the various events related to PD pathophysiology. We have corroborated the modeling data by examining the effects of alpha-syn expression in the absence and presence of proteasome inhibition on GSH metabolism in dopaminergic neuronal cultures. We report here that the expression of the mutant A53T form of alpha-syn is neurotoxic and causes GSH depletion in cells after proteasome inhibition, compared to wild-type alpha-syn-expressing cells and vector control. Modeling data predicted that GSH depletion in these cells was due to ATP loss associated with mitochondrial dysfunction. ATP depletion elicited by combined A53T expression and proteasome inhibition results in decreased de novo synthesis of GSH via the rate-limiting enzyme gamma-glutamyl cysteine ligase. Based on these data and other recent reports, we propose a novel dynamic model to explain how the presence of mutated alpha-syn protein or proteasome inhibition may individually impact on mitochondrial function and in combination result in alterations in GSH metabolism via enhanced mitochondrial dysfunction.     

Catalytic and regulatory properties of sulphur metabolizing enzymes in cyanobacterium Synechococcus elongatus PCC 7942

Synechococcus elongatus PCC 7942 was able to grow with several S sources. The sulphur metabolizing enzymes viz. ATP sulphurylase, cysteine synthase, thiosulphate reductase and L- and D-cysteine desulphydrases were regulated by sulphur sources, particularly by sulphur amino acids and organic sulphate esters. Sulphur starvation reduced ATP sulphurylase and cysteine synthase whereas reduced glutathione appreciated Cys degradation activity. With partially purified enzymes apparent Km values for sulphate, ATP, D- and L-Cys, thiosulphate, sulphide and O-acetyl serine were in a range of 12-50 microM. p-Nitrophenyl sulphate inhibited ATP sulphurylase competitively. Met was a feedback inhibitor of several key enzymes.     

Glutamate synthase: a possible role in nitrogen metabolism of the developing maize endosperm

Glutamate synthase activity was demonstrated in the developing endosperm of maize (Zea mays L.). The enzyme shows specificity for glutamine and alpha-ketoglutarate as amino donor and acceptor, respectively. Both NADH and NADPH function as electron donors although lower activities were often, but not always, obtained with NADPH. The apparent K(m) values for glutamine, alpha-ketoglutarate, and NADH were 1.35 mm, 0.57 mm, and 7 mum, respectively.The pattern of activity during endosperm development revealed a well defined peak coinciding with the period of most active N accumulation. Activity in general was related to the rates of accumulation throughout development. Maximum glutamate synthase activity was the order of 56 nmoles of glutamate formed per minute per endosperm compared with a rate of N accumulation of 9.5 nmoles per minute.It is suggested that glutamate synthase plays a key role in the N nutrition of the maize endosperm providing a means whereby N transported in the form of glutamine is made available for the synthesis of other seed protein amino acids via transaminase reactions. Transaminase activity involving glutamate, the product of the glutamate synthase reaction, was also demonstrated.     

Unique characteristics of rat spleen lymphocyte, L1210 lymphoma and HeLa cells in glutathione biosynthesis from sulfur-containing amino acids

Suspensions of rat spleen lymphocyte, murine L1210 lymphoma and HeLa cells were partially depleted of glutathione (GSH) with diethyl maleate and allowed to utilize either [35S]methionine, [35S]cystine or [35S]-cysteine for GSH synthesis. Lymphocytes preferentially utilized cysteine, compared to cystine, at a ratio of about 30 to 1, which was not related to differences in the extent of amino acid uptake. Only HeLa cells displayed a slight utilization of methionine via the cystathionine pathway for cysteine and GSH biosynthesis. HeLa and L1210 cells readily utilized either cystine or cysteine for GSH synthesis. The three cell types accumulated detectable levels of intracellular cysteine glutathione mixed disulfide when incubated in a medium containing a high concentration of cystine. Various enzyme activities were measured including gamma-glutamyl transpeptidase, GSH S-transferase and gamma-cystathionase. These results support the concept of a dynamic interorgan relationship of GSH to plasma cyst(e)ine that may have importance for growth of various cell types in vivo.     

Glutathione as an endogenous sulphur source in the yeast Saccharomyces cerevisiae.

Glutathione-deficient mutants (gshA) of the yeast Saccharomyces cerevisiae, impaired in the first step of glutathione (GSH) biosynthesis were studied with respect to the regulation of enzymes involved in GSH catabolism and cysteine biosynthesis. Striking differences were observed in the content of the sulphur amino acids when gshA mutants were compared to wild-type strains growing on the same minimal medium. Furthermore, all mutants examined showed a derepression of gamma-glutamyltranspeptidase (gamm-GT), the enzyme initiating GSH degradation. However, gamma-cystathionase and cysteine synthase were unaffected by the GSH deficiency as long as the nutrient sulphate source was not exhausted. The results suggest that the mutants are probably not impaired in the sulphate assimilation pathway, but that the gamma-glutamyl cycle could play a leading role in the regulation of the sulphur fluxes. Studies of enzyme regulation showed that the derepression of gamma-GT observed in the gshA strains was most probably due to an alteration of the thiol status. The effectors governing the biosynthesis of cysteine synthase and gamma-cystathionase seemed different from those playing a role in gamma-GT regulation and it was only under conditions of total sulphate deprivation that all these enzymes were derepressed. As a consequence the endogenous pool of GSH was used in the synthesis of cysteine. GSH might, therefore, fulfil the role of a storage compound.     

Survival and cell death in cells constitutively unable to synthesize glutathione

We examined the role of GSH in survival and cell death using GCS-2 cells that are deficient in glutamate cysteine ligase (gamma-glutamyl cysteine synthetase, gammaGCS), an enzyme essential for GSH synthesis. Cells maintained in 2.5 mM GSH have GSH levels that are approximately 2% of wild type and grow indefinitely; however, they express both pro- and anti-apoptotic Bcl-2 family members and have detectable levels of cytoplasmic cytochrome C. Withdrawal of GSH from the medium results in a fall in intracellular GSH to undetectable levels, decreased mitochondrial dehydrogenase activity, decreased anti-apoptotic factor RNAs, increased pro-apoptotic factor RNAs, additional cytochrome C release, and a fall in ATP levels; however, cells continue to grow for another 24h. At 48 h, these trends continue with the exception that mitochondrial membrane potential and ATP levels rise; DNA fragmentation begins at 48 h. Thus, severe reduction of GSH to 2% of wild type produces a metastable state compatible with survival, but complete absence of GSH triggers apoptosis.     

Effect of aurothioglucose on glutathione and glutathione-metabolizing and related enzymes in rat liver and kidney

The antirheumatic drug aurothioglucose is an inhibitor of the selenoenzyme GSH peroxidase. During chrysotherapy, the decreased levels of erythrocyte GSH and serum sulfhydryls of rheumatoid arthritis patients are normalized concomitant with clinical efficacy. This investigation examined the in vivo and in vitro effect of gold(I) as aurothioglucose on enzymes related to the GSH redox cycle or metabolism. The enzymes measured were GSH peroxidase, GSSG reductase, gamma-glutamyl transpeptidase, gamma-glutamylcysteine synthetase, GSH S-transferase, GSH thiotransferase, glucose-6-phosphate dehydrogenase, superoxide dismutase and catalase. Rats were injected with 30 mumol aurothioglucose/kg body wt. daily for 7 days by intramuscular injection. GSH levels in aurothioglucose-treated rats were 68% higher in erythrocytes (P less than 0.005) and 45% higher in kidney (P less than 0.001) than in control rats. Treatment with aurothioglucose did not elevate plasma or liver GSH. The enzyme activities that were changed by aurothioglucose treatment were GSH peroxidase in kidney (41% decreased, P = 0.005) and liver (13% decreased, P less than 0.05), gamma-glutamyl transpeptidase in kidney (15% decreased, P less than 0.05), and catalase in kidney (58% decreased, P less than 0.001). Kidney glucose-6-phosphate dehydrogenase activity was increased 50% (P less than 0.005) and GSH S-transferase was increased 72% (P less than 0.001). In vitro the only liver enzymes inhibited more than 50% by concentrations of less than 50 microM aurothioglucose were GSH peroxidase (50% inhibited by 25 microM aurothioglucose) and GSH thiotransferase (50% inhibited by 5 microM aurothioglucose). Studies of in vitro enzyme inhibition by aurothioglucose could not be used to predict decreased enzyme activities in vivo. Although decreased activities of two major enzymes that utilize GSH, GSH peroxidase and gamma-glutamyl transpeptidase, coincided with elevated GSH in kidneys of aurothioglucose-treated rats, a direct cause and effect relationship remains speculative.     

Regulation of sulphate assimilation by glutathione in poplars (Populus tremula x P. alba) of wild type and overexpressing gamma-glutamylcysteine synthetase in the cytosol

Glutathione (GSH) is the major low molecular weight thiol in plants with different functions in stress defence and the transport and storage of sulphur. Its synthesis is dependent on the supply of its constituent amino acids cysteine, glutamate, and glycine. GSH is a feedback inhibitor of the sulphate assimilation pathway, the primary source of cysteine synthesis. Sulphate assimilation has been analysed in transgenic poplars (Populus tremula x P. alba) overexpressing gamma-glutamylcysteine synthetase, the key enzyme of GSH synthesis, and the results compared with the effects of exogenously added GSH. Although foliar GSH levels were 3-4-fold increased in the transgenic plants, the activities of enzymes of sulphate assimilation, namely ATP sulphurylase, adenosine 5'-phosphosulphate reductase (APR), sulphite reductase, serine acetyltransferase, and O-acetylserine (thiol)lyase were not affected in three transgenic lines compared with the wild type. Also the mRNA levels of these enzymes were not altered by the increased GSH levels. By contrast, an increase in GSH content due to exogenously supplied GSH resulted in a strong reduction in APR activity and mRNA accumulation. This feedback regulation was reverted by simultaneous addition of O-acetylserine (OAS). However, OAS measurements revealed that OAS cannot be the only signal responsible for the lack of feedback regulation of APR by GSH in the transgenic poplars.     

Sulfur assimilation and glutathione metabolism under cadmium stress in yeast, protists and plants

Glutathione (gamma-glu-cys-gly; GSH) is usually present at high concentrations in most living cells, being the major reservoir of non-protein reduced sulfur. Because of its unique redox and nucleophilic properties, GSH serves in bio-reductive reactions as an important line of defense against reactive oxygen species, xenobiotics and heavy metals. GSH is synthesized from its constituent amino acids by two ATP-dependent reactions catalyzed by gamma-glutamylcysteine synthetase and glutathione synthetase. In yeast, these enzymes are found in the cytosol, whereas in plants they are located in the cytosol and chloroplast. In protists, their location is not well established. In turn, the sulfur assimilation pathway, which leads to cysteine biosynthesis, involves high and low affinity sulfate transporters, and the enzymes ATP sulfurylase, APS kinase, PAPS reductase or APS reductase, sulfite reductase, serine acetyl transferase, O-acetylserine/O-acetylhomoserine sulfhydrylase and, in some organisms, also cystathionine beta-synthase and cystathionine gamma-lyase. The biochemical and genetic regulation of these pathways is affected by oxidative stress, sulfur deficiency and heavy metal exposure. Cells cope with heavy metal stress using different mechanisms, such as complexation and compartmentation. One of these mechanisms in some yeast, plants and protists is the enhanced synthesis of the heavy metal-chelating molecules GSH and phytochelatins, which are formed from GSH by phytochelatin synthase (PCS) in a heavy metal-dependent reaction; Cd(2+) is the most potent activator of PCS. In this work, we review the biochemical and genetic mechanisms involved in the regulation of sulfate assimilation-reduction and GSH metabolism when yeast, plants and protists are challenged by Cd(2+).     

A novel shrunken endosperm mutant of barley

Although mutations affecting several enzymes of the starch synthetic pathway in developing cereal endosperm have been isolated, none has a major effect on soluble starch synthase We report a new recessive shrunken endosperm mutant in barley ( Hordeum vulgare L. cv. Bomi-like), shx , which has 25% of normal starch content. We have assayed the activity of sucrose synthase (EC 2.4.1.13), ADP and UDP-glucose pyrophosphorylases (EC 2.7.7.27 and 2.7.7.9), branching enzyme (EC.2.4.1.18), and granule-bound and soluble starch synthase (EC 2.4.1.21) in shx. Sucrose synthase activity is reduced by 49% and UDP-glucose pyrrphosphorylase is 80% of the normal level. Branching enzyme and starch-bound starch synthase activities are normal, but ADP-glucose pyrophosphorylase activity is reduced by 72%. The soluble starch synthase that is primer-independent in the presence of sodium citrate shows 14% of normal activity in shx. whereas the primer-dependent form is unaffected. This lower starch synthase activity in shx cannot be explained by inhibition, substrate destruction or lack of primer. Although several starch-synthetic enzymes are affected, it is suggested that the primer independent from of soluble starch synthase may be the primary-site of the mutation in shx.     

Elevation of glutathione levels by ammonium ions in primary cultures of rat astrocytes

It is well established that ammonia is detoxified in the brain to form glutamine and that astrocytes play a major role in this process. The synthesis of glutamine requires glutamate and ATP. Since glutamate and ATP are also required for the synthesis of glutathione (GSH), we examined the effect of pathophysiological concentrations of ammonia on levels of GSH in primary cultures of astrocytes. GSH content in the medium increased in a dose- and time-dependent manner in the presence of ammonia. After an initial decrease, cellular GSH content increased in a similar manner. The levels of glutathione disulfide (GSSG) were also increased. A linear relationship was observed between ammonia concentration and the increase in GSH levels. An increase in the efflux of GSH from cells into medium was also observed under these conditions. Buthionine sulfoximine and acivicin, but not methionine sulfoximine, blocked the ammonia induced increase in GSH levels. No, or minor, changes in the activities of enzymes (gamma-glutamyl transpeptidase, GSH reductase and GSH-peroxidase) that might influence GSH levels were identified and thus could not account for the ammonia induced increase in GSH levels in astrocytes. These findings indicate that pathophysiological concentrations of ammonium ions result in increased astroglial levels of GSH which may affect the metabolism and function of astrocytes.     

The phagocytosis-associated respiratory burst in human monocytes is associated with increased uptake of glutathione

During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [35S]glutathione ([35S]GSH) after 20-30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was inhibited by catalase, which removes hydrogen peroxide (H2O2), and micromolar H2O2 stimulated GSH uptake effectively in monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H2O2 and glutathione peroxidase prevented uptake. Acivicin, which inhibits GSH breakdown by gamma-glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a Km (8.5 microM) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter that is triggered by the release of H2O2 during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism has the potential to protect the phagocyte against oxidant damage.     

Cooperative binding of gamma-glutamyl substrate to human glutathione synthetase.

Human glutathione synthetase is responsible for catalyzing the final step in glutathione biosynthesis. It is a homodimer with a monomer subunit MW of 52 kDa. Kinetic analysis reveals a departure from linearity of the Lineweaver-Burk double reciprocal plot for the binding of gamma-glutamyl substrate, indicating cooperative binding. The measured apparent K(m) values for gamma-glutamyl-alpha-aminobutyrate (an analog of gamma-glutamyl-alpha-aminobutyrate) are 63 and 164 microM, respectively. Neither ATP (K(m) of 248 microM) nor glycine (K(m) of 452 microM) exhibits such cooperative binding behavior. Although ATP is proposed to play a key role in the sequential binding of gamma-glutamyl substrate to the enzyme, the cooperative binding of the gamma-glutamyl substrate is not affected by alterations of ATP concentration. Quantitative analysis of the kinetic results for gamma-glutamyl substrate binding gives a Hill coefficient (h) of 0.75, indicating negative cooperativity. Our studies, for the first time, show that human glutathione synthetase is an allosteric enzyme with cooperative binding for gamma-glutamyl substrate.     

Interaction of glutathione analogues with Hydra attenuata gamma-glutamyltransferase.

gamma-Glutamyltransferase activity was studied in extracts of the cnidarian Hydra attenuata. The binding of gamma-glutamyl peptide analogues to the enzyme was studied by observing their effects on heat denaturation and their inhibition of p-nitroaniline release from gamma-glutamyl p-nitroanilide. Neither position-1 analogues, in which the gamma-glutamyl moiety was changed to a beta-aspartyl (beta-Asp-Abu-Gly) or an alpha-glutamyl (Glu-Abu-Gly) linkage, nor glutamate protected the enzyme against inactivation at 58 degrees C. GSH (reduced glutathione), gamma-Glu-Abu-Gly and gamma-Glu-Met on the other hand did prevent heat denaturation. GSH and analogues of GSH were competitive inhibitors of p-nitroaniline release, but those analogues in which glycine was replaced by 2-aminoisobutyrate, phenylalanine, leucine or tyrosine had Ki values that were approximately five times those of analogues with the cysteine residue replaced.