Prolyl oligopeptidase acts as a link between chaperone-mediated autophagy and macroautophagy.

The accumulation of aggregated α-synuclein (α-syn) has been identified as the primary component of Lewy bodies that are the pathological hallmarks of Parkinson's disease (PD). Several preclinical studies have shown α-syn aggregation, and particularly the intermediates formed during the aggregation process to be toxic to cells. Current PD treatments only provide symptomatic relief, and α-syn serves as a promising target to develop a disease-modifying therapy for PD. Our previous studies have revealed that a small-molecular inhibitor for prolyl oligopeptidase (PREP), KYP-2047, increases α-syn degradation by accelerating macroautophagy (MA) leading to disease-modifying effects in preclinical PD models. However, α-syn is also degraded by chaperone-mediated autophagy (CMA). In the present study, we tested the effects of PREP inhibition or deletion on CMA activation and α-syn degradation. HEK-293 cells were transfected with α-syn and incubated with 1 & 10 µM KYP-2047 for 24 h. Both 1 & 10 µM KYP-2047 increased LAMP-2A levels, induced α-syn degradation and reduced the expression of Hsc70, suggesting that the PREP inhibitor prevented α-syn aggregation by activating the CMA pathway. Similarly, KYP-2047 increased the LAMP-2A immunoreactivity and reduced the Hsc70 levels in mouse primary cortical neurons. When LAMP-2A was silenced by a siRNA, KYP-2047 increased the LC3BII/LC3BI ratio and accelerated the clearance of α-syn. Additionally, KYP-2047 induced CMA effectively also when MA was blocked by bafilomycin A1. Based on our results, we suggest that PREP might function as a core network node in MA-CMA crosstalk, and PREP inhibition can reduce α-syn levels via both main autophagy systems.

Prolyl oligopeptidase inhibition reduces oxidative stress via reducing NADPH oxidase activity by activating protein phosphatase 2A.

Oxidative stress (OS) is a common toxic feature in various neurodegenerative diseases. Therefore, reducing OS could provide a potential approach to achieve neuroprotection. Prolyl oligopeptidase (PREP) is a serine protease that is linked to neurodegeneration, as endogenous PREP inhibits autophagy and induces the accumulation of detrimental protein aggregates. As such, inhibition of PREP by a small-molecular inhibitor has provided neuroprotection in preclinical models of neurodegenerative diseases. In addition, PREP inhibition has been shown to reduce production of reactive oxygen species (ROS) and the absence of PREP blocks stress-induced ROS production. However, the mechanism behind PREP-related ROS regulation is not known. As we recently discovered PREP's physiological role as a protein phosphatase 2A (PP2A) regulator, we wanted to characterize PREP inhibition as an approach to reduce OS. We studied the impact of a PREP inhibitor, KYP-2047, on hydrogen peroxide and ferrous chloride induced ROS production and on cellular antioxidant response in HEK-293 and SH-SY5Y cells. In addition, we used HEK-293 and SH-SY5Y PREP knock-out cells to validate the role of PREP on stress-induced ROS production. We were able to show that absence of PREP almost entirely blocks the stress-induced ROS production in both cell lines. Reduced ROS production and smaller antioxidant response was also seen in both cell lines after PREP inhibition by 10 µM KYP-2047. Our results also revealed that the OS reducing mechanism of PREP inhibition is related to reduced activation of ROS producing NADPH oxidase through enhanced PP2A activation. In conclusion, our results suggest that PREP inhibition could also provide neuroprotection by reducing OS, thus broadening the scope of its beneficial effects on neurodegeneration.

Prolyl oligopeptidase colocalizes with α-synuclein, ß-amyloid, tau protein and astroglia in the post-mortem brain samples with Parkinson's and Alzheimer's diseases.

Prolyl oligopeptidase (EC 3.4.21.26, PREP) is a serine protease that hydrolyzes proline-containing peptides shorter than 30-mer but it has also nonhydrolytic functions. PREP has been shown to accelerate aggregation of wild-type α-synuclein (α-syn) under cell-free conditions, and PREP inhibitors can block this aggregation both in vitro and in vivo. α-syn is the main component of Lewy bodies in Parkinson's disease (PD) and Lewy body dementia. To clarify the possible interaction of PREP with other markers of neurodegenerative diseases, we studied colocalizations of PREP and (1) α-syn, (2) ß-amyloid, (3) tau protein and (4) astroglial and microglial cells in human post-mortem brain samples from PD, Alzheimer's disease (AD) patients and in healthy control brain samples. In the substantia nigra of PD brains, an intense colocalization with PREP and α-syn was evident. PREP colocalized also with ß-amyloid plaques in AD brains and with tau protein in AD and in healthy brains. PREP was also found in astroglial cells in PD, AD and control brains, but not in the microglia. Our findings are the first ones to demonstrate colocalization of PREP and pathological proteins in the human brain and support the view that, at least in spatial terms, PREP could be associated with pathogenesis of neurodegenerative diseases.

Comparison of motor performance, brain biochemistry and histology of two A30P α-synuclein transgenic mouse strains.

Three point mutations in the SNCA gene encoding α-synuclein (aSyn) have been associated with autosomal dominant forms of Parkinson's disease. To better understand the role of the A30P mutant aSyn, we compared two transgenic mouse strains: a knock-in mouse with an introduced A30P point mutation in the wild-type (WT) gene (Snca(tm(A30P))) and a transgenic (Tg) mouse overexpressing the human A30P aSyn gene under the prion promoter [tg(Prnp-SNCA A30P)]. The brain aSyn load, motor performance, brain dopamine (DA) and sensitivity to 6-hydroxydopamine (6-OHDA) were studied in these mice. aSyn was evidently accumulating with age in all mice, particularly in tg(Prnp-SNCA A30P) Tg mice. There were no robust changes in basal locomotor activities of the mice of either line at 6 months, but after 1 year, tg(Prnp-SNCA A30P) Tg mice developed severe problems with vertical movements. However, the younger Tg mice had a reduced locomotor response to 1mg/kg of d-amphetamine. Snca(tm(A30P)) mice with the targeted mutation (Tm) were slightly hyperactive at all ages. Less 6-OHDA was required in tg(Prnp-SNCA A30P) Tg (1 µg) than in WT (3µg) mice for an ipsilateral rotational bias by d-amphetamine. That was not seen with the Snca(tm(A30P)) strain. A small dose of 6-OHDA (0.33 µg) led to contralateral rotations and elevated striatal DA in Tg/Tm mice of both lines but otherwise 6-OHDA-induced striatal DA depletion was similar in all mice, indicating no A30P-aSyn-related toxin sensitivity. 3,4-Dihydroxyphenylacetic acid/DA-ratio was elevated in tg(Prnp-SNCA A30P) mice, suggesting an enhanced DA turnover. This ratio and homovanillic acid/DA-ratio were declined in Snca(tm(A30P)) mice. Our results demonstrate that the two differently constructed A30P-aSyn mouse strains have distinct behavioral and biochemical characteristics, some of which are opposite. Since the two lines with the same background were not identically produced, the deviations found may be partially caused by factors other than aSyn-related genetic differences.

Different interactions of prolyl oligopeptidase and neurotensin in dopaminergic function of the rat nigrostriatal and mesolimbic pathways.

Prolyl oligopeptidase (PREP) is an intracellular enzyme digesting small proline-containing peptides. Since PREP resides the same brain areas as neurotensin in the nigrostriatal and mesolimbic dopaminergic pathways, we were interested to study if there is an intracellular interaction between them. A colocalization of PREP with neurotensin and neurotensin receptor 1 (NTS1) in the rat striatum, nucleus accumbens (NAcc), substantia nigra (SN) and ventral tegmental area (VTA) was studied with immunofluorescence. From the same brain areas, the levels of dopamine and its metabolites were measured 1 h after the injection of saline, NTS1 ligands (JMV-449; 5 µg) or antagonist (SR142948; 5 µg) to the rat striatum or NAcc. We also studied whether an intraperitoneal injection of a PREP inhibitor (KYP-2047; 5 mg/kg) affects the levels of dopamine and its metabolites alone or modifies the effects of the NTS1 ligands. PREP was highly colocalized with neurotensin and NTS1 in the VTA, and with NTS1 in the SN. Colocalization was moderate or low in other brain areas. When injected to the striatum, JMV-449 had a tendency to increase dopamine (p = 0.052) and metabolite levels in the striatum and SN, whereas SR142948 did not. After the injection to the NAcc, JMV-449 but not SR142948, increased dopamine levels in the VTA and dopamine metabolite levels in the NAcc and VTA. KYP-2047 decreased the dopamine levels in the striatum, but increased dopamine metabolite levels in the NAcc and VTA. Our results suggest a novel role for PREP in the modulation of dopaminergic transmission, which may be different in nigrostriatal and mesolimbic pathways.

A prolyl oligopeptidase inhibitor, KYP-2047, reduces α-synuclein protein levels and aggregates in cellular and animal models of Parkinson's disease.

BACKGROUND AND PURPOSE: The aggregation of α-synuclein is connected to the pathology of Parkinson's disease and prolyl oligopeptidase (PREP) accelerates the aggregation of α-synuclein in vitro. The aim of this study was to investigate the effects of a PREP inhibitor, KYP-2047, on α-synuclein aggregation in cell lines overexpressing wild-type or A30P/A53T mutant human α-syn and in the brains of two A30P α-synuclein transgenic mouse strains. EXPERIMENTAL APPROACH: Cells were exposed to oxidative stress and then incubated with the PREP inhibitor during or after the stress. Wild-type or transgenic mice were treated for 5 days with KYP-2047 (2 × 3 mg·kg(-1) a day). Besides immunohistochemistry and thioflavin S staining, soluble and insoluble α-synuclein protein levels were measured by Western blot. α-synuclein mRNA levels were quantified by PCR. The colocalization of PREP and α-synuclein,and the effect of KYP-2047 on cell viability were also investigated. KEY RESULTS: In cell lines, oxidative stress induced a robust aggregation of α-synuclein,and low concentrations of KYP-2047 significantly reduced the number of cells with α-synuclein inclusions while abolishing the colocalization of α-synuclein and PREP. KYP-2047 significantly reduced the amount of aggregated α-synuclein,and it had beneficial effects on cell viability. In the transgenic mice, a 5-day treatment with the PREP inhibitor reduced the amount of α-synuclein immunoreactivity and soluble α-synuclein protein in the brain. CONCLUSIONS AND IMPLICATIONS: The results suggest that the PREP may play a role in brain accumulation and aggregation of α-synuclein, while KYP-2047 seems to effectively prevent these processes.

Vascular endothelial growth factor C acts as a neurotrophic factor for dopamine neurons in vitro and in vivo.

Neurotrophic factors regulate the development and maintenance of the nervous system and protect and repair dopaminergic neurons in animal models of Parkinson's disease (PD). Vascular endothelial growth factors A (VEGF-A) and B have also neurotrophic effects on various types of neurons, including dopaminergic neurons. We examined the ability of the key lymphangiogenic factor VEGF-C to protect dopaminergic cells in vitro and in vivo. The study was initiated by a finding from microarray profiling of Neuro2A-20 cells which revealed up-regulation of VEGF-C by glial cell-line-derived neurotrophic factor (GDNF). Next, we observed that VEGF-C can rescue embryonic dopaminergic neurons and activate the mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway in vivo. VEGF receptors 1-2 and co-receptors, neuropilins 1-2, were expressed both in mouse embryonic cultures and adult midbrains. In vivo, VEGF-C had a robust functional effect in the rat unilateral 6-hydroxydopamine (6-OHDA) model of PD and there was a small additive effect on the survival of tyrosine hydroxylase (TH)-positive cells with GDNF. The neuroprotective effect of VEGF-C is most likely due to a combination of direct and indirect neurotrophic effects because, VEGF-C, unlike GDNF, induced also angiogenesis in the striatum following 6-OHDA insult as it did in human umbilical vein endothelial cells (HUVEC). However, we detected activation of astroglia and microglia as well as blood-brain barrier disruption after intracerebral delivery of VEGF-C, raising a concern of its safe usage as a therapeutic molecule. Our results provide evidence of VEGF-C as a neurotrophic factor that influences the dopaminergic system through multiple mechanisms.

Prolyl oligopeptidase induces angiogenesis both in vitro and in vivo in a novel regulatory manner.

BACKGROUND AND PURPOSE A serine protease, prolyl oligopeptidase (POP) has been reported to be involved in the release of the pro-angiogenic tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (Ac-SDKP) from its precursor, 43-mer thymosin ß4 (Tß4). Recently, it was shown that both POP activity and the levels of Ac-SDKP are increased in malignant tumours. The aim of this study was to clarify the release of Ac-SDKP, and test if POP and a POP inhibitor, 4-phenyl-butanoyl-L-prolyl-2(S)-cyanopyrrolidine (KYP-2047), can affect angiogenesis. EXPERIMENTAL APPROACH We used HPLC for bioanalytical and an enzyme immunoassay for pharmacological analysis. Angiogenesis of human umbilical vein endothelial cells was assessed in vitro using a 'tube formation' assay and in vivo using a Matrigel plug assay (BD Biosciences, San Jose, CA, USA) in adult male rats. Moreover, co-localization of POP and blood vessels was studied. KEY RESULTS We showed the sequential hydrolysis of Tß4: the first-step hydrolysis by proteases to <30-mer peptides is followed by an action of POP. Unexpectedly, POP inhibited the first hydrolysis step, revealing a novel regulation system. POP with Tß4 significantly induced, while KYP-2047 effectively prevented, angiogenesis in both models compared with Tß4 addition itself. POP and endothelial cells were abundantly co-localized in vivo. CONCLUSIONS AND IMPLICATIONS We have now revealed that POP is a second-step enzyme in the release of Ac-SDKP from Tß4, and it has novel autoregulatory effect in the first step. Our results also advocate a role for Ac-SDKP in angiogenesis, and suggest that POP has a pro-angiogenic role via the release of Ac-SDKP from its precursor Tß4 and POP inhibitors can block this action.

Importance of membrane-bound catechol-O-methyltransferase in L-DOPA metabolism: a pharmacokinetic study in two types of Comt gene modified mice.

BACKGROUND AND PURPOSE: Catechol-O-methyltransferase (COMT) metabolizes compounds containing catechol structures and has two forms: soluble (S-COMT) and membrane-bound (MB-COMT). Here we report the generation of a mouse line that expresses MB-COMT but not S-COMT. We compared the effects of deleting S-COMT only or both COMT forms on the pharmacokinetics of oral L-DOPA. EXPERIMENTAL APPROACH: L-DOPA (10 mg kg(-1)) and carbidopa (30 mg kg(-1)) were given to mice by gastric tube, and samples were taken at various times. HPLC was used to measure L-DOPA in plasma and tissue samples, and dopamine and its metabolites in brain. Immunohistochemistry and Western blotting were used to characterize the distribution of COMT protein isoforms. KEY RESULTS: Lack of S-COMT did not affect the levels of L-DOPA in plasma or peripheral tissues, whereas in the full COMT-knock-out mice, these levels were increased. The levels of 3-O-methyldopa were significantly decreased in the S-COMT-deficient mice. In the brain, L-DOPA levels were not significantly increased, and dopamine was increased only in females. The total COMT activity in the S-COMT-deficient mice was 22-47% of that in the wild-type mice. In peripheral tissues, female mice had lower COMT activity than the males. CONCLUSIONS AND IMPLICATIONS: In S-COMT-deficient mice, MB-COMT in the liver and the duodenum is able to O-methylate about one-half of exogenous L-DOPA. Sexual dimorphism and activity of the two COMT isoforms seems to be tissue specific and more prominent in peripheral tissues than in the brain.

Spatial association of prolyl oligopeptidase, inositol 1,4,5-triphosphate type 1 receptor, substance P and its neurokinin-1 receptor in the rat brain: an immunohistochemical colocalization study.

Prolyl oligopeptidase (POP) is a serine endopeptidase which hydrolyzes proline-containing peptides shorter than 30 amino acids. It has been suggested that POP is associated with cognitive functions, possibly via the cleavage of neuropeptides such as substance P (SP). Recently, several studies have also linked POP to the inositol 1,4,5-triphosphate (IP(3)) signaling. However, the neuroanatomical interactions between these substances are not known. We used double-labeled immunofluorescence to determine the POP colocalization with SP, SP receptor (neurokinin-1 receptor, NK-1R) and IP(3) type 1 receptor (IP(3)R1) in the rat brain. Furthermore, since striatal and cortical GABAergic neurons are involved in SP neurotransmission, we studied the coexpression of POP, SP and GABA by triple-labeled immunofluorescence. POP was moderately present in IP(3)R1-containing cells in cortex; the colocalization was particularly high in the thalamus, hippocampal CA1 field and cerebellar Purkinje cells. Colocalization of POP with SP and NK1-receptor was infrequent throughout the brain, though some POP and SP coexpression was observed in cerebellar Purkinje cells. We also found that POP partially colocalized with SP-containing GABAergic neurons in striatum and cortex. Our findings support the view that POP is at least spatially associated with the IP(3)-signaling in the thalamus, hippocampus and cerebellar Purkinje cells. This might point to a role for POP in the regulation of long-term potentiation and/or depression. Moreover, the low degree of colocalization of POP, SP and its NK-1R suggests that a transport system is needed either for POP or SP to make hydrolysis possible and that POP may act both intra- and extracellularly.

Clinical features and a follow-up study in a family with X-linked progressive cone-rod dystrophy.

PURPOSE: To study a large family with X-linked progressive cone-rod dystrophy. METHODS: There were 128 members in the family. Of these, 45 had an ophthalmological examination and 3 gave their permission to use the results of their recent ophthalmological examination. In addition to the usual eye examination, visual fields, colour vision, dark adaptation and electroretinogram (ERG) were examined. RESULTS: Ten affected men aged 6 to 81 years were found in the family. The visual acuities varied from counting fingers (cf) 10 cm to 0.5 in the right eye (RE) and from cf 30 cm to 0.4 in the left eye (LE). The refraction was myopic in all affected members, varying from -1.5 to -24.0 D (RE) and from -2.0 to -20.25 D (LE). In visual functions, central scotomas and concentric constriction in the visual fields, red or red-green defects in colour vision, abnormal cone and rod dark adaptation and affected cone response in ERG were found. The 6 obligate carriers were aged 17 to 77 years. Their visual acuities varied from 0.05 (strabismic amblyopia) to 1.25(RE) and from 0.7 to 1.25 (LE), and refraction from +/-0 to +6.0 D (RE) and from -0.5 to +5.0 D (LE). Their visual fields and colour vision were normal. The non-affected men were aged 13 to 55 years, their visual acuity was normal in both eyes, and refraction varied from -5.0 to +1.5 D (RE) and from -5.5 to +1.75 (LE). The result of the eye examination was normal except in colour vision: two men were congenitally deuteranomalous. The women who were not obligate carriers were aged 10 to 77 years, their visual acuity was from 0.3 to 1.6 in both eyes, and refraction from -5.5 to +4.75 (RE) and from -5.25 to +4.0 (LE). Two women had one amblyopic eye. Otherwise the eye examination was normal. CONCLUSIONS: The clinical diagnosis of X-linked cone dystrophy 1 (COD1) is based on progressive loss of visual acuity, moderate or high myopia, red colour vision defect and affected cone response or cone and rod response in ERG. The future identification of the COD1 gene will confirm the diagnosis of the disease and help in genetic counseling of the family.

Observations on the use of three different contrast sensitivity tests in children and young adults.

Forty-one children and young adults aged 4 to 25 years (mean 14.5 +/- 6.9, SD) with normal eyes were examined with three different contrast sensitivity tests: the Vistech distance and near test, the Cambridge Low Contrast Gratings test, and the LH-5 Contrast test. In different age groups, the youngest children aged 4 to 9 years had the lowest result values. The results of the older children aged 10 to 15 years and young adults aged 16 to 25 years were close to each other. The range of the results in all tests was large in every age group. The values of contrast sensitivity could not be compared from one test to another; in the Vistech tests the values varied from 10 to 200, in the Cambridge test from 170 to 560, and in the LH-5 test from 5 to 50. Most of the children liked the LH-5 test best, while most of the young adults preferred the Vistech distance test. It is useful to examine children and adults with different contrast sensitivity tests; however, the same test should be used in follow-up examinations.

Color vision in diabetic school children.

The color vision of 64 diabetic school children was studied. Acquired color vision defects due to diabetes could not be found in any of the children. Two of the children had a congenital red-green color vision defect. In the examination, three different pseudoisochromatic plate tests (Isihara, Standard Pseudoisochromatic Plates part 2, and Lanthony Tritan Album) were used as well as the Nagel anomaloscope and three different cap arrangement tests (Panel D 15, Lanthony Desaturated Panel, and Farnsworth-Munsell 100 hue). The plate tests and the anomaloscope examination were fast, reliable, and well accepted by the children. The cap arrangement tests took more time, and many of the children neither liked nor properly performed these tests. Twelve color dependent glucose strip tests for diabetes care at home were also studied. A few of the youngest school children made mistakes in interpreting the colors of these strips, although their color vision was normal.

Comparison of six colour vision tests for occupational screening.

Screening of red-green colour vision defects was done for 52 school children (22 boys and 30 girls) and 231 trade school students (226 boys and 5 girls) with three different kinds of pseudo-isochromatic plates: Ishihara (1983), Boström-Kugelberg (1972), and Standard Pseudoisochromatic Plates part 1 (SPP 1) 1978, and with three different kinds of vision screeners: Keystone View Model DVS 2, Bausch and Lomb Vision Tester, and Rodenstock Farbentestscheibe 3040.173. After these tests, each subject was examined with the Nagel Anomaloscope; this revealed 26 red-green defectives in the study group. Ishihara found 20/26 (76.9%), Boström-Kugelberg 24/26 (92.3%), and SPP 1 17/26 (65.4%) of the defectives. None of the normals were diagnosed as defectives with Ishihara or SPP 1. With Boström-Kugelberg four normals were diagnosed as defectives. Keystone found 24/26 (92.3%), Bausch and Lomb 26/26 (100%), and Rodenstock 25/26 (96.2%) of the defectives. But 9, 21, and 112 normals, respectively, were diagnosed as defective. In the present study, the Boström-Kugelberg and Ishihara plates as well as Keystone Vision Screener and Bausch and Lomb Vision tester came close to an effective screening test and could be recommended for screening red-green colour vision defects in occupational health care.

Comparison of cinnarizine, cyproheptadine, doxepin, and hydroxyzine in treatment of idiopathic cold urticaria: usefulness of doxepin.

Randomized double-blind trials using doxepin and several conventional antihistamines were carried out for treatment of patients with idiopathic cold urticaria. In the first double-blind trial, eight of nine patients preferred doxepin (10 mg three times daily) to cinnarizine (10 mg three times daily). In the second double-blind trial, the results of ice cube tests suppressing the effect of cyproheptadine (4 mg three times daily), doxepin (10 mg three times daily), and hydroxyzine (10 mg three times daily) did not statistically differ. However, doxepin was subjectively the most effective and it had fewer side effects than other treatments that were compared. Doxepin effectively suppressed the wheal and itching responses and shortened the duration of the wheal response in the ice cube test in all patients with cold urticaria who were studied.

Affinity chromatography of yeast alpha-glucosidase using ligand-mediated chromatography on immobilized phenylboronic acids.

The synthesis of 3-nitro-4-(6-aminohexylamido)phenylboronic acid is described. The properties of two novel forms of immobilized phenylboronate agarose adsorbents [m-aminophenylboronic acid-Matrex Gel and 3-nitro-4-(6-aminohexylamido)phenylboronic acid-Sepharose CL-6B] were investigated. Both gels bind and selectively retard the glycoprotein alpha-glucosidase from yeast. The retardation is affected by following parameters: (i) pH, (ii) presence of sugar, (iii) concentration of sugar and (iv) buffer species (especially triethanolamine). Five sugars were studied, namely sorbitol, fructose, ribose, glucose and maltose. The concentration of sugar required to produce significant retardation increased in the above order, whereas the ability of sugar to form a complex with boron decreases in the same order. These effects were observed with crude as well as pure enzyme. Since alpha-glucosidase is a glycoprotein, it is proposed that this protein is mainly bound to these immobilized phenylboronates via sugar (glyco) residues. Displacement of the enzyme from the column is effected by the sugar in the buffer (or in a preincubation mixture). However, the marked pH-dependence (this retardation effect could only be observed at pH 7.4) suggests that these results are not due solely to hydrophobic or ionic mechanisms and are more complex than simple sugar-phenylboronic acid interactions.

Evidence for the importance of cysteine and arginine residues in Pseudomonas fluorescens UK-1 pantoate dehydrogenase.

Homogeneous pantoate dehydrogenase (D-pantoate:NAD+ 4-oxidoreductase, EC 1.1.1.106) was shown to be sensitive to inactivation by p-chloromercuribenzoate (100 microM), 5,5'-dithiobis(2-nitrobenzoic acid) (1 mM), iodoacetic acid (1 mM) and phenylglyoxal (5.3 mM). Potassium D-pantoate and NAD protected against inactivation by p-chloromercuribenzoate, 5,5'-dithiobis (2-nitrobenzoic acid) and iodoacetic acid. NAD and D-pantoate also provided substantial protection against inactivatrion by phenylglyoxal. Titration of the sulphydryl groups by 5,5'-dithiobis(2-nitrobenzoic acid) and incorporation of [14C]carboxymethyl revealed that there are two cysteine residues which are modified and one of those is essential for activity. In the presence of NAD and D-pantoate, incorporation of [14C]phenylglyoxal was decreased by 0.42 mol/mol of subunit.