Localization of mitochondria in plant cells by vital staining with rhodamine 123

The positively-charged fluorescent dye rhodamine 123 (r-123) specifically stains mitochondria in living plant protoplasts, suspensionculture cells, and root hairs. This dye functions as a vital stain and permits visualization of the localization, distribution and movement of the mitochondria. Dehydration of root hairs caused mitochondria to aggregate into clumps. Mitochondria were either homogenous or heterogeneous and were frequently seen to accumulate in the perinuclear regions of suspension-culture cells but not in those of protoplasts or root-hair cells. Dinitrophenol and high concentrations of ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid and KCl immediately eliminated fluorescence in r-123-stained mitochondria whereas ionomycin enhanced it. Treatment of seedlings with r-123 resulted in differential brightness of fluorescence in different tissues. Meristematic tissues, such as root and shoot tips, exhibited the brightest fluorescence. The cytotoxicity of r-123 in both germinating seedlings and suspension-culture cells was low. The specificity, sensitivity and low toxicity of r-123 should make it a useful tool in experiments designed to examine agents and conditions which affect the location, the physiological status or the viability of mitochondria.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - DAPI 46-diamidino-2-phenylindole - r-123 rhodamine 123     

The effect of fluorescent labeling on calcium-induced fusion of fusogenic carrot protoplasts

Various fluorescent compounds — carboxyfluorescein, scopoletin, fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC), rhodamine 123, and rhodamine B ethyl ester — were used to study their effects on calcium-induced fusion of fusogenic carrot (Daucus carota L.) protoplasts. These protoplasts normally fused at a high percentage (50–60%) in response to 10 mM calcium, pH 6.0; however, if cells had been labeled with scopoletin, FITC, or RITC, fusion was greatly reduced. In contrast, labeling with carboxyfluorescein, rhodamine 123, or rhodamine B ethyl ester had no detectable effect on calcium-induced fusion. The two rhodamine dyes are shown to be localized in mitochondria.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - FITC fluorescein isothiocyanate - RITC rhodamine isothiocyanate - PE phosphatidylethanolamine     

Immunological and biochemical analysis of a null mutant of α-glycerolphosphate dehydrogenase from Drosophila melanogaster

Summary A Drosophila null mutant(BO-1-4) of -glycerolphosphate dehydrogenase induced by ethylmethane sulfonate(EMS) was analyzed by double immunodiffusion, enzyme immuno-inactivation, immunoelectrophoresis and two-dimensional electrophoresis. Based on all the immunological evidence, this mutant appears to express no protein that can cross-react with the antiserum specific to -glycerolphosphate dehydrogenase. A protein spot corresponding to -glycerolphosphate dehydrogenase was identified on two-dimensional gels of the soluble fly homogenates. The absence of this protein spot on two-dimensional gels of this null mutant further supported the immunological data. The activities of seven other enzymes in the related metabolic pathways were determined for the mutant and the control Drosophila. The null mutant does not show significant alterations in activities of these enzymes. The relationship between the deficiency of this enzyme and the inability for the sustained flight of the null mutant was discussed in terms of cellular metabolic regulations.Abbreviations used -GPD -glycerolphosphate dehydrogenase (EC 1.1.1.8) - EMS ethylmethane sulfonate - TEMED N,N,N,N-tetramethylene diamine - pI isolectric point - CRM immunological cross-reacting material     

Steroid synthesizing cellular sites in the ovary of the domestic pigeon Columba livia (Gmelin): a histochemical study.

Synopsis The ovary of the domestic pigeon,Columba livia, has been assayed histochemically for the localization of ⁵-3-hydroxysteroid dehydrogenase (⁵-3-HSDH), 17-hydroxysteroid dehydrogenase (17-HSDH), 11-hydroxysteroid dehydrogenase (11-HSDH), glucose-6-phosphate dehydrogenase (G6P-DH) and NADH-diaphorase activities during different periods of the reproductive cycle. ⁵-3-HSDH, 17-HSDH, 11-HSDH, G6P-DH and NADH-diaphorase activity was found in the theca interna of growing, atretic and postovulatory follicles, the granulosa of ovulatory, atretic and postovulatory follicles, and interstitial gland cells during the pre-incubation and the laying periods. During the incubation and squab feeding periods only ⁵-3-HSDH, G6P-DH and NADH-diaphorase activities were observed in the above mentioned cells. The steroidogenic potential of atretic follicles depends upon the type of atresia a follicle undergoes.     

Localization of adenosine 5′-phosphosulfate sulfotransferase in spinach leaves

Roots of spinach (Spinacia oleracea L.) seedlings contained only a very low activity of adenosine 5-phosphosulfate sulfotransferase compared to the cotyledons. Adenosine 5-phosphosulfate sulfotransferase activity increased about tenfold in cotyledons during greening. Preparation of organelle fractions from spinach leaves by a combination of differential and isopycnic density gradient centrifugation showed that adenosine 5-phosphosulfate sulfotransferase banded with NADP-glyceraldehyde-3-phosphate dehydrogenase, a marker enzyme for intact chloroplasts. In the fractions of peroxisomes, mitochondria and broken chloroplasts virtually no adenosine 5-phosphosulfate sulfotransferase activity was measured. Comparison with the chloroplast enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase indicates that in spinach, adenosine 5-phosphosulfate sulfotransferase is localized almost exclusively in the chloroplasts.Abbreviations APS Adenosine 5-phosphosulfate - APSSTase Adenosine 5-phosphosulfate sulfotransferase - BSA Bovine serum albumin - BRIJ58 Polyethylene glycolmonostearylether - DTE Dithioerythritol - DTT Dithiothreitol - EDTA Ethylenediaminetetraacetic acid - ME 2-Mercaptoethanol - NADP-GPD NADP-linked glyceraldehyde-3-phosphate dehydrogenase - PAPS Adenosine 3-phosphate 5-phosphate 5-phosphosulfate - POPOP 1,4 Di [2-(5-phenyloxazolyl)]-benzene - PPO 2,5-Diphenyloxazol The results presented in this paper are taken from the Ph. D. thesis of H.F.     

A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid

Summary Cholic acid (3,7,12-trihydroxy-5-cholanoic acid) was completely and selectively transformed into 12-ketoursodeoxycholic acid (3,7-dihydroxy-12-oxo-5-cholanoic acid) by means of two consecutive enzymatic steps catalyzed, the first, by 7- and 12-hydroxysteroid dehydrogenase and, the second, by 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with -ketoglutarate-glutamate dehydrogenase and glucose-glucose dehydrogenase, respectively.     

The effects of mitochondrial energetics inhibitors on the fluorescence of potential-sensitive dyes rhodamine 123 and DiS-C3-(5) in lymphocyte suspensions

The effects of uncouplers (FCCP, DNF), oligomycin, and rotenone on the fluorescence of potential-sensitive dyes, rhodamine 123 and diS-C₃-(5), in lymphocyte suspensions were compared. The fluorescence of these optical probes gradually increased at higher FCCP concentrations. The dependences of fluorescence intensities and FCCP concentrations were similar for both dyes, and only diS-C₃-(5) fluorescence started increasing at lower FCCP concentrations. Rotenone (1 µM) significantly increased rhodamine 123 fluorescence. TMPD-induced and uncoupler-induced diS-C₃-(5) fluorescence changes increased 1.5- to 2-fold if the incubation mixture was supplemented with oligomycin (0.1–0.2 µg/ml). The fluorescence responses of the dyes in the lymphocyte suspension correlate with the effects of mitochondrial energetics inhibitors on _m in isolated mitochondria. The results suggest the possibility of using these dyes for estimating the direction of the _m changes in the lymphocyte suspension.Abbreviations _m difference in electrical potential across the mitochondrial inner membrane - _p difference in electrical potentials across the plasma membrane - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - DNP 2,4-dinitrophenol - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - diS-C₃-(5) 3,3-dipropylthiodicarbocyanine - MOPS morpholinopropane sulfonic acid     

Ammonia assimilation in the fission yeast Schizosaccharomyces pombe 972

Glutamine synthetase (GS) activity of Schizosaccharomyces pombe 972 was high in ammonia-limited cultures, low in phosphate-and sulphate-limited cultures and not detected in glucose-limited cultures. When ammonia was pulsed into an ammonia-limited culture then GS activity decreased at a rate faster than that calculated if enzyme synthesis ceased and enzyme was diluted out by growth. Enzyme activity increased in ammonia-starved, phosphate-limited cultures and in the ammonia pulse system when the added ammonia had been utilised. These increases in enzyme activity were prevented by the presence of 100 g/ml cycloheximide. GS activity was inversely related to the intracellular concentration of glutamate.Abbreviations Gs Glutamine synthetase, EC 6.3.1.2 - GOGAT Glutamine: 2-oxo-glutarate amino transferase, EC 2.6.1.53 - GDH Glutamate dehydrogenase, EC 1.4.1.3     

Histochemical studies on the distribution of β-glucuronidase and succinic dehydrogenase in young and old spinal ganglion cells of the rat

Summary The present study compares the distribution of -glucuronidase and succinic dehydrogenase in young and old spinal ganglion cells of rat. In young cells there are indications of cyclic activity of these enzymes, i.e., in some stages there are perinuclear concentrations of the enzymes, at other times -glucuronidase and succinic dehydrogenase are uniformly distributed throughout the cytoplasm. These stages have been discussed with the identical distribution of mitochondria. However, in old spinal ganglion cells both -glucuronidase and succinic dehydrogenase become mainly concentrated in the pigment areas, suggesting thereby their possible role in the production of pigment, through the medium of the mitochondria.     

The effect of gyrase inhibitors and cyclic AMP on induction and glucose repression of the 6-hydroxy-nicotine oxidases in Arthrobacter oxidans

The induction by d,l-nicotine of the enantiozymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase in Archrobacter oxidans was differently affected by the inhibitors of Escherichia coli gyrase, novobiocin and nalidixic acid. These compounds inhibited 6-hydroxy-L-nicotine oxidase induction slightly, but led to an increase in the level of 6-hydroxy-D-nicotine oxidase activity. Furthermore, the specific repression by glucose of 6-hydroxy-D-nicotine oxidase synthesis was not abolished by the addition of cAMP but by that of novobiocin.Abbreviations 6-HDNO 6-hydroxy-D-nicotine oxidase - 6-HLNO 6-hydroxy-L-nicotine oxidase - cAMP cyclic 3,5-adenosine monophosphate - Enzymes Adenylate cyclase - ATP pyrophosphate-lyase (cyclizing) (EC 4.6.1.1) - cAMP-phosphodiesterase 3:5-cyclic-nucleotide 5-nucleotido-hydrolase (EC 3.1.4.17) - DNA gyrase DNA topoisomerase II (EC 5.99) - DNA polymerase deoxynucleosidetriphosphate: DNA desoxynucleotidyl-transferase (EC 2.7.7.7) - 6-hydroxy-L-nicotine oxidase 6-hydroxy-L-nicotine: oxygen oxidoreductase (EC 1.5.3.5) - 6-hydroxy-D-nicotine oxidase 6-hydroxy-D-nicotine: oxygen oxidoreductase (EC 1.5.3.6) - -lactamase penicillin amido--lactamhydrolase (EC 3.5.2.6) - nicotine dehydrogenase nicotine: (acceptor)6-oxidoreductase (hydroxylating) (EC 1.5.99.4)     

Plant regeneration from nucllar tissues of Aegle marmelos through organogenesis

A protocol for organogenesis from nucellar explants excised from fertilized ovules of immature fruits of Aegle marmelos Corr. was developed. Adventitious buds were initiated on Murashige and Skoog's (MS) medium containing various combinations of 6-benzyladenine (BA), -naphthalene-acetic acid (NAA), 3-indoleacetic acid and gibberellic acid. Medium containing 4.4 m BA and 2.7 M NAA produced the maximum number of adventitious buds per explant. Shoots were elongated by transferring explants with shoot buds to medium with a low concentration of BA (0.44 M). Rooting of in vitro-regenerated shoots was obtained in half-strength MS medium with 4.9 M indole-3-butyric acid. This is the first report of plant regeneration from nucellar explants of A. marmelos.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - GA₃ gibberellic acid     

Interaction between Tubulin and Na2+,K2+-ATPase in Brain Stem Neurons

Functionally active Na²⁺,K²⁺-ATPase isozymes containing three types of the catalytic subunits (1, 2, and 3) were obtained from calf brain by two methods: selective removal of contaminating proteins according to Jorgensen (1974) and selective solubilization of the enzyme with subsequent reformation of the membrane structure according to Esmann (1988). All preparations were characterized with respect to ouabain-inhibition constants. The presence of the cytoskeleton protein tubulin (3 isoform) in the high-molecular-weight complex of Na²⁺,K²⁺-ATPase 31 isozyme from brain stem axolemma and the junction between Na²⁺,K²⁺-ATPase 3 subunit and tubulin 3 subunit are shown for the first time.     

Isozyme patterns in zygotic and somatic embryogenesis of carrot

Isozyme patterns of carrot (Daucus carota L.) zygotic embryos between the torpedo stage up to 5-day-old seedlings have been compared with those of the similar stages from the embryogenic cell suspension culture to the late somatic plantlet. Somatic embryos blocked at the torpedo stage by -cyclodextrine have also been analyzed. All these stages have been analyzed with respect to seven different enzyme systems: arylesterase, glucosephosphate isomerase, phosphogluconate dehydrogenase, alcohol dehydrogenase, isocitrate dehydrogenase, aspartate aminotransferase and phosphoglucomutase (EC 2.7.5.1, PGM). The relationships between the different stages of both types of embryogenesis have been visualized using an unrooted tree. Generally, profiles of somatic embryos were different from those of zygotic embryos. Interestingly however, a typical zygotic embryo pattern was found in the cyclodextrine-blocked somatic embryos. Only aspartate aminotransferase patterns revealed a similarity between zygotic and somatic torpedo embryos. Both plantlet types showed close patterns with common isozymes. Moreover, similarities were evident between somatic plantlets and cell suspensions. A few isozymes appeared to be stage specific markers: esterase 10–11 were specific to achenes and early germination, phosphogluconate dehydrogenase 8 was specific to 4–5 day-old seedlings and phosphoglucomutase 1 and 7 and alcohol dehydrogenase 4 were markers for zygotic embryos. No somatic embryogenesis specific isozyme could be found. We show that patterns can be associated with particular tissue formation: mainly, aspartate aminotransferase 2 and 1, phosphoglucomutase 8 and 9 and phosphogluconate dehydrogenase 7 coincided with apical meristem initiation and phosphoglucomutase 4 and 5, zones b and d of esterase and zone b of phosphogluconate dehydrogenase coincided with vascular bundle formation.Abbreviations ADH alcohol dehydrogenase - CD -cyclodextrine - CS cell suspension culture - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediaminetetraaeetie acid - LiBo lithium hydroxide/boric acid - PEG polyethylene glycol - PVP polyvinylpyrrolidone - SEg somatic embryo at the globular stage - SEh heart stage - SEte early torpedo stage - SEtl late torpedo stage - SEce early cotyledonary stage - SEcl late cotyledonary stage - SECD somatic embryo blocked at the torpedo stage with -cyclodextrine - EST esterase - GOT aspartate aminotransferase - IDH isocitrate dehydrogenase - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) - PMS phenazonium methosulfate - PGD phosphogluconate dehydrogenase - PGI glucosephosphate isomerase - PGM phosphoglucomutase - SO dry seed - S1–3 seed after 1–3 days of germination - SP1–2 young and old somatic plantlets - ZE zygotic embryo - ZP4–5 4–5 day-old seedlings     

Enzymatic synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid in a membrane reactor

Summary Dehydrocholic acid (3,7,12-trioxo-5-cholanic acid) (0.5% concentration) was completely and selectively reduced to 12-ketoursodeoxycholic acid (3, 7-dihydroxy-12-oxo- 5-cholanic acid) in a membrane reactor by means of 3-hydroxysteroid dehydrogenase and 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with the glucose-glucose dehydrogenase system.     

The histochemical distribution of hydroxysteroid dehydrogenases in the human foetal membranes at term

Summary The histochemical distribution of various hydroxysteroid dehydrogenases in human, term, foetal membranes has been investigated using the tetrazolium dye, Nitro-B.T.The trophoblastic layer was the most active, showing 3-, 3-, 11-, 16- and 17-hydroxysteroid dehydrogenase activities, a pattern of activity similar to that of the placental villous trophoblast.The amniotic epithelium showed weak 3-, 3-, 16- and 17-hydroxysteroid dehydrogenase activity; weak 3- and 3-hydroxysteroid dehydrogenase activity was noted in the connective tissue layers.All activity demonstrated was N.A.D.-linked.     

Mitochondrial factors involved in Parkinson's disease by MPTP toxicity inMacaca fascicularis and drug effect

The maximal rates (V_max) of some mitochondrial enzyme activities related to energy transduction (citrate synthase, succinate dehydrogenase, malate dehydrogenase, NADH-cytochrome c reductase, cytochrome oxidase) and amino acid metabolism (glutamate dehydrogenase, glutamate-pyruvate-and glutamate-oxaloacetate-transaminases) were evaluated in non-synaptic (free) and intrasynaptic light and heavy mitochondria fromhippocampus ofMacaca fascicularis (Cynomolgus monkey). The different mitochondrial populations were isolated from thehippocampus of monkeys treated p.o. with dihydroergocriptine at a dose of 12 mg/kg/day before and during the induction of a Parkinson's-like syndrome by MPTP administration (i.v., 0.3 mg/kg/day for 5 days). The MPTP administration modified the activity of some enzymes related to the metabolism of glutamate and the activity of succinate dehydrogenase on selected types of mitochondria. Pharmacological treatment by dihydroergocriptine promoted return to the steady-state levels of most enzymes, demonstrating a protective effect on these biochemical parameters.     

Template-directed reactions of aminonucleosides

Summary We have studied the reactions between adenosine 5-phosphorimidazolide and 9-(2-amino-2-deoxyxylofuranosyl) adenine (I) or 3-methylamino-3-deoxyadenosine (II), both with and without a poly (U) template. We find that both amino compounds react much more rapidly than does adenosine, in the absence of a template. The rate of reaction is greatly enhanced by a poly (U) template in the case of I, but the enhancement is slight in the case of II.Abbreviations A adenosine - xylo A_NH2 9-(2-amino-2-deoxy--D-xylofuranosyl) adenine - A_NHMe 3-methylamino-3-deoxyadenosine - ImpA adenosine 5-phosphorimidazolide - A³ pA adenylyl-[35]-adenosine - A² pA adenylyl-[25]-adenosine - U_NPA adenylyl-[52]-2-amino-2-deoxyuridine - xylo A_NPA 9-[adenylyl-(52)-2-amino-2-deoxy--D-xylofuranosyl]adenine - A_(NMe)pA adenylyl-[53]-3-methylamino-3-deoxyadenosine - pA adenosine 5phosphate - AppA P₁, P₂-diadenosine 5pyrophosphate - (pA)_n n = 2, 3 [2-5]-linked oligomers of pA - A² pA² pA [2-5]-linked trinucleoside diphosphate of A - poly (U) polyuridylic acid     

Regulation of adenosine 5′-phosphosulfate sulfotransferase activity by H2S and cyst(e)ine in primary leaves of Phaseolus vulgaris L.

During chloroplast development in the primary leaves of Phaseolus vulgaris, the extractable activity of adenosine 5-phosphosulfate sulfotransferase increased ten-fold. When chloroplast development took place in air enriched with 3.5 l H₂S·l^-1 there was a decrease in adenosine 5-phosphosulfate sulfotransferase activity. Cyst(e)ine in concentrations up to 1 mM (in the external medium) did not affect the increase in adenosine 5-phosphosulfate sulfotransferase activity in intact plants. In plants with excised roots, 0.75 mM cyst(e)ine inhibited this increase. In green primary leaves, H₂S or cyst(e)ine treatment resulted in a decrease of extractable adenosine 5-phosphosulfate sulfotransferase activity. In intact plants, this effect of cyst(e)ine was observed at a concentration of 1 mM, and in plants with excised roots, 0.25 mM had a comparable effect.In developing plants, the extractable activities of O-acetyl-L-serine sulfhydrylase (EC 4.2.99.9) and ribulosebisphosphate carboxylase (EC 4.1.1.39.) were not affected by H₂S or cyst(e)ine.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5phosphosulfate sulfotransferase - BSA bovine serum albumin - DTE dithioerythritol - EDTA ethylenediaminetetra-acetic acid - OASSase O-acetyl-L-serine sulfhydrylase - PAPS adenosine 3-phosphate 5-phosphosulfate - POPOP 1,4 Di 2-(5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazol - RubP ribulose-bisphosphate - RubPCase ribulosebiphosphate carboxylase This is no. 8 in the series Regulation of Sulfate Assimilation in Plants. The term cysteine is used when it is clear that cystine is not involved; cyst(e)ine is used for an undefined mixture of cysteine and cystine. The concentrations are expressed in all cases relative to cysteine     

Polymerization of nucleotide analogues I: Reaction of nucleoside 5′-phosphorimidazolides with 2′-amino-2′-deoxyuridine

Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - U_{N 3} 2-azido-2-deoxyuridine - U_{NH 2} 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpU_{N 3} 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpU_{NH 2} 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pU_{N 3} 2-azido-2-deoxyuridine 5-phosphate - pU_{NH 2} 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - U_NpA adenylyl-[52]-2-amino-2-deoxy-uridine - U_NpU uridylyl-[52]-2-amino-2-deoxyuridine (pU_N)_n n=2,3,4 [25]-linked oligomers of pU_{NH 2} poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole     

Experimental phenylketonuria: Metabolic studies in rat liver

Summary The in vivo effects of L-phenylalanine on the gluconeogenic pathway in the liver of fasted rats with experimentally induced phenylketonurialike characteristics have been investigated. Significant increases of the fructose 6-phosphate, glucose 6-phosphate and glucose concentrations were observed. The study of the effect of L-phenylalanine on the cytoplasmic and mitochondrial redox state and energy charge showed an increase in the mitochondrial NAD⁺/NADH ratio while the energy charge was virtually unchanged.The effects of phenylalanine and its metabolic derivatives (phenylacetate, phenylethylamine, phenyl-lactate, o-hydroxyphenylacetate and phenylpyruvate) on the activity of lactate de-hydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37) and 3-hydroxybutyrate de-hydrogenase (EC 1.1.1.30) in rat liver have been also investigated. Phenylpyruvate inhibited the lactate dehydrogenase activity with a Ki of 5.3mm. Phenylpyruvate also inhibited both the mitochondrial (Ki = 4mm) and cytoplasmic (Ki = 5mm) malate dehydrogenase activities. Phenyl-pyruvate, phenylacetate and o-hydroxyphenylacetate inhibited the 3-hydroxybutyrate dehydrogenase activity with Ki values of 0.7, 6.0 and 9.5mm respectively.