Promotion of murine antitumour activity by prothymosin α treatment: I. Induction of tumoricidal peritoneal cells producing high levels of tumour necrosis factor α

Summary The effect of prothymosin (ProT) on the survival of DBA/2 mice inoculated with syngeneic tumour cells was studied. DBA/2 mice inoculated intraperitoneally (i.p.) with 2×10⁵ syngeneic leukaemic L1210 cells developed ascites within 8–12 days and died 10–14 days later. Treatment with ProT consistently inhibited the development of ascites in 20% of the treated animals and prolonged the survival of 40%–60% of the animals up to 70 days. The most effective treatment schedule of ProT was 300 ng/mouse given i.p. at 2-day intervals for 3 weeks followed by a rest period of 7 days, prior to tumour cell inoculation. Peritoneal exudate (PE) cells collected from mice treated with the optimal dose of ProT produced, in the absence of exogenous stimulus, six- to eightfold higher levels of tumour necrosis factor (TNF) than PE cells from control mice. Furthermore these cells exhibited cytotoxic activity against several tumour cell lines including the syngeneic L1210, the TNF-insensitive P815 mastocytoma, the human MOLT-4 lymphoblastic leukaemia, as well as the murine TNF-sensitive L929 fibroblast cell line. Kinetic studies revealed that both production of TNF and tumoricidal activity peaked 7 days after the last injection of ProT and were maintained at high levels over a period of 1 month. Injections with 150 ng ProT slightly improved the survival of mice whereas higher (500 ng and 1000 ng) doses of ProT and a wide range of thymosin 1 doses remained without any effect. PE cells collected from these mice produced extremely low levels of TNF and exhibited negligible tumoricidal activity. Our data demonstrate that ProT has a protective effect in vivo against the growth of adoptively transfered tumour cells and suggest that this effect is, at least in part, mediated by ProT-activated PE cells. These cells were demonstrated to produce high levels of TNF in vitro and to exhibit activity against both TNF-sensitive and TNF-resistant cell lines.Supported by a CEC grant to Dr. M. Papamichail     

Phase I study of TNFα AutoVaccIne in Patients with metastatic cancer

We evaluated the safety and immunogencity of a novel vaccine directed against autologous TNF in a Phase I fixed dose escalation trial. The vaccine consisted of two recombinant TNF proteins, with specific peptides replaced by foreign immunodominant T cell epitopes from tetanus toxoid. The main objectives were to establish a safe dose and evaluate the vaccines ability to raise neutralising TNF antibodies. Secondary objectives were improvements in body weight and tumour response. Thirty-three patients were vaccinated with three doses (20, 100, or 400 g) of TNF vaccine at 2-weekly intervals adjuvanted with aluminium hydroxide. Anti-TNF antibody titres were measured by both a RIA, using soluble native TNF as the antigen, and by an ELISA using immobilized partly denatured TNF. Eleven patients (33%) had mild grade1/2 injection site reactions at the higher doses. In 10 of 20 patients, serum antibodies recognize denatured TNF in the ELISA, whereas, antibody titres against native TNF in the RIA were undetectable. This suggests that the production process had partly denatured the vaccine preventing the formation of cross-reacting antibodies to native TNF. In conclusion, TNF vaccine was able to elicit vaccine specific antibodies. However, since the antibodies were only able to cross-react with partly denatured TNF, evaluation of safety and tumour responses to the TNF vaccine was compromised.     

Relationship between soluble tumor necrosis factor (TNF) receptors and TNFα during immunotherapy with interleukin-2 and/or interferon α

Eleven metastatic cancer patients were studied during three different regimens of immunotherapy with interleukin-2 (IL-2) and/or interferon (IFN): group A received 4 days of IL-2 i.a. infusion (n=3), group B IFN s.c. during 5 days (n=4), followed on day 3 by 5 days of a continuous IL-2 i.v. infusion, and group C had 4 days of IL-2 i.v. infusion together with s.c. IFN on days 1 and 4 (n=4). Soluble tumor necrosis factor receptors (sTNFR) p55 and p75 and TNF concentrations in serum were analyzed before therapy and daily during 8 days of the first therapy cycle. sTNFR was measured by radioimmunoassay. sTNFR p55 increased in all patient groups from a baseline value of 5.2±0.9 ng/ml to a maximum of 13.6±1.2 ng/ml by days 3–4 (P=0.003). sTNFR p75 increased from 7.6±1.1 ng/ml to peak values of 30.1±2.6 ng/ml in groups A and B (P=0.02). In group C the sTNFR p75 response was weak (NS). In group B, the increase of both p55 and p75 occurred only after addition of IL-2 to IFN. TNF increased weakly during treatment with IFN alone (group B); it rose strongly during IL-2 and the combined treatment (groups A-C) from 8±2 pg/ml to 115±13 pg/ml (P=0.003). In group B, it reached the maximum 24 h after addition of IL-2 to IFN and decreased thereafter. there was a significant relationship between TNF and sTNFR p55 or sTNFR p75 in groups A and C, (P=0.001), but not in group B. Group C was also investigated during the third therapy cycle. The increase of sTNFR p75 was stronger (P=0.01) and that of TNF weaker than in the first cycle; the sTNFR p55 response was similar in both cycles. In conclusion sTNFR p55 and p75 are rapidly induced during IL-2 and IL-2+IFN treatment, the increase of sTNF receptors parallels or exceeds that of TNF and may influence the immunomodulatory effects of TNF during cytokine therapy.     

Isolation and partial sequence of goat spleen prothymosin α

Goat prothymosin , a highly acidic polypeptide of pl 3.5, 109 amino acid residues, has been isolated from lymphoid and non-lymphoid tissues of young female goats. Unlike rat, murine and porcine prothymosins , goat prothymosin appears at a higher concentration in the spleen compared with the thymus. The sequence of segments of the polypeptide involving known mutations has been determined, by automatic sequencing of its tryptic peptide fragments. The acidic amino acid-rich segment in the middle of the molecule, including residues 49–83, has not been sequenced. Goat prothymosin closely resembles bovine prothymosin , with only one substitution, proline for alanine at position 85. It also resembles human prothymosin , with only three substitutions. It differs more significantly from rat and murine prothymosins , by two deletions and three substitutions. The results show the highly conserved nature of the molecule, with substitutions at given positions only.Abbreviations ProT Prothymosin - T₁ Thymosin ₁ - MLR Mixed Lymphocyte Response - HPLC High Performance Liquid Chromatography - RIA Radioimmunoassay - B Aspartic acid or Asparagine - Z Glutamic acid or Glutamine     

Tnf-α and IL-1α inhibit both pyruvate dehydrogenase activity and mitochondrial function in cardiomyocytes: Evidence for primary impairment of mitochondrial function

Cytokines such as tumor necrosis factor (TNF) and Interleukin-1 (IL1) are known to influence energy metabolism and mitochondrial function in tumor and vascular smooth muscle cells. The aim of the present study was to investigate whether in cardiomyocytes mitochondrial function and PDH activity may also be impaired by TNF and IL1. Pyruvate dehydrogenase (PDH) activity and mitochondrial oxygen consumption of cultured cardiomyocytes were determined after subchronic exposure (24 h) to TNF (1, 10, 100, 1000 I.U./ml) and IL1 (0.1, 1, 10, 100 I.U./ ml).TNF- and IL1- exposure of the cardiomyocytes resulted in a concentration dependent decrease of PDH activity up to 38%. In parallel, selective oxygen consumption of the respiratory chain complexes I (NADH:ubiquinone oxidoreductase) and II (succinate:ubiquinone oxidoreductase) decreased by up to 45%. Addition of the PDH activator dichloracetate (0.01 M) resulted in complete restoration of PDH activity but not of mitochondrial function. The results suggest a primary inhibition of the mitochondrial respiratory chain by TNF and IL1 and a subsequent down regulation of PDH activity.     

Evaluation of hydrophobicity versus chaperonelike activity of bovine alphaA- and alphaB-crystallin

Calf lens A-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than B-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to A-crystallin than to B-crystallin at room temperature. Bis-ANS binding to both A- and B-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of A-crystallin is lower than that of B-crystallin whereas at higher temperatures, A-crystallin shows significantly higher protection against aggregation of substrate proteins compared to B-crystallin. Therefore, calf lens A-crystallin is more hydrophobic than B-crystallin and chaperone-like activity of -crystallin subunits is not quantitatively related to their hydrophobicity.     

Proteolytic digestion of α-lactalbumin: Physiological implications

The kinetics of the partial digestion of bovine -lactalbumin (-LA) by trypsin, -chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of -LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)--LA>Zn(II), Ca(II)--LA>apo--LA. The HPLC digestion patterns of Ca(II)--LA and Zn(II), Ca(II)--LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo--LA were quite different, reflecting the existence of the thermally induced denaturation states of apo--LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded -LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of -LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II))-specific site does not cause any drastic changes in the overall structure of -LA. The acidic form of -LA (atpH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or -chymotrypsin at neutralpH. Complexation of -LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to -LA may have physiological significance (e.g., for nutritional transport).On leave from the Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR.     

Study ofO-sialylation of glycoproteins in C6 glioma cells treated with retinoic acid

When treated with retinoic acidin vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase activity. A 300kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide -O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the 2,3 sialyltransferase but to thede novo synthesis of the polypeptide chain of this glycoprotein.Abbreviations RA retinoic acid - Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5 monophosphosialate - 2,3 ST CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase - GalNAc-O-benzyl N-acetylgalactosaminide -O-benzyl - Gal1-3GalNAc-O-benzyl Galactosyl 1-3N-acetylgalactosaminide -O-benzyl - TBS Tris-HCl buffer 50mm pH 7.5 containing NaCl 0.15m and Tween 20 0.05% - B1 buffer TBS containing MgCl₂ 1mm, MnCl₂ 1mm and CaCl₂ 1mm     

β-Amyloid Peptide Binds Equivalently to Binary and Ternary α<Subscript>2</Subscript>-Macroglobulin–protease Complexes

₂-Macroglobulin (₂M) is a protease inhibitor that has separate binding sites for transforming growth factor- (TGF-) and -amyloid peptide (A), both of which have been identified in the ₂M sequence. In the 3D-structure of ₂M, TGF- occupies the ₂M central cavity, overlapping with the space that can accommodate up to two molecules of protease. As a result, ternary ₂M–protease complexes (2 mol protease/mol ₂M) have been reported to not bind TGF-. The goal of the present study was to test whether binding of A to ₂M is controlled by steric constraints imposed by associated proteases, similarly to TGF-. We confirmed that binary ₂M–trypsin complex (1 mol trypsin/mol ₂M) binds increased amounts of TGF-1, compared with native ₂M, while ternary ₂M–trypsin complex binds substantially decreased amounts of TGF-1. By contrast, A-binding to binary and ternary ₂M–trypsin complex was equivalent. In both cases, binding was substantially increased compared with the negligible level observed with native ₂M. Plasmin is a large protease (Mr ~82,000) that substantially occupies the ₂M central cavity; however, ₂M–plasmin complex also bound increased amounts of A, compared with native ₂M. We conclude that A accesses its binding site, in ₂M, from outside the ₂M central cavity. The TGF--and A-binding sites are spatially separated not only in the primary sequence of ₂M, but also in the 3D-structure.     

Purification and partial characterisation of and α-tocopherol-binding protein from rabbit heart cytosol

An -tocopherol-binding protein has been isolated and purified from rabbit heart cytosol. The purified protein had an apparent molecular mass of 14,200, as derived from SDS-PAGE. The content of the protein in rabbit heart was around 11.8 g per g of tissue. The binding of -tocopherol to the purified protein was rapid, reversible, and saturable. Neither nor tocopherol could displace the bound -tocopherol from the protein, suggesting a high specificity for -tocopherol. -Tocopherol-binding protein did not bind oleate. Transfer of -tocopherol from liposomes to mitochodria was stimulated 8-fold in the presence of the binding protein, suggesting that this protein may be involved in the intracellular transport of -tocopherol in the heart.     

Carvedilol prevents cardiac hypertrophy and overexpression of hypoxia-inducible factor-1α and vascular endothelial growth factor in pressure-overloaded rat heart

Summary The use of -blockers has emerged as a beneficial treatment for cardiac hypertrophy. Hypoxia-inducible factor-1 (HIF-1) is tightly regulated in the ventricular myocardium. However, the expression of HIF-1 in cardiac hypertrophy due to pressure overload and after treatment with -blocker is little known. To evaluate the effect of carvedilol on both myocardial HIF-1 expression and cardiac hypertrophy, infra-renal aortic banding was performed for 4 weeks in adult Sprague-Dawley rats to induce cardiac hypertrophy. Carvedilol at 50 mg/kg body weight per day after surgery was given. Heart weight and the ratio of heart weight and body weight increased significantly after aortic banding for 4 weeks in the absence of drug treatment. Mean arterial pressure increased from 80 ± 9 mmHg in the sham group to 94 ±5 mmHg (p < 0.001) in the banding group. Echocardiography showed concentric hypertrophy after aortic banding. Mean arterial pressure decreased after treatment with carvedilol. The increased wall thickness and heart weight was reversed to normal by carvedilol. Western blot showed that HIF-1, vascular endothelial growth factor (VEGF) and brain natriuretic peptide (BNP) proteins were up-regulated and nerve growth factor- (NGF-) down-regulated in the banding group. Treatment with valsartan, doxazosin, or N-acetylcysteine did not significantly affect HIF-1 and VEGF proteins expression in the banding groups. Real-time polymerase chain reaction showed that mRNA of HIF-1, VEGF and BNP increased and mRNA of NGF- decreased in the banding group. Treatment with carvedilol reversed both protein and mRNA of HIF-1, VEGF, BNP, and NGF- to the baseline values. Increased immunohistochemical labeling of HIF-1, VEGF, and BNP in the ventricular myocardium was observed in the banding group and carvedilol again normalized the labeling. In conclusion, HIF-1, VEGF, and BNP mRNA and protein expression were up-regulated, while NGF- mRNA and protein was downregulated in the rat model of pressure-overloaded cardiac hypertrophy. Treatment with carvedilol is associated with a reversal of abnormal regulation of HIF-1,VEGF, BNP, and NGF- in the hypertrophic myocardium.     

Evaluation of two new coupling agents for incorporation of α,α-dialkylamino acids, such as α-methylalanine, in solid-phase peptide synthesis

Summary The introduction of solid-phase peptide synthesis (SPPS) has greatly facilitated the preparation of peptides containing proteinaceous amino acids. Less common, sterically hindered ,-dialkylamino acids, such as -methylalanine (MeA, aminoisobutyric acid, Aib), have proven a synthetic challenge for incorporation by this approach, especially when present in contiguous sequences. Solution protocols, utilizing highly reactive intermediates such as oxazalones, are generally used during the preparation of peptaibol antibiotics such as alamethicin, emerimicin, etc. which contain such contiguous sequences. Two recently developed coupling strategies (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, HATU, and Fmoc-protected amino acid fluorides) allow peptides comprising contiguous sequences of ,-dialkylamino acids to be prepared using SPPS. The present study evaluates the relative merits of these two methods on a set of difficult peptides containing oligo-MeA sequences.     

Inhibition of protein synthesis enhances the lytic effects of tumor necrosis factor α and interferon γ in cell lines derived from gynecological malignancies

Summary Few clinical responses have occurred in preliminary studies using the cytokines tumor necrosis factor (TNF) or interferon (IFN) in cancer patients. This may be related to the observation that many malignant cell lines are resistant to lysis by these cytokinesin vitro. Resistance to lysis by TNF or IFN in many cells is controlled by a protein-synthesis-dependent mechanism, such that when protein synthesis is inhibited cells become sensitive to lysis by these cytokines. Because there is some evidence that TNF and IFN act through different lytic mechanisms and are opposed by different resistance mechanisms, we treated a panel of eight cell lines, five derived from human cervical carcinomas (ME-180, MS751, SiHa, HT-3, and C-33A) and three derived from ovarian carcinomas (Caov-3, SK-OV-3, and NIH: OVCAR-3) with both TNF and IFN to determine whether such combination treatment might maximizein vitro cell lysis. Our results showed that pretreatment with IFN followed by exposure to TNF in the presence of protein synthesis inhibitors increased lysis of seven of the eight cell lines above that seen with either TNF or IFN and inhibitors of protein synthesis. Only the cell line C-33A was resistant to lysis by TNF and IFN, when exposed to these agents both alone and in combination with protein synthesis inhibitors. Clinically, combining the cytokines TNF and IFN with protein synthesis inhibitors may maximize thein vivo lytic effects of these cytokines.Supported by American Cancer Society Career Development Award 90-221     

A model for cleavage ofO-glycosidic bonds in glycoproteins

The present work investigated the possibility of cleavage of -linkages between mannose or galactose and serine/threonine residues by -mannosidase and -galactosidase. The study was carried out initially with model synthetic compounds imitating theO-glycosidic bond in glycoproteins, and further with glucoamylase. It was shown that -mannosidase and -galactosidase can hydrolyse these linkages after proteolytic digestion of glucosamylase.     

A Simple Method for Preparation of 18α-Glycyrrhetinic Acid and Its Derivatives

Treatment of 18-glycyrrhizic acid with a methanolic solution of HCl resulted in 1 : 1 mixture of methyl esters of 18- and 18-glycyrrhetinic acids. Benzoylation of the mixture led to methyl esters of 3-benzoyl-18-glycyrrhetinic acid and 3-benzoyl-18-glycyrrhetinic acid, which were separated by chromatography on silica gel. 18-Glycyrrhetinic acid was prepared by alkaline hydrolysis of methyl 3-benzoyl-18-glycyrrhetinate and was further used for the syntheses of 3-keto-18-glycyrrhetinic acid and methyl esters of 18-glycyrrhetinic acid and 3-keto-18-glycyrrhetinic acid.     

Phylogeny and evolutionary rates of G protein α subunit genes

Summary Rooted phylogenetic trees for a total of 34 genes encoding the stimulatory (s), inhibitory (i), transducin (t), G_x (x), G_z (z), G₁₁ (11), G₁₂ (12), G₁₃ (13), G₁₆ (16), G_q (q), and other (o) G protein a subunits have been constructed. The analysis shows that the G₁₂ (12 and 13), G_q (11, 16, and q), and G_s (s genes) groups form one cluster, and the G_x (x and z genes), G_i (i genes), G_t (t1 and t2), and G_o (o genes) groups form another cluster. During mammalian evolution, the rates of synonymous substitutions for these genes were estimated to be between 1.77 × 10^–9/site/year and 5.63 × 10^–9/site/year, whereas those of nonsynonymous substitutions were between 0.008 × 10^–9/site/year and 0.067 × 10^–9/site/year. These evolutionary rates are similar to those for histone genes, suggesting equally important biological functions of the G protein a subunits. Offprint requests to: S. Yokoyama     

Chemical induction of interleukin-8, a proinflammatory chemokine,in human epidermal keratinocyte cultures and its relation to cytogenetic toxicity

Tumor promoters, proinflammatory cytokines, endotoxins, and protein synthesis inhibitors can modulate cell cycle kinetics of various cell types, stimulate production of reactive oxygen species, and induce keratinocytes to produce interleukin-8 (IL-8), a potent chemotactant for polymorphonuclear neutrophils and T lymphocytes. The aim of this study was to determine whether perturbations of cytogenetic responses correlated with the induction of IL-8 expression. Cultures of primary human keratinocytes were grown in serum-free medium with 5 mol/L bromodeoxyuridine to label DNA and exposed either to phorbol-13-myristate-12-acetate (PMA) (0.0001–100 ng/ml), cycloheximice (CHX) (0.01–50 g), lipopolysaccharide (0.1–100 g/ml), tumor necrosis factor- (TNF) (3.13–50 ng/ml), or interleukin-1 (IL-1) (1–182 pg/ml). Metaphase chromosome preparations were stained by a fluorescence-plus-Giemsa technique to differentiate sister chromatids. For IL-8 production, keratinocytes were grown to 70% confluency and then exposed to chemicals for 24 h. Immunoreactive IL-8 was quantitated from the supernatants by ELISA. With the exception of benzo(a)pyrene used as a positive control, none of the agents induced sister chromatid exchanges. However, PMA and TNF induced IL-8 production that coincided with significant cell cycle inhibition. IL-1 had no effect on cytogenetic endpoints, yet stimulated a 6.3-fold increase in IL-8. CHX inhibited cell cycle progression and mitotic activity at concentrations that were 200 times lower than required for IL-8 induction; however, puromycin (0.31–10 g/ml), another protein synthesis inhibitor, did not induce IL-8. At all concentrations tested, TNF reduced the mitotic index by 45%, slowed cell cycle progression by 3.5 h, and induced a flat, albeit large, IL-8 response at concentrations 12.5 ng/ml. These agent-specific response patterns suggest that induction of IL-8 production is not always the inevitable result of cell cycle perturbations or genetic damage.Abbreviations B(a)P benzo(a)pyrene - BrdU 5-bromo-2-deoxyuridine - CHX cycloheximide - ICAM intercellular adhesion molecules - IL-1 interleukin-1 - IL-8 interleukin-8 - KGM keratinocyte growth medium - LPS lipopolysaccharide - PKC protein kinase C - PMA phorbol-13-myristate-12-acetate - PMN polymorphonuclear neutrophil - ROS reactive oxygen species - SCE sister chromatid exchange - TNF tumor necrosis factor      

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

Fungal thermostable α-dialkylamino acid aminotransferase: occurrence,purification and characterization

-Dialkylamino acid aminotransferase was found in various fungi; this is the first evidence for the occurrence of the enzyme in eukaryotes. The enzyme was purified from Fusarium solani and shown to be composed of four subunits with an identical molecular weight of 42,000. -Aminoisobutyrate and cycloleucine served as amino donors, and pyruvate, -ketobutyrate, -ketovalerate, -ketoisovalerate, and glyoxylate as amino acceptors. The K _m values for -aminoisobutyrate and -ketobutyrate were 28 and 0.3 mM, respectively. -Ketobutyrate inhibited the enzyme noncompetitively with -aminoisobutyrate, and showed K _i value of 8 mM. The significant inhibitory effect of l-cycloserine was observed, but d-cycloserine did not inhibit the enzyme. The pH and temperature optima for transamination of -aminoisobutyrate with pyruvate were about 8.0 and 60°C, respectively. Despite the production of this enzyme by the mesophile, the enzyme was thermostable; it retained its full activity upon heating at 60°C for 30 min.Abbreviations ACPC 1-aminocyclopropane-1-carboxylic acid - AIB -aminoisobutyrate - PLP pyridoxal 5-phosphate