Comparison of the properties of purified mitochondrial and cytosolic rat kidney transamidinase

1. Mitochondrial rat kidney transamidinase was solubilized by two extractions with the surfactant Zwittergent 3-14. 2. Mitochondrial and cytosolic forms of rat kidney transamidinase were purified by chromatography on DEAE-Trisacryl M, phenyl-Sepharose Cl-4B and hydroxylapatite columns. 3. The specific activity of purified mitochondrial enzyme was significantly higher than purified cytosolic enzyme. 4. The subunit molecular mass, the electrophoretic mobility under nondenaturing conditions, and the activation energy were similar for purified mitochondrial and cytosolic transamidinase.     

Formation of 2-guanidinoethanol by a transamidination reaction from arginine and ethanolamine by the rat kidney and pancreas

The formation of 2-guanidinoethanol (GEt) from L-arginine (Arg) and ethanolamine (EA) was studied using rat kidney homogenates. Maximum GEt formation was observed between pH 8.7 and 9.1, and the enzyme catalyzing the GEt synthesis was stable between pH 5.6 and 9.1. The rate of GEt formation from Arg and EA by rat kidney homogenates obeyed simple Michaelis-Menten type kinetics. L-Ornithine and glycine inhibited GEt formation by rat kidneys. Both of them inhibited GEt formation in a linear mixed-type inhibitory manner when Arg concentrations were varied at a fixed concentration of EA, while they showed competitive inhibition when EA concentrations were varied at a fixed concentration of Arg. L-Canavanine and guanidinoacetic acid as well as Arg acted as an amidine donor for GEt formation, but L-homoarginine, 3-guanidinopropionic acid and 4-guanidinobutyric acid did not. GEt synthesis was also observed in the rat pancreas. It had almost half of the activity of rat kidney to form GEt. This ratio of kidney to pancreas was approximately equal to that of L-arginine:glycine amidinotransferase (transamidinase, EC 2.1.4.1) in kidney and pancreas. These results suggest that GEt may be synthesized from Arg and EA by a transamidinase catalyzing reaction.     

Arginine:glycine amidinotransferase. A comparative study in lizard and mouse tissues

Kidney transamidinase activity in the lizard (Calotes versicolor), like that in the mouse, showed the pH optimum at 7.4. The lizard enzyme was inhibited to a greater degree than the mouse enzyme at high concentrations (greater than 20 mM) of L-arginine and glycine. Kidney and liver in the lizard and kidney and pancreas in the mouse were the tissues with high transamidinase activity. While transamidinase activity was widely distributed in mouse tissues, the enzyme was found to be restricted only to a few tissues in the lizard. Hydrocortisone administration into male lizards did not significantly alter the transamidinase levels in kidney and liver.     

Characterization of rat adipose tissue lipoprotein lipase using a monospecific antibody

An antibody to a highly pure enzyme preparation was developed to facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation. Lipoprotein lipase was purified by heparin-Sepharose affinity chromatography followed by preparative isoelectric focusing. The enzyme migrated as a single broad band on SDS disc gel and two-dimensional gel electrophoresis with an apparent molecular mass of 67 000 and 62 000 Da, respectively. The amino acid composition of the purified rat enzyme was virtually identical to that of bovine milk. A major protein component with no lipase activity co-eluted with the enzyme from the affinity column, but was separated by the isoelectric focusing step. The molecular mass was slightly lower (58 000 Da) but the amino acid composition of this protein was similar to that of the enzyme. An antibody raised against the purified rat enzyme was highly potent and was effective in inhibiting rat heart lipoprotein lipase, but not the salt-resistant hepatic lipase. Analysis of crude acetone-ether adipose tissue preparation on SDS slab polyacrylamide gel coupled to Western blotting revealed five protein bands = (62 000, 56 000, 41 700, 22 500, 20 000 Da). Similarly, following affinity purification by immunoadsorption, the purified antibody reacted with five equivalent protein bands. Fluorescent concanavalin A binding data indicated that the 56 kDa band is a glycosylated form of lipoprotein lipase. Pretreatment of adipose tissue with proteinase inhibitors revealed that the lower molecular mass proteins (41 700 and 20 000 Da) were degradation products of lipoprotein lipase, and the 22 500 Da band could be accounted for by non-specific binding.     

Purification and characterization of aminopeptidase N from human plasma

Human plasma aminopeptidase N (EC 3.4.11.2) was homogeneously purified from outdated bank plasma. Purification procedures included ammonium sulfate fractionation, immunoaffinity chromatography, DEAE-cellulose column chromatography, hydroxyapatite column chromatography and Sephadex G-200 gel filtration. The final recovery of the enzyme was 18% and its specific activity was 71.6 mumol/min/mg protein. SDS-polyacrylamide gel disc electrophoresis and analytical ultracentrifugation showed the homogeneity of the enzyme. Equilibrium ultracentrifugation showed a molecular weight of 210,800. SDS-polyacrylamide gel disc electrophoresis indicated that the enzyme was a dimer consisting of two identical subunits. The isoelectric point of the enzyme was 3.9 at 4 degrees C. The amino acid composition of the enzyme was very similar to those of aminopeptidase N from human kidney, small intestine, and placenta which we have reported previously. Neutral sugar accounted for 11.6%. The Km, Vmax and Kcat values and hydrolytic coefficient (Kcat/Km) of the enzyme with L-alanyl-beta-naphthylamide as substrate were 8.7 X 10(-5) mol/l, 85.9 mumol/min/mg protein, 303/s and 3,483/mmol/l/s, respectively. The enzyme was activated by cobalt ions and markedly inhibited by amastatin. Plasma aminopeptidase N was immunologically indistinguishable from kidney aminopeptidase N.     

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver

The bile acid-conjugating enzyme, bile acid-CoA: amino acid N-acyltransferase, was purified 480-fold from the soluble fraction of homogenized frozen human liver. Purification was accomplished by a combination of anion exchange chromatography, chromatofocusing, glycocholate-AH-Sepharose affinity chromatography, and high performance liquid chromatography (HPLC) gel filtration. Following purification, the reduced, denatured enzyme migrated as a single 50-kDa protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar molecular mass was obtained for the native enzyme by HPLC gel filtration. Elution from the chromatofocusing column suggested an apparent isoelectric point of 6.0 (+/- 0.2). Using a rabbit polyclonal antibody raised against the purified enzyme, Western blot analysis using 100,000 x g human liver supernatant confirmed that the affinity-purified polyclonal antibody was specific for human liver bile acid-CoA:amino acid N-acyltransferase. The purified enzyme utilized glycine, taurine, and 2-fluoro-beta-alanine (a 5-fluorouracil catabolite), but not beta-alanine, as substrates. Kinetic studies revealed apparent Km values for taurine, 2-fluoro-beta-alanine, and glycine of 1.1, 2.2, and 5.8 mM, respectively, with corresponding Vmax values of 0.33, 0.19, and 0.77 mumol/min/mg protein. These data demonstrate that a single monomeric enzyme is responsible for the conjugation of bile acids with glycine or taurine in human liver.     

Repression of rat kidney L-arginine:glycine amidinotransferase synthesis by creatine at a pretranslational level

The first committed reaction in the biosynthesis of creatine is catalyzed by the enzyme L-arginine:glycine amidinotransferase, commonly called transamidinase. Creatine, the end product of the biosynthetic pathway, is known to alter the levels of kidney transamidinase activity. Rats fed a diet containing 0.3% creatine had 26% of the kidney transamidinase activity of the rats fed a creatine-free diet. This reduction in transamidinase activity was correlated with a decrease in transamidinase protein in the creatine-fed rats. The relative synthetic rates and mRNA functional activities of transmidinase were measured in control and creatine-fed rats. The relative synthetic rate of transamidinase in creatine-fed rats was 21% of that found in the control animals. The functional transamidinase mRNA in creatine-fed rats was correspondingly reduced to 37% of the amount in the control animals. Thus, creatine affects transamidinase activity by altering its rate of synthesis at a pretranslational step and represents an example of end-product repression in a higher eukaryote.     

Isolation and characterization of angiotensin converting enzyme from human kidney

Angiotensin converting enzyme [EC 3.4.15.1] was solubilized from the membrane fraction of human kidney cortex using trypsin and purified to homogeneity by DEAE-cellulose, hydroxylapatite and DEAE-Sephadex A-50 column chromatographies, preparative isoelectric focusing, and Sephadex G-200 gel filtration. The final recovery of the enzyme was 13.9%. The molecular weight of the enzyme was estimated to be 199,000 by a sedimentation equilibrium method. A value of 170,000 was obtained for the reduced and denatured enzyme by dodecylsulfate-polyacrylamide gel electrophoresis. The enzyme was a glycoprotein consisting of a single polypeptide chain with an isoelectric point of 5.10. Neutral sugar accounted for 13% per weight of the enzyme. The purified enzyme had a specific activity of 96.9 mumol/min/mg protein for hippurylhistidylleucine. The Km value, Kcat value and hydrolytic coefficient (Kcat/Km) of the enzyme for hippurylhistidylleucine were 2.0 mM, 545 s-1 and 273 mM-1 . s-1, respectively. Rabbit antibody against the human kidney converting enzyme inhibited the activities of the enzymes from human lung and serum as equally as that from human kidney, but not those from sheep, dog, or rat sera. The human kidney and lung converting enzymes were immunologically identical on double immunodiffusion analysis.     

Multiple forms of human renin. Purification and characterization.

Human renin was purified from a juxtaglomerular cell tumor with a high renin content, 24.2 Goldblatt units/mg of protein. The purification procedure comprised three steps: gel filtration, DEAE-cellulose chromatography, and preparative isoelectric focusing. Five forms of renin amounting to 5.3 mg of enzyme were obtained with isoelectric points of 4.95, 5.10, 5.35, 5.55, and 5.70. They were all glycoproteins. The three major fractions had very similar specific activities, 868, 860, and 809 Goldblatt units/mg of protein. These fractions produced a single band on analytical isoelectric focusing and a single arc on immunoelectrophoresis. On polyacrylamide gel electrophoresis at pH 7.8, each fraction consisted of two renin bands with the same molecular weight, but different net charges. The molecular weight determined by gel filtration and Fergusson plot analysis on polyacrylamide gel was 38,000 to 42,000. The optimum pH determined on N-acetyltetradecapeptide substrate was 6.5, and the Km was 6.8 x 10(-6) M. These parameters were identical with those for standard human kidney renin. Antibodies raised against tumor renin completely inhibited the activity of both tumor and standard renin. Under dissociating conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel electrophoresis in the presence of 6 M urea), part of the purified enzyme dissociated into two smaller fragments (Mr = 20,000 and 25,000) containing renin activity.     

Effects of Arginine and Methionine on the Growth Depression of Rats Fed Diets High in Glycine

In order to determine if the growth retardation by dietary exceess glycine could be prevented by the addition of arginine and/or methionine, weanling rats were fed a 25% casein diet (standard) or a 10% casein diet (low protein diet) with a supplement of several combinations of glycine, arginine, or methionine.

The changes in body weight, urinary creatinine, and kidney transamidinase activity were determined. The growth depression effect by excess glycine was prevented considerably in animals receiving standard diet and completely in animals receiving low protein diet by the addition of arginine and methionine to the high glycine diets.

The total urinary creatinine was increased by the supplement of both glycine and arginine, while the growth rate was not invariably raised and kidney transamidinase activity had a tendency to decrease.     

Human tissue kallikrein. I. Isolation and characterization of human pancreatic kallikrein from duodenal juice

Human pancreatic kallikrein was purified from duodenal juice by ion exchange chromatography on DEAE-Sepharose and immunoaffinity chromatography. Thus, an enzyme preparation with a specific activity (using Ac-Phe-Arg-OEt as substrate) of 1 000 U/mg protein was obtained. A specific biological activity of 1310 KE/mg protein was measured in the dog blood pressure assay and of 0.361 HMW kininogen-U/mg, corresponding to the liberation of 383 micrograms bradykinin-equivalents per mg enzyme per min from HMW kininogen in the rat uterus assay. In dodecyl sulfate gel electrophoresis one protein band corresponding to a molecular mass of 27 kDa was obtained. Using gel filtration on Ultrogel AcA-44 a molecular mass of 40 kDa was measured. The amino-acid composition was determined and isoleucine and alanine were identified as the only N-terminal amino-acid residues. On isoelectric focusing four protein bands with isoelectric points of 5.60, 5.65, 5.70 and 5.85 were separated. The bimolecular velocity constant for the inhibition by diisopropyl fluoro phosphate was determined as 10.5 l x mol-1 x min-1. The dissociation constant Ki of the human pancreatic kallikrein-aprotinin complex was calculated to be 1.5 x 10(-10)M. The kinetic constants for the kallikrein-catalysed hydrolysis of Ac-Phe-Arg-OEt and D Val-Leu-Arg-Nan were determined. Immunological studies showed a close relationship between the human pancreatic kallikrein and other human tissue kallikreins, especially with human urinary kallikrein. Detergents such as Triton X-100, Tween 20 and lysolecithin, as well as human serum albumin, activated the human pancreatic kallikrein preparation.     

Isolation and characterization of human urinary kallikrein

Human urinary kallikrein was purified by gel filtration on Sephacryl S-200 and affinity chromatography on aprotinin-Sepharose, followed by ion exchange chromatography on DEAE-Sepharose. Thus an enzyme preparation with a specific activity (using AcPheArgOEt as substrate) of 1 100 U/mg protein was obtained. A specific bioligical activity of 2 300 KE/mg was measured in the dog blood pressure assay and of 0.742 HMW kininogen-U/mg, corresponding to the liberation of 787 micrograms bradykinin per mg enzyme per min from HMW-kininogen, in the rat uterus assay. In dodecyl sulfate electrophoresis two protein bands with apparent molecular weights of 41 000 and 34 000 were separated. The amino acid composition was determined and isoleucine was identified as the only aminoterminal amino acid. On isoelectric focusing six protein bands with isoelectric points of 3.75, 3.80, 3.90, 4.00, 4.05 and 4.25 were separated. The kinetic constants for the kallikrein-catalyzed hyrdolysis of AcPheArgOEt and D ValLeuArgNHNp were determined. The bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 9 +/- 2l x mol-1 x min-1. Immunological studies showed that a close relationship exists between the urinary enzyme and other human glandular kallikreins. Deoxycholate, lysolecithin and other amphiphiles activated human urinary kallikrein.     

Acidic and basic forms of glutathione S-transferases from human placenta and comparison with human kidney glutathione S-transferase

Glutathione S-transferase (GSH-transferase) was purified from human placenta and kidney by affinity chromatography on S-glutathione-carbamidomethyl-epsilon-aminolysyl-Sepharose CL 4B and gel filtration chromatography on Sephades G-75. Electrophoretically pure enzyme with the specific activities of 50.7 and 55.9 U/mg, respectively, were obtained. In addition to the known acidic isoenzyme from human placenta (isoelectric point, pI, 4.5), we describe here for the first time the presence of 6 basic forms with pI values between 8.0 and 9.0. The kidney GSH-transferase contained 2 acidic forms with isoelectric points at 4.6 and 4.65, and 6 basic forms with pI values between 8.7 and 9.4. The basic and acidic isoenzymes from placenta were separated by ion exchange chromatography on Sephadex DEAE A-25. The acidic form accounted for 36% of the total GSH-transferase activity from placenta. Antibodies against the kidney enzyme were raised in rabbit. Total cross-reactivity of placental GSH-transferase with antikidney-GSH-transferase antibodies was obtained, suggesting that the kidney and placental enzymes are immunologically closely related.     

Purification and Characterization of Aromatic L-Amino Acid Decarboxylase from Rat Kidney and Monoclonal Antibody to the Enzyme

Aromatic L-amino acid decarboxylase was purified from rat kidney to homogeneity, as judged by polyacrylamide gel electrophoresis, in the presence and absence of sodium dodecyl sulfate (SDS). The final preparation showed an activity of 3,4-dihydroxyphenylalanine (dopa) decarboxylation of approximately 11,000 nmol/min/mg of protein at 37 degrees C. The purified enzyme also catalyzed the decarboxylation of 5-hydroxytryptophan, tyrosine, tryptophan, and phenylalanine. The enzyme appeared to be composed of two identical subunits, each possessing a molecular weight of 48,000. The isoelectric point of the enzyme was estimated to be 6.7 in the presence of 8 M urea and 5.60-5.85 in its absence. To examine the identity of aromatic L-amino acid decarboxylase from various tissues, a monoclonal antibody directed against the enzyme from rat kidney was prepared. Immunotitration and analysis by antibody-affinity chromatography followed by SDS-polyacrylamide gel electrophoresis revealed that the enzymes from the striatum, adrenal medulla, pineal gland, liver, and kidney were indistinguishable with respect to immunological cross-reactivity and molecular size.     

Characterization of dehydropeptidase I in the rat lung

The activity of dehydropeptidase I in rat tissues decreases in the order of lung greater than kidney greater than liver-spleen greater than other tissues, while aminopeptidase activity is high in the kidney, and lower in the lung than in other tissues. Dehydropeptidase I was solubilized from the membrane fraction of rat lung by treatment with papain and purified by DEAE-cellulose column chromatography, affinity chromatography on concanavalin-A-Sepharose and high-performance liquid chromatography gel filtration. The purified preparation was found to be homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The relative molecular mass was estimated to be 150,000 by gel filtration, comprising a homodimer of two 80,000-Mr subunits. The enzyme activity was inhibited by cilastatin, o-phenanthroline and ATP. This enzyme catalyzed the hydrolysis of S(substituent)-L-cysteinyl-glycine adducts such as L-cystinyl-bis(glycine) and N-ethylmaleimide-S-L-cysteinyl-glycine, as well as the conversion of leukotriene D4 to E4. Furthermore it catalyzed a hydrolytic splitting of L-Leu-L-Leu, but not S-benzyl-L-cysteine p-nitroanilide, which is a good substrate for aminopeptidase. Our enzyme preparation was immunologically identical to the rat renal dehydropeptidase I. The physiological significance of the pulmonary dehydropeptidase I on the metabolism of glutathione and its adducts is discussed.     

Heterogeneity of hypoxanthine guanine phosphoribosyl transferase from human erythrocytes.

Hypoxanthine guanine phosphoribosyl transferase (E.C.: 2.4.2.8) has been purified 4000- to 4500-fold from normal human erythrocytes by three different schemes of protein fractionation. In one scheme, the enzyme was separated by preparative polyacrylamide gel electrophoresis in an LKB Uniphor system and purified by affinity column chromatography employing Sepharose/phosphoribosyl/pyrophosphate. In the second, the enzyme was isolated by isotachophoresis in the presence of Amphiline carrier ampholytes employing a Tris/phosphate/β-alanine ion system. The enzyme was then purified by isotachophoresis in the presence of carrier ampholytes using a Tris/acetate/glycine ion system. The hypoxanthine guanine phosphoribosyl transferase purified by affinity chromatography and isotachophoresis consisted, on immunoelectrophoresis, mainly of one component and had less than 5% impurities. When subjected to analytical polyacrylamide gel electrophoresis, such preparations were resolved into four isoenzymes. In the third scheme, the enzyme was isolated by isoelectric focusing. In this system, the enzyme was also resolved into four isoenzymes. Their isoelectric points were: 5.47, 5.63, 5.74, and 5.84. When subjected to analytical polyacrylamide gel electrophoresis each isoenzyme migrated at a different rate. In sodium dodecyl sulfate gel electrophoresis each isoenzyme yielded one major and one minor band. Protein appearing in the major and minor bands migrated at rates consistent with a molecular size of 33,500 and 26,500, respectively.     

Purification and characterization of acid trehalase from the yeast suc2 mutant

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.     

Purification and characterization of human placental microsomal aminopeptidase: immunological difference between placental microsomal aminopeptidase and pregnancy serum cystyl-aminopeptidase

Human placental microsomal aminopeptidase (microsomal PAP) was purified 3,880-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100 and also trypsin digestion, and subjected to zinc sulfate fractionation, chromatographies with DE-52, hydroxylapatite, Sephacryl S-300 and lentil lectin-Sepharose 4B, and finally affinity chromatography with bestatin-Sepharose 4B. Microsomal PAP was separated from aminopeptidase A (AAP) by affinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 220,000 by high-performance liquid chromatography with an aqueous gel column. The purified enzyme gave almost a single band with a molecular mass of 140,000 by sodium dodecyl sulfate (SDS) gel electrophoresis. The isoelectric point of the enzyme was 5.2. The purified enzyme was most active at pH 8.0 with L-leucine-p-nitroanilide as substrate; the Km value for this substrate was 1.1 mmol/l. The microsomal PAP was immunologically different from the pregnancy serum cystyl aminopeptidase (serum PAP).     

Multiple forms of gamma-butyrobetaine hydroxylase (EC 1.14.11.1).

gamma-Butyrobetaine hydroxylase [4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1] from human kidney was resolved into three forms by chromatofocusing. After further chromatography on an anion-exchanger, each form appeared as a single band on electrophoresis in polyacrylamide gel containing sodium dodecyl sulphate. The isoelectric points of isoenzymes 1, 2 and 3 were 5.6, 5.7 and 5.8 respectively, as estimated by isoelectric focusing. Their specific activities were 17-29 mu kat/g of protein. The concentrations of the three isoenzymes were about equal, possibly slightly lower for isoenzyme 1. The requirement for Fe2+ and the Km values for gamma-butyrobetaine and 2-oxoglutarate were about the same for the different enzyme forms. L- and D-Carnitine caused decarboxylation of 2-oxoglutarate to the same extent (8 and 29%) with the three forms. The enzyme forms had the same mass, 64 kDa, as determined by gel filtration in nondenaturing media. The same subunit mass, 42 kDa, was obtained for the multiple forms by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Isoenzyme 2 was resolved into two protein bands by isoelectric focusing in polyacrylamide gels containing urea. Isoenzyme 1 contained only one of these bands and isoenzyme 3 the other. The three enzyme forms of gamma-butyrobetaine hydroxylase thus appear to be dimeric combinations of two subunits differing in charge but not in size. gamma-Butyrobetaine hydroxylase from crude extracts of human, rat and calf liver was also separated into multiple forms by a chromatofocusing technique. The isoenzyme pattern was the same in human liver and kidney. The technique used to resolve the mammalian enzymes gave no evidence for the presence of multiple forms of the bacterial enzyme from Pseudomonas sp. AK 1.     

Purification of ferrichrome synthetase from Aspergillus quadricinctus and characterisation as a phosphopantetheine containing multienzyme complex

Aspergillus quadricinctus was grown under iron limitation to induce the enzymes for ferrichrome biosynthesis. The mycelium was disintegrated by ultraturrax homogenization, and ferrichrome synthetase was purified by column chromatography on DEAE cellulose, hydroxyapatite and Bio-Gel A-5m. The enzyme was almost homogeneous in single fractions as shown in gel electrophoresis under non-denaturating conditions. By fast-protein liquid chromatography on Superose 6, the purified ferrichrome synthetase (molecular weight 9.6.10(5) dissociated partly into an enzyme complex with reduced ferrichrome synthetase activity of 8 x 10(5) Da, one acetylhydroxyornithine (AHO) activating protein of 5.5 x 10(5) Da and one glycine activating protein of 4 x 10(5) Da. After SDS treatment the AHO activating protein dissociated into subunits of 9 x 10(4) Da, while the glycine activating protein dissociated into subunits of 5 x 10(4) Da and 4 x 10(4) Da in a molar ratio of 6:1. No subunits were found after SDS treatment of the larger of the two ferrichrome synthetizing enzyme complexes. Pantetheine was detected in protein bands of defined molecular weights (4 x 10(4), 9 x 10(4) and greater than 3.4 x 10(5) after SDS polyacrylamide gel electrophoresis. Gel slices were cut out, and the growth factor activity for Lactobacillus plantarum ATCC 8014 was analyzed. The calculated content was 2 mol of pantetheine per mol of ferrichrome synthetase of 9.6 x 10(5) Da.