Oxidation-induced increase in activity of angiotensin converting enzyme in the rat kidney

The activity of angiotensin converting enzyme(ACE) in crude extracts of the rat renal cortex was increased when the oxidizing agent diamide was added to the extract. The maximal activity was obtained at concentrations over 1 mM, and the value was twice or more the activity in the absence of the pretreatment. The activity of ACE was also increased by the diamide-pretreatment of the isolated membrane fraction of the renal cortex, thereby indicating that the increase in activity was not due to oxidation of endogenous glutathione (GSH) that may lower the ACE activity, but rather that ACE itself was oxidized. When O2 was included in the extract for 2 h, the ACE activity also increased to about twice the original activity. Lineweaver-Burk plots analysis demonstrated that, after oxidation with diamide and O2, the Vmax was increased but the Km remained unchanged. We conclude that the action of ACE in the kidney functions may differ in relation to oxidation of the tissue.     

Angiotensin I-converting enzyme inhibitory peptides isolated from tofuyo fermented soybean food

Angiotensin I-converting enzyme (ACE) inhibitory activity was observed in a tofuyo (fermented soybean food) extract with an IC(50) value of 1.77 mg/ml. Two ACE inhibitors were isolated to homogeneity from the extract by adsorption and gel filtration column chromatography, and by reverse-phase high-performance liquid chromatography (HPLC). The purified substances reacted with 2,4,6-trinitrobenzensulfonic acid sodium salt. The amino acid sequences of these inhibitors determined by Edman degradation were Ile-Phe-Leu (IC(50), 44.8 microM) and Trp-Leu (IC(50), 29.9 microM). The Ile-Phe-Leu sequence is found in the alpha- and beta-subunits of beta-conglycinin, while the Trp-Leu sequence is in the B-, B1A- and BX-subunits of glycinin from soybean. Both of the peptides are non-competitive inhibitors. The inhibitory activity of Trp-Leu was completely preserved after a treatment with pepsin, chymotrypsin or trypsin. Even after successive digestion by these gastrointestinal proteases, the activity remained at 29% of the original value.     

Isolation and characterization of a novel angiotensin I-converting enzyme inhibitory peptide derived from the edible mushroom Tricholoma giganteum

The fruiting body of Tricholoma giganteum has many pharmaceutical uses and has long been utilized as a home remedy in Asia. This study describes the extraction and characterization of the first angiotensin I-converting enzyme (ACE) inhibitory peptide from T. giganteum. The maximum ACE inhibitory activity (IC50: 0.31 mg) was obtained when the fruiting body of T. giganteum was extracted with distilled water at 30 degrees C for 3 h. After the purification of ACE inhibitory peptides with ultrafiltration, Sephadex G-25 column chromatography, and reverse-phase HPLC, an active fraction with an IC50 of 0.04 mg and a yield of 0.3% was obtained. The ACE inhibitory peptide was a novel tripeptide, showing very low similarity to other ACE inhibitory peptide sequences, and was sequenced as Gly-Glu-Pro. The purified ACE inhibitor from T. giganteum competitively inhibited ACE, and it maintained inhibitory activity even after incubation with proteases. ACE inhibitor from T. giganteum showed a clear antihypertensive effect in spontaneously hypertensive rats (SHR), at a dosage of 1 mg/kg.     

The relationship between ACE inhibitory activity and degradations of sulfur containing materials in Dolsan leaf mustard juice

This study was carried out to investigate the relationship ACE inhibitory activity and degradations of sulfur containing materials in Dolsan leaf mustard juice (DLMJ). The changes of sulfur containing materials which were treated with autolysis, myrosinase, ascorbate and papain were studied, as well as the changes of ACE inhibitory activity in DLMJ. At 37°C, sulfur containing materials by autolysis decreased most rapidly from 0.43% to 0.13% in the second day. Conversely, ACE inhibitory activity increased most from 66% to 87%, in the second day at 37°C. As myrosinase concentrations increased more, sulfur containing materials in DLMJ decreased more. The ACE inhibitory activities at 0, 0.5, 1, 2, and 4 Units of myrosinase for 240 min later were 70, 74, 75, 82, and 85%, respectively. At 1 mM ascorbate, concentrations of sulfur containing materials in DLMJ decreased more significantly on the second day than on the other days. At 1 mM ascorbate for 6 days, ACE inhibitory activity reached a maximum of about 92%. And, an increase of papain concentration was noted in accordance with a decreased sulfur containing materials. The maximum rate of ACE inhibitory activity at control, 3, 6, and 12 Units of papains treatments was shown as 70, 70, 75, and 78% at 60 min, respectively. These results suggested that the degradation of sulfur containing materials led to the increase of ACE inhibitory activity. Consequently, it was suggested that ACE inhibiting was significantly related to the degradatives of sulfur containing materials.     

Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays

Angiotensin-converting enzyme 2 (ACE2) is a component of the renin-angiotensin system, and its expression and activity have been shown to be reduced in cardiovascular diseases. Enzymatic activity of ACE2 is commonly measured by hydrolysis of quenched fluorescent substrates in the absence or presence of an ACE2-specific inhibitor, such as the commercially available inhibitor DX600. Whereas recombinant human ACE2 is readily detected in mouse tissues using 1 μM DX600 at pH 7.5, the endogenous ACE2 activity in mouse tissues is barely detectable. We compared human, mouse, and rat ACE2 overexpressed in cell lines for their sensitivity to inhibition by DX600. ACE2 from all three species could be inhibited by DX600, but the half maximal inhibitory concentration (IC(50)) for human ACE2 was much lower (78-fold) than for rodent ACE2. Following optimization of pH, substrate concentration, and antagonist concentration, rat and mouse ACE2 expressed in a cell line could be accurately quantified with 10 μM DX600 (>95% inhibition) but not with 1 μM DX600 (<75% inhibition). Validation that the optimized method robustly quantifies ACE2 in mouse tissues (kidney, brain, heart, and plasma) was performed using wild-type and ACE2 knockout mice. This study provides a reliable method for measuring human, as well as endogenous ACE2 activity in rodents. Our data underscore the importance of validating the effect of DX600 on ACE2 from each particular species at the experimental conditions employed.     

Isolation and characterization of a specific endogenous Na+,K+-ATPase inhibitor from bovine adrenal

In order to identify a specific endogenous Na+,K+-ATPase inhibitor which could possibly be related to salt-dependent hypertension, we looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na+,K+-ATPase; (ii) competitive displacing activity against [3H]ouabain binding to the enzyme; (iii) inhibitory activity for 86Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C18 open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na+,K+-ATPase such as unsaturated free fatty acids, lysophosphatidylcholines, vanadate, dihydroxyeicosatrienoic acid, dehydroepiandrosterone sulfate, dopamine, lignan, ascorbic acid, etc. This substance was further purified by using an additional five steps of high-performance liquid chromatography with five different types of columns. Molecular mass was estimated as below 350 by fast atom bombardment mass spectroscopy and ultrafiltration. Heat treatment at 250 degrees C for 2 h and acid treatment with 6 N HCl at 115 degrees C for 21 h almost completely destroyed the inhibitory activity of the purified substance for Na+ pump activity. Additionally, alkaline treatment with 0.2 N NaOH at 23 degrees C for 2 h destroyed approximately 70% of the inhibitory activity, whereas boiling for 10 min and various enzyme digestion did not destroy the activity. The dose dependency for the four types of the activities for this substance paralleled those of ouabain, spanning 2 orders of magnitude in concentration range. The inhibitory potencies of the purified substance for Na+,K+-ATPase, Na+ pump, and ouabain binding activities were diminished with increasing K+ concentration, exhibiting a characteristic typical of cardiac glycosides. This substance had no effect on the Ca2+-ATPase activity or the Ca2+ loading rate into the vesicle prepared from skeletal muscle sarcoplasmic reticulum. These results strongly suggest that this water-soluble nonpeptidic Na+,K+-ATPase inhibitor may be a specific endogenous regulator for the ATPase.     

Angiotensin I converting enzyme (ACE) inhibitory peptide derived from the sauce of fermented blue mussel, Mytilus edulis

The angiotensin I converting enzyme (ACE) inhibitory activity of fermented blue mussel sauce (FBMS) was investigated. Blue mussels were fermented with 25% NaCl (w/w) at 20 degrees C for 6 months and the resultant mixture was passed through a 40-mesh sieve, desalted using an electrodialyzer and then lyophilized. The IC(50) value of FBMS for ACE activity was 1.01 mg/ml. An ACE inhibitory peptide was purified from FBMS using Sephadex G-75 gel chromatography, SP-Sephadex C-25 ion exchange chromatography and reversed-phase high-performance liquid chromatography on a C(18) column. The IC(50) value of purified ACE inhibitory peptide was 19.34 microg/ml, and 10 amino acid residues of the N-terminal sequence was EVMAGNLYPG. The purified peptide was evaluated for antihypertensive effect in spontaneously hypertensive rats (SHR) following oral administration. Blood pressure significantly decreased after peptide ingestion. This result suggested that FBMS may have beneficial effects on hypertension.     

A Na pump inhibitor from bovine posterior pituitary: purification, structure determination and its cardiovascular effect in rat.

We examined the hypothesis that hypothalamo-hypophysial tissue contains an endogenous Na pump inhibitor. From bovine posterior pituitary, we purified a substance which inhibits Rb uptake by human erythrocytes. This inhibitory activity was found in the eluate of 10% acetonitrile from a C18 flash column and purified by subsequent three steps of reversed-phase high-performance liquid chromatography (HPLC). Sequence analysis revealed that this substance was identical to joining peptide, one of the major products of proopiomelanocortin (POMC). This peptide had hypertensive and tachycardiac effects in spontaneously hypertensive rats (SHR) after central administration, with weak Na,K-ATPase inhibitory activity (IC50 = 0.5 mM).     

Optimization of submerged culture conditions for the production of angiotensin converting enzyme inhibitor from <Emphasis Type="Italic">Flammulina velutipes</Emphasis>

The angiotensin-converting enzyme (ACE) inhibitory effect was tested in the culture broth from submerged mycelial cultures of 20 basidiomycetes. The ACE inhibitory effect of culture broth from Flammulina velutipes strain 414 was the highest (52.8%), followed by Lentinus edodes strains 2 (44.4%) and 16 (41.3%). Nutritional requirements for the production of ACE inhibitory substance from F. velutipes were studied. Sucrose, ammonium acetate, and glutamic acid were chosen for the maximum production of ACE inhibitory substance. The optimal medium composition was (g/l): sucrose 20, ammonium acetate 5, glutamic acid 2, KH₂PO₄ 3, MgSO₄·7H₂O 0.8, and yeast extract 0.5. Under optimal culture conditions, the ACE inhibitory effect was more than 80%. Received 04 May 2002/ Accepted in revised form 11 June 2002     

Partial characterization of an endogenous inhibitor of a calcium-dependent form of insulin protease

Insulin protease activity has resisted high-yield purification to homogeneity, due to its low amount in tissues, its instability, and its erratic recovery from several types of chromatography. This report outlines the preliminary characterization of a naturally-occurring insulin protease inhibitor that accounts for some of these problems in rat skeletal muscle. In these experiments, inhibitory activity was assayed by its effect upon hydrolysis of 125I-(A14)-insulin by the partially purified insulin protease activity of rat skeletal muscle cytosol. During Sephadex G-200 chromatography of cytosol at pH 7.5, inhibitory activity copurifies with insulin protease activity, and the incomplete resolution of the two activities contributes to the impression that insulin protease exists in distinct 180,000-dalton and 80,000-dalton forms. By contrast, during DEAE-Sephacel chromatography of cytosol at pH 7.5, inhibitory activity and insulin protease activity are resolved by eluting the resin with 50 mM NaCl and 200 mM NaCl, respectively. Post-DEAE-Sephacel inhibitor has an Mr(app) of 67,000 daltons or 80,000-120,000 daltons, as determined by high-performance liquid chromatography or Sephadex G-150 chromatography, respectively. Post-DEAE-Sephacel insulin protease activity exhibits a Km for insulin of 15 nM and resides in a 200,000-dalton neutral thiol protease which requires 50 micromolar calcium for its maximum insulin-degrading activity. The inhibitor reduces the enzyme's activity reversibly, nonprogressively, and non-competitively with respect to insulin, but it does not alter the enzyme's sensitivity to calcium ion. These observations suggest that calcium and an endogenous protease inhibitor may influence cellular degradation of insulin via previously unrecognized effects upon cytosolic insulin protease activity.     

Antihypertensive Effects of Peptides in Autolysate of Bonito Bowels on Spontaneously Hypertensive Rats

An autolysate of bonito bowels was treated with ultrafiltration, loose R0 concentration, ion-exchange chromatography, and reverse phase chromatography to increase its potency to inhibit angiotensin I-converting enzyme (ACE) activity by 16-fold. Oral administration of the partially purified autolysate decreased the systolic blood of spontaneously hypertensive rats (SHR) in a dose-dependent manner at the doses of 1g peptides/kg or higher. The relationship between the antihypertensive activity (in vivo) of the partially purified preparation and its ACE inhibitory activity (in vitro) in comparison with previously reported ACE inhibitory peptides is discussed.     

Three angiotensin-converting enzyme inhibitors from Rabdosia coetsa

The EtOAc extract of Rabdosia coetsa showed angiotensin-converting enzyme (ACE) inhibitory activity. Bioassay-guided isolation of this extract yielded ethyl caffeate (1), rosmarinic acid (2) and methyl rosmarinate (3), which inhibited ACE activity by 32.42%, 55.19% and 39.50% respectively, at the concentration of 10 μg/ml.     

The antihypertensive activity of angiotensin-converting enzyme inhibitory peptide containing in bovine lactoferrin

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the bovine lactoferrin hydrolysate using peptic hydrolysis by 2-step of reverse-phase high-performance liquid chromatography. This peptide was identified as Leu-Arg-Pro-Val-Ala-Ala and it produced a concentration-dependent inhibition of ACE activity in vitro with an IC50 value of about 4.14 microM. Also, this inhibition was identified as noncompetitive from the Lineweaver-Burk plot. Moreover, the antihypertensive activity of Leu-Arg-Pro-Val-Ala-Ala was investigated by the intravenous injection into spontaneously hypertensive rats (SHRs). A dose-dependent reduction of systolic blood pressure by this peptide was observed at 60 min after injection and it maximally decreased the blood pressure at a rate of 1 nmol/ml/kg. The blood pressure lowering activity of this peptide was calculated as 210% of captopril (10 pmol/ml/kg) that was used as positive control. Otherwise, identification of this peptide in the blood of SHRs was carried out chromatographically. Reduction of blood pressure coincides with the peak peptide concentration in the serum. Thus, we conclude that this peptide inhibits ACE activity in vitro and lowers systolic blood pressure in spontaneously hypertensive rat.     

Natural killer cell cytolytic granule-associated enzymes. I. Purification, characterization, and analysis of function of an enzyme with sulfatase activity

An enzyme with sulfatase activity has been isolated from the granules of a rat NK leukemia cell line, CRNK-16. The enzyme has been purified from crude preparation, with a specific activity of 52 nmol/min/mg of protein, by DEAE ion exchange and Con A-Sepharose affinity chromatography, resulting in a specific activity of 230 nmol/min/mg of protein. The molecular mass of the purified enzyme was estimated to be 40 kDa by gel filtration chromatography at pH 7.4, but the enzyme had the ability to complex to molecular masses of greater than 300 kDa at low pH when crude granule extract was used as the starting sample, suggesting that it associates with other granule components. The enzyme was determined to be an arylsulfatase by its ability to (a) hydrolyze p-nitrophenyl sulfate (Km = 26.0 mM) and p-nitrocatechol sulfate (pNC sulfate) (Km = 1.1 mM) and (b) be inhibited by sulfite (Ki = 6.0 x 10(-7) M), sulfate (Ki = 1 x 10(-3) M), and phosphate (Ki = 4 x 10(-5) M) in a competitive manner. The pH optimum for enzymatic activity was determined to be 5.6. The role of this enzyme in cytolytic function was investigated by examining the effect of its substrates and inhibitors on granule- and cell-mediated lysis. pNC sulfate was shown to cause a dose-dependent inhibition of target cell lysis by isolated cytolytic granules (complete inhibition at 12.5 mM). Sulfite induced an incomplete inhibition (50% at 1 mM), whereas phosphate was essentially without inhibitory effect. Sulfate, on the other hand, altered lytic activity in a biphasic manner, inasmuch as it induced an inhibition of lysis at high concentrations and an increase of lysis at low concentrations. Cell-mediated lysis was inhibited by pNC sulfate in a dose-dependent fashion at concentrations greater than 2.5 mM, with nearly complete inhibition at 50 mM. Sulfate also altered the lytic activity by intact cells in a biphasic manner, although the effect was much less pronounced. Sulfite and phosphate caused only a 30% inhibition of lytic activity. These results suggest that the sulfatase enzyme is involved in NK cytolytic function, presumably at the lethal hit stage.     

Modification of the furanacryloyl-L-phenylalanylglycylglycine assay for determination of angiotensin-I-converting enzyme inhibitory activity

Angiotensin-I-converting enzyme (ACE, EC 3.4.15.1) plays a central role in the regulation of blood pressure in man. The objective of this study was to evaluate and modify the furanacryloyl-L-phenylalanylglycylglycine (FAPGG) assay method for quantification of ACE activity. The fixed time conditions developed for assay of ACE activity were as follows: 0.8 mM FAPGG, 175 + or - 10 units l(-1) ACE, incubation at 37 degrees C for 30 min and enzyme inactivation with 100 mM ethylenediaminetetra-acetic acid (EDTA). Hydrolysis of FAPGG to FAP and GG was quantified by measuring the decrease in absorbance at 340 nm. It was shown that increasing the level ACE activity in the assay from 155 to 221 + or - 15 units l(-1) resulted in a corresponding increase in the apparent IC(50) value for Captopril from 9.10 to 39.40 nM. Similar trends in the apparent IC50 values for a whey protein hydrolysate were obtained. The results demonstrate the requirement for carefully controlling ACE activity levels in the assay in order to obtained comparable and reproducible values for the inhibitory potency of ACE inhibitors.     

Purification and characterization of angiotensin I-converting enzyme inhibitory peptide from enzymatic hydrolysates of Styela clava flesh tissue

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the Styela clava flesh tissue. Nine proteases (Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, pepsin, trypsin, α-chymotrypsin and papain) were used, and their respective enzymatic hydrolysates and an aqueous extract were screened to evaluate their potential ACE inhibitory activity. Among all of the test samples, Protamex hydrolysate possessed the highest ACE inhibitory activity, and the Protamex hydrolysate of flesh tissue showed relatively higher ACE inhibitory activity compared with the Protamex hydrolysate of tunic tissue. We attempted to isolate ACE inhibitory peptide from the Protamex hydrolysate of S. clava flesh tissue using ultrafiltration, gel filtration on a Sephadex G-25 column and high performance liquid chromatography (HPLC) on an ODS column. The purified ACE inhibitory peptide exhibited an IC₅₀ value of 37.1 μM and was identified as non-competitive inhibitor of ACE. Amino acid sequence of the peptide was identified as Ala-His-Ile-Ile-Ile, with a molecular weight 565.3 Da. The results of this study suggested that the peptides derived from enzymes-assisted extracts of S. clava would be useful new antihypertension compounds in functional food resource.     

A rat brain isozyme of angiotensin-converting enzyme. Unique specificity for amidated peptide substrates

We have purified angiotensin-converting enzyme (ACE, EC 3.4.15.1) from rat brain corpus striatum and rat lung. The brain enzyme has Mr 165,000 by sodium dodecyl sulfate gel electrophoresis, whereas the lung enzyme is 175,000. This difference is not an artifact of preparation since mixture of the two tissues prior to purification results in isolation of two proteins with Mr 165,000 and 175,000. Separation of tryptic fragments of 125I-labeled lung and brain ACE by reverse-phase chromatography yields distinct but similar patterns. No differences between the native enzymes are detected in dansyl-tripeptide cleavage specificity, inhibitor profile, immunological properties, sucrose gradient sedimentation, or gel filtration of ACE from the two tissues. However, lung and brain ACE can be differentiated in their ability to cleave amidated peptides. Both lung and brain ACE cleave Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (substance P) via two pathways. In one pathway, ACE first releases Gly-Leu-Met-NH2 and then dipeptides sequentially from the carboxyl terminus. The other first produces Leu-Met-NH2, and then releases dipeptides to leave substance P 1-5. Lung ACE favors initial tripeptide release 3:1, while the striatal enzyme acts via the two pathways to a similar extent. Lung and striatal ACE also differ in their ability to degrade other amidated peptides. His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 (substance K) and bombesin are degraded by striatal but not lung ACE. Physalaemin and luteinizing hormone-releasing hormone are cleaved by both enzymes, while eledoisin, kassinin, thyrotropin-releasing hormone, and substance P 5-11 are not cleaved by either enzyme. Physalaemin is degraded more rapidly by the lung enzyme. The coincidence of an ACE isozyme with substance P and substance K in the descending striatonigral pathway and the unique ability of this isozyme to cleave substance P and substance K suggest that one or both of these peptides is a physiological substrate for striatonigral ACE.     

Analysis of novel angiotensin-I-converting enzyme inhibitory peptides from protease-hydrolyzed marine shrimp Acetes chinensis.

Acetes chinensis is an underutilized shrimp species thriving in the Bo Hai Gulf of China. In a previous study, we had used the protease from Bacillus sp. SM98011 to digest this kind of shrimp and found that the oligopeptide-enriched hydrolysate possessed antioxidant activity and high angiotensin I-converting enzyme (ACE) inhibitory activity with an IC50 value of 0.97 mg/ml. In this paper, by ultrafiltration, gel permeation chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC), five peptides with high ACE inhibitory activity were purified from the shrimp hydrolysates and their sequences were identified by amino acid composition analysis and molecular weight (MW) analysis. Three of them, FCVLRP (a), IFVPAF (f) and KPPETV (j), were novel ACE inhibitory peptides. Their IC50 values were 12.3 microM, 3.4 microM and 24.1 microM, respectively, and their recoveries were 30 mg/100 g (solid basis of shrimp), 19 mg/100 g and 33 mg/100 g, respectively. Lineweaver-Burk plots for the three novel peptides showed that they are all competitive inhibitors. To test the ACE inhibitory activity of peptide a, f, j after they were digested by digestive enzymes in vivo, 12 derived peptides from FCVLRP and IFVPAF were synthesized based on their amino acid sequences and the cleavage sites of digestive enzymes. No digestive enzyme cleavage site was found in KPPETV. The IC50 values of the derived peptides were determined and the result showed that except for VPAF, FC and FCVL, the ACE inhibitory activity of the other nine derived peptides did not significantly change when compared with their original peptides. Surprisingly, five peptides had lower IC50 values than their original peptides, particularly for RP (IC50 value = 0.39 microM), which is about 30 times lower than its original peptide and almost the lowest IC50 value for ACE inhibitory peptides reported. Therefore, the novel peptides identified from A. chinensis hydrolysates probably still maintain a high ACE inhibitory activity even if they are digested in vivo. This is the first report about novel ACE inhibitory peptides from hydrolysates of marine shrimp A. chinensis. The novel peptides from hydrolysate of A. chinensis and some of their derived peptides with high ACE inhibitory activity probably have potential in the treatment of hypertension or in clinical nutrition.     

Purification of carnitine palmitoyltransferase from heart mitochondria by hydrophobic affinity chromatography

Carnitine palmitoyltransferase II of rat heart mitochondria was purified to homogeneity by a rapid method exploiting the hydrophobic nature of the protein. The method involves solubilization of mitochondrial membrane proteins by detergents and subsequent fractionation by hydrophobic affinity chromatography. Sepharose, cross-linked via a primary amino group of 1,omega-diaminoalkane, 4-aminobutyric acid, 6-aminocaproic acid, or 6-aminohexanol, was found to reversibly bind carnitine palmitoyltransferase under nondenaturing conditions. A homologous series of n-alkyl-agarose resins with n = 2 to 8 and phenyl-Sepharose were also found to reversibly bind the enzyme. Alkyl-Superose, phenyl-Superose, and Superose 12 chromatographies were also very useful in fractionating the enzyme. Successive chromatography on three or four hydrophobic columns yielded a highly pure enzyme preparation. The purified preparation appeared as one major protein band on polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate (M(r) 68,000). The isolated enzyme had significant activity (sp act = 15.0 mumol/min/mg protein when 80 microM palmitoyl-CoA and 20 mM carnitine were used as substrates). Antibodies against this protein recognized (in immunoblots) one major protein band in crude preparations of rat heart mitochondria (M(r) 68,000), indistinguishable from purified carnitine palmitoyltransferase II. L-Palmitoylcarnitine (0.1 mM) and coenzyme A (0.1 mM), products of the enzyme-catalyzed reaction, inhibited carnitine palmitoyltransferase activity 66 and 71%, respectively. D-Palmitoylcarnitine (0.1 mM), however, did not inhibit the activity. Malonyl-CoA, a specific inhibitor of membrane-bound carnitine palmitoyltransferase I, did not show significant inhibition.