Hydroxylapatite Column Chromatography Research Articles

Ca 2+-dependent protein kinase from the halotolerant green alga Dunaliella tertiolecta: Partial purification and Ca 2+-dependent association of the enzyme to the microsomes

Ca 2+-dependent protein kinase (CDPK) was purified 900-fold from the soluble fraction of Dunaliella tertiolecta cells by ammonium sulfate precipitation, DEAE—Toyopearl, phenyl-Sepharose, and hydroxylapatite column chromatography. The CDPK was activated by micromolar concentration of Ca 2+ and required neither calmodulin nor phospholipids for its activation. The enzyme phosphorylated casein, myosin light chain, and histone type III-S (histone H-1), but did not phosphorylate protamine and phosvitin. The K m values for ATP and casein were 11 μ m and 300 μg/ml, respectively. Phosphorylation of casein was inhibited by calmodulin antagonists, calmidazolium, trifluoperazine, and compound 48 80 , but not affected by calmodulin. CDPK bound to phenyl-Sepharose in the presence of Ca 2+ and was eluted by ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA). This suggests that hydrophobicity of the enzyme was increased by Ca 2+. CDPK was also bound to the microsomes isolated from Dunaliella cells in the presence of micromolar concentration of Ca 2+ and released in the presence of EGTA, suggesting the possibility of in vivo Ca 2+-dependent association of the enzyme. The enzyme phosphorylated many proteins in the microsomes but few in the cytosol, if at all.

A thiocyanate hydrolase of Thiobacillus thioparus. A novel enzyme catalyzing the formation of carbonyl sulfide from thiocyanate.

A thiocyanate hydrolase that catalyzes the first step in thiocyanate degradation was purified to homogeneity from Thiobacillus thioparus, an obligate chemolithotrophic eubacterium metabolizing thiocyanate to sulfate as an energy source. The thiocyanate hydrolase was purified 52-fold by steps involving ammonium sulfate precipitation, DEAE-Sephacel column chromatography, and hydroxylapatite column chromatography. The enzyme hydrolyzed 1 mol of thiocyanate to form 1 mol of carbonyl sulfide and 1 mol of ammonia as follows: SCN- + 2H2O----COS + NH3 + OH-. This is the first report describing the hydrolysis of thiocyanate to carbonyl sulfide by an enzyme. The enzyme had a molecular mass of 126 kDa and was composed of three different subunits: alpha (19 kDa), beta (23 kDa), and gamma (32 kDa). The enzyme exhibited optimal activities at pH 7.5-8.0 and at temperatures ranging from 30 to 40 degrees C. The Km value for thiocyanate was approximately 11 mM. Immunoblot analysis with polyclonal antibodies against the purified enzyme suggested that it was induced in T. thioparus cells when the cells were grown with thiocyanate.

A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases.

A peptide derived from p34cdc2, cdc2(6-20)NH2 with the amino acid sequence of KVEKIGEGTYGVVYK-amide, was found to be a specific and efficient substrate for a pp60c-src-related protein tyrosine kinase from bovine spleen (STK). Glu-12 and Thr-14 were identified to be substrate specificity determinants in this peptide (Cheng, H.-C., Litwin, C. M. E., Hwang, D. M., and Wang, J. H. (1991) J. Biol. Chem. 266, 17919-17925). In this study, we demonstrated the presence of cdc2(6-20)NH2 peptide tyrosine kinase activity in the membrane fractions of bovine brain, spleen, thymus, lung, liver, and kidney. Hydroxylapatite column chromatography of thymus membrane extract revealed four protein tyrosine kinases, TK-I, TK-II, TK-III, and TK-IV, with different relative activities toward cdc2(6-20)NH2 and a general tyrosine kinase substrate, poly(Glu/Tyr). Only TK-I and TK-II showed significant activity toward cdc2(6-20)NH2, they were suggested as belonging to the src-family by virtue of their cross-reactivity with an antibody against a synthetic peptide corresponding to a conserved sequence of src-family kinases. Further immunological characterization using antibodies specific to individual src-related protein tyrosine kinases suggested that TK-I, TK-II, and STK are bovine homologs of p56lck, p55fyn, and p56lyn, respectively. Substrate specificity and kinetic characterization of src-family tyrosine kinases including human platelet pp60c-src, bovine p56lyn, p56lck, and p55fyn, as well as several non-src-related tyrosine kinases including epidermal growth factor receptor, p43v-abl, TK-III, and TK-IV showed that all the src-family tyrosine kinases but none of the other kinases displayed efficient cdc2(6-20)NH2 phosphorylation. In all cases, the high efficiency of cdc2(6-20)NH2 peptide phosphorylation could be markedly attenuated when Glu-12 and Thr-14 of the peptide were substituted, respectively, by valine and serine.

Recognition of a 56 kDa protein in partially purified rat hepatic nuclear thyroid hormone receptor by anti-human c-erb A beta antibody.

Human beta thyroid hormone receptor (c-erb A beta protein) produced by an Escherichia coli expression system was purified by sequential column chromatography followed by electroelution from an electrophoresis gel and an antibody was prepared. The antibody recognized a 56 kDa protein band in a partially purified rat hepatic nuclear thyroid hormone receptor fraction on Western blotting. Although multiple bands appeared on Western blotting of crude rat hepatic receptor preparations, a 56 kDa band was the most prominent and preadsorption of the antibody by purified c-erb A protein resulted in almost complete disappearance of the 56 kDa band, indicating that the 56 kDa band was formed by a specific antigen-antibody interaction. Furthermore, the 56 kDa protein appeared to co-elute with 3, 5, 3'-triiodo-L-thyronine binding activity in hydroxylapatite, Sephacryl S-200, and DNA-cellulose column chromatography of rat hepatic nuclear receptor, and sequential column purification resulted in selective enrichment of the 56 kDa band. These results suggest that the 56 kDa protein may be the major component of the rat hepatic thyroid hormone receptor.

Ca(2+)-regulated actin and phospholipid binding protein (68 kD-protein) from bovine liver: identification as a homologue for annexin VI and intracellular localization.

An F-actin binding protein was purified from bovine liver by means of DNase I affinity, hydroxylapatite and DEAE-cellulose column chromatographies. It consisted of a single polypeptide chain having an apparent molecular weight of 68,000 with a Stokes radius of 35 A. Electron microscopy of rotary shadowed specimens showed that the 68 kD protein is a globular protein. This protein showed a higher affinity for F-actin in the presence of Ca2+ than in its absence, which is opposite to the actin-binding property shown by nonmuscle alpha-actinin or fimbrin. The 68 kD protein had no F-actin severing and capping activity. Interestingly, the 68 kD protein was found to aggregate liposomes at micromolar Ca2+ concentrations. Immunoblot analysis and partial protein sequence data identified the 68 kD protein as an annexin VI (p68) homologue. Immunocytochemical studies showed that the 68 kD protein was localized along stress fibers as well as membrane ruffles, microspikes and focal contacts, raising the possibility that annexin VI may contribute to control membrane-microfilament interaction in the cell.

Purification and Properties of Reduced Nicotinamide Adenine Dimucleotide Dehydrogenase from Photobacterium phosphoreum.

Reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (EC 1.6.99.3) of Photobacterium phosphoreum was solubilized from membrane vesicles with 2% sucrose monolaurate, and was purified almost to homogeneity by use of diethylaminoethyl (DEAE)-Sephacel, hydroxylapatite, and Butyl-Toyopearl column chromatography in the presence of 0.5% sucrose monolaurate. About 100-fold purification was achieved, and the purified enzyme is composed of a single polypeptide with a molecular weight of 49000, as determined by gel electorophoresis in the presence of sodium dodecyl sulfate. The NADH dehydrogenase contains about one mol flavin adenine dinucleotide (FAD) as a prosthetic group per mol enzyme, but does not contain acid labile sulfide or non-heme iron. This NADH dehydrogenase can use ubiquinone-1, menadione, potassium ferricyanide, and cytochrome c as electron acceptors. The enzyme barely catalyzes the oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor. The enzyme is not susceptive to rotenone, 2-n-heptyl-4-hydroxyquinoline N-oxide, capsaicin, or N, N'-dicyclohexylcarbodiimide, which were reported as inhibitors of NADH dh I. On the other hand, flavone, reported as an inhibitor of NADH dh II of yeast and plant mitochondria, does inhibit the NADH dehydrogenase activity. These results indicate that the NADH dehydrogenase of P. phosphoreum belongs to NADH dh II.The NADH oxidase activity of the membrane vesicles of P. phosphoreum is remarkably stimulated by monovalent cations such as Na+ and K+, but is inhibited by divalent or trivalent cations. The NADH dehydrogenase activities (especially NADH-ferricyanide and NADH-ubiquinone-1 oxidoreductase) of the purified enzyme and membrane were also enhanced by Na+ and K+. However, the ubiquinol-1 oxidase activity of terminal oxidase complex (cytochrome bd complex) was not enhanced largely by the monovalent cations. These results suggest that in the respiratory chain of P. phosphoreum, NADH dehydrogenase contributes to the enhancement of electron transfer activity by monovalent cations.

Identification and characterization of a 3' to 5' exonuclease associated with spinach chloroplast DNA polymerase.

Spinach chloroplast DNA polymerase was shown to copurify with a 3' to 5' exonuclease activity during DEAE-cellulose, hydroxylapatite, and heparin-agarose column chromatography. In addition, both activities comigrated during nondenaturing polyacrylamide gel electrophoresis and cosedimented through a glycerol gradient with an apparent molecular weight of 105,000. However, two forms of exonuclease activity were detected following velocity sedimentation analysis. Form I constituted approximately 35% of the exonuclease activity and was associated with the DNA polymerase, whereas the remaining activity (form II) was free of DNA polymerase and exhibited a molecular weight of approximately 26,500. Resedimentation of form I exonuclease generated both DNA polymerase associated and DNA polymerase unassociated forms of the exonuclease, suggesting that polymerase/exonuclease dissociation occurred. The exonuclease activity (form I) was somewhat resistant to inhibition by N-ethylmaleimide, whereas the DNA polymerase activity was extremely sensitive. Using in situ detection following SDS-polyacrylamide activity gel electrophoresis, both form I and II exonucleases were shown to reside in a similar, if not identical, polypeptide of approximately 20,000 molecular weight. Both form I and II exonucleases were equally inhibited by NaCl and required 7.5 mM MgCl2 for optimal activity. The 3' to 5' exonuclease excised deoxyribonucleoside 5'-monophosphates from both 3'-terminally matched and 3'-terminally mismatched primer termini. In general, the exonuclease preferred to hydrolyze mismatched 3'-terminal nucleotides as determined from the Vmax/Km ratios for all 16 possible combinations of matched and mismatched terminal base pairs. These results suggest that the 3' to 5' exonuclease may be involved in proofreading errors made by chloroplast DNA polymerase.

The unique feature of dog liver cytosolic glutathione S-transferases. An isozyme not retained on the affinity column has the highest activity toward 1,2-dichloro-4-nitrobenzene.

In the adult dog liver cytosol we identified four glutathione S-transferase (GST) subunits, Yd1 (Mr 26,000), Yd2 (Mr 27,000), Yd3 (Mr 28,000), and Ydf (Mr 27,400), and purified GST forms comprising Yd1, Yd2, and Yd3, to apparent homogeneity. Unlike rat transferases the enzyme activity toward 1,2-dichloro-4-nitrobenzene (DCNB) was not retained on the affinity column. Thus the DCNB-active enzyme, GST YdfYdf, from the flow-through fraction of the affinity column was also purified to homogeneity by gel filtration, DE52 chromatography, chromatofocusing, and hydroxylapatite column chromatography. Immunoblot analysis of dog GSTs revealed that the subunits Yd1, Yd2, and Yd3 belong to the pi, alpha, and mu class, respectively. On the contrary, Ydf had no reactivity with antibodies raised against any of the three classes of GST. Each subunit, Yd1, Yd2, Yd3, and Ydf, was distinguishable by its own retention time on reverse-phase high performance liquid chromatography. N-terminal amino acid sequences of the dog GSTS Yd1Yd1 and Yd3Yd3 revealed a high degree of homology to the pi and mu class transferases from rat, human, and mouse, respectively, while the N terminus of Yd2Yd2 is blocked. N-terminal amino acid sequences of GST YdfYdf showed no homology to any of the three classes of GST. The most significant property noted of GST YdfYdf is the high specific activity toward DCNB, exceeding by 1 order of magnitude the corresponding values for the known mu class GSTs. The present results strongly suggest that dog GST YdfYdf is a unique enzyme distinct from the hitherto characterized GST isozymes.

Fractionation of cellulases from the ruminal fungus Neocallimastix frontalis EB188.

Cellulases from the ruminal fungus Neocallimastix frontalis EB188 were separated by using hydroxylapatite column chromatography. Seven carboxymethylcellulases, six avicelases, and four beta-glucosidases accounted for the majority of the activities. The separation of enzymes was confirmed by using polyacrylamide gel electrophoresis. Electrophoretic migration, analysis of hydrolysis products, and substrate specificity measurements suggested that several different cellulases were secreted in N. frontalis EB188. The possible relationship of cellulase diversity to protein glycosylation is discussed.

Purification of an ultraviolet-inducible, damage-specific DNA-binding protein from primate cells.

A UV-inducible, damage-specific DNA-binding (DDB) protein with high affinity for double-stranded UV-irradiated DNA has been identified recently in monkey kidney (CV-1) cells (Hirschfeld, S., Levine, A. S., Ozato, K., and Protić, M. (1990) Mol. Cell. Biol. 10, 2041-2048). We have now purified the DDB protein from extracts of CV-1 cells using hydroxylapatite, phosphocellulose, Mono S, and DNA-affinity column chromatography. The DDB activity, either from mock-treated or UV-induced cells, is heterodisperse in column chromatography, and separation of three forms of the protein was obtained on a phosphocellulose column. Analysis of purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that greater than 90% of all three forms is a protein of approximately 126 kDa. The size of the native DDB protein was deduced from gel filtration and native polyacrylamide gel electrophoresis to be approximately 210 kDa, which suggests that the native DDB protein in solution is a homodimer. Preparations of partially purified DDB protein from UV-treated cells have enhanced levels of DDB activity and the protein when compared with similar preparations from mock-treated cells. This damage-recognition protein, alone or in conjunction with other subunits, may be of general importance for the initial recognition of DNA damage in mammals.

Purification of human c-erb A beta protein.

Human thyroid hormone receptor (c-erb A protein) produced by Escherichia coli expression vector plasmid was purified sequentially using polyethylenimine precipitation of DNA, hydroxylapatite column chromatography, ammonium sulphate precipitation, Sephacryl S-300 gel filtration and mono Q-Sepharose column chromatography. These column procedures resulted in 41.3-fold purification of 3,5,3'-tri-iodo-L-thyronine (T3) binding activity over the initial E. coli extract. Purified protein as well as crude preparation showed high-affinity binding to T3. The c-erb A protein enriched by column purification was further purified by electroelution after electrophoresis. Rabbit antibody against the c-erb A protein was prepared and used for the Western blotting analysis. The antibody recognized c-erb A protein but not the bacterial proteins in crude E. coli extract. When partially purified rat hepatic nuclear thyroid hormone receptor was analysed, a 56 kDa receptor was specifically recognized by the antibody.

Purification and characterization of ADP-ribosyltransferases (exoenzyme C3) of Clostridium botulinum type C and D strains

By cation-exchange column chromatography followed by gel filtration or hydroxylapatite column chromatography, ADP-ribosyltransferases (exoenzyme C3) were isolated from culture supernatants of Clostridium botulinum type C strains Stockholm (CST) and 6813 (C6813) and from type D strains South African (DSA) and 1873 (D1873), and their molecular properties were compared. The purified C3 enzymes were homogeneous in polyacrylamide gel electrophoresis. The C3 enzymes existed as single-chain polypeptides with molecular masses of 25.0 to 25.5 kDa and transferred ADP-riboses to the same substrates in rat brain membrane extract. The C3 enzymes could be roughly classified into two groups with respect to amino acid composition, amino-terminal sequence, and antigenicity. One group contains the C3 enzymes of strains C6813 and DSA, and the other contains those of strains CST and D1873. The specific activity of the C3 enzyme of strain C6813 was about 15 times higher than that of the C3 enzyme of strain CST. These results indicate that the classification of the C3 molecules differs from that of the neurotoxin molecules.

Characterization of a female-specific hepatic mitochondrial cytochrome P-450 whose steady-state level is modulated by testosterone

Polyclonal antibody to mitochondrial P-450c27/25 reacted with two proteins of apparent molecular masses of 52 kilodaltons (kDa) and 50 kDa from the female rat liver mitochondrial proteins bound to an omega-octylaminoagarose column. The two proteins were purified to greater than 85% homogeneity by DEAE-Sephacel and hydroxylapatite column chromatography, and both were found to be P-450 as judged by dithionite-reduced CO difference spectra. Both of the P-450 forms required mitochondrial-specific ferredoxin and ferredoxin reductase for in vitro reconstitution of enzyme activities, suggesting that they are mitochondrial forms. The 52-kDa P-450 exhibited the properties of mitochondrial 27/25-hydroxylase with respect to high vitamin D3 25-hydroxylase activity [1.4 nmol (nmol of P-450)-1 min-1] and N-terminal amino acid sequence. The 50-kDa P-450, on the other hand, lacked significant vitamin D3 25-hydroxylase activity, but showed 17 beta-reductase [0.380-0.400 nmol (nmol of P-450)-1 min-1] and 17 beta-oxidase [0.1-0.16 nmol (nmol of P-450)-1 min-1] activities with both androgens and estrogens as substrates. Immunoblot analysis of proteins using a monoclonal antibody specific for P-450c27/25 showed a 2-3-fold higher level of this enzyme in the female liver mitochondria than in the males. Similarly, use of a polyclonal antibody in the immunoblot analysis showed that the 50-kDa P-450 is female-specific. The relative level of P-450c27/25 was reduced significantly in castrated females, while the level of the female-specific 50-kDa P-450 was increased. However, the levels of both enzymes were increased in castrated males.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of Monoclonal Antibodies to Bovine Follicle-Stimulating Hormone and Application in an Enzyme Immunoassay

Twenty monoclonal antibodies (MAbs) to bovine follicle-stimulating hormone (bFSH) were generated by using the hybridoma technique. Twelve of the MAbs have been shown to bind specifically to bFSH and seven of them were purified from the respective ascitic fluids with the methods of caprylic acid precipitation and hydroxylapatite column chromatography. The purity of the MAbs was in the range of 86-95%. Three of the purified MAbs have been coupled to horseradish peroxidase (HRP). By using a matrix cross-matching procedure, two MAbs reacted with discrete antigenic determinants are selected for a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The designed ELISA procedure could be performed within 60 min in a two-stage incubation and has a minimum sensitivity of 1ngFSH/ml. For serum samples, its mean intra- and inter-assay coefficients of variation were 3.4% and 7.3% respectively. The DAS-ELISA can be used to establish a complete FSH profile in the field of in vitro diagnosis.

Ca 2+-independent, phospholipid-activated protein kinase in 3Y1 cells

We obtained a Ca 2+-independent but 12- O-tetradecanoyl phorbol ester (TPA) · phospholipid-activated protein kinase from rat embryo fibroblast 3Y1 cells by succeeding steps of DEAE-cellulose, H-9 affinity, and hydroxylapatite chromatography. This kinase was separated from a conventional PKC (Type III), by H-9 affinity column chromatography. The major peak from H-9 affinity column was eluted at 0.4 m of arginine and on the following step of hydroxylapatite column chromatography, at the KPO 4 concentration of 0.1 m. The enzyme could be stimulated by phospholipids and by the tumor promoter TPA, but did not respond to calcium. The Ca 2+-independent, phospholipid-activated protein kinase activity was susceptible to the protein kinase C inhibitors H-7 and K252a, but showed a phospholipid dependency and substrate specificity distinct from the conventional types of PKC. This protein kinase did not react with monoclonal antibodies against Types I, II, and III PKC. The activity of this enzyme was specifically reduced by immunoprecipitation, depending on the concentration of the polyclonal antibody, PC-δ, which was raised against a peptide synthesized according to a sequence of rat brain nPKCδ. The enzyme had a M r of 76,000 as estimated by Western blotting. These results provide evidence for a unique type of Ca 2+-independent, phospholipid-activated kinase, as expressed in 3Y1 cells.

Overproduction and purification of McrC protein from Escherichia coli K-12

The McrC protein, encoded by one of the two genes involved in the McrB restriction system, was produced in Escherichia coli cells by using a T7 expression system. Following sequential DEAE-Sepharose and hydroxylapatite column chromatography, the protein was purified to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified McrC protein agreed exactly with the one deduced from the DNA sequence by Ross et al. (J. Bacteriol. 171:1974-1981, 1989).

Characterization of calcium-independent forms of protein kinase C-beta in phorbol ester-treated rabbit platelets

The subcellular distribution, size, and activation state of protein kinase C (PKC) were studied after short term exposure of rabbit platelets to a saturating dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Cytosolic and Nonidet P-40-solubilized particulate extracts prepared from TPA-treated platelets were subjected to analytical column chromatography on Mono Q, hydroxylapatite, and Superose 6/12. PKC activity was assayed according to the ability of the enzyme to phosphorylate (i) histone H1 in the presence of the activators calcium, diacylglycerol, and phosphatidylserine; (ii) histone H1 after proteolytic activation of PKC with trypsin; and (iii) protamine in the absence of calcium and lipid. Within 1 min of TPA treatment of platelets, greater than 95% of the PKC activity was particulate associated, as assessed by all three methods. The particulate PKC activity from 1-min TPA-treated cells eluted from Mono Q with approximately 0.35 M NaCl (peak I), and it was highly dependent upon Ca2+ and lipid for optimal histone H1 phosphorylation. With longer exposure times of platelets to TPA, the disappearance of the Mono Q peak I form of PKC was correlated with the production of new PKC species that were released from Mono Q with approximately 0.4 M NaCl (peak II), approximately 0.5 M NaCl (peak III), and approximately 0.6 M NaCl (peak IV). These last forms of PKC were still lipid activated but exhibited little Ca2+ dependence. The Mono Q peak III form displayed a particularly high level of histone H1 phosphorylating activity in the absence of lipid and Ca2+. All of these forms behaved as approximately 65-kDa proteins on Superose 6/12, but on sodium dodecyl sulfate-polyacrylamide gels, Western blotting with anti-PKC-beta antibodies revealed immunoreactive polypeptides of approximately 79 kDa (Mono Q peaks I, II, and IV) and approximately 100-kDa (Mono Q peak III). Hydroxylapatite column chromatography permitted partial resolution of the Mono Q peaks I and II forms, which were eluted within a concentration range of potassium phosphate (100-150 mM) which was typical of the beta isozyme of PKC. Treatment of the Mono Q peak III and IV PKC forms with alkaline phosphatase resulted in the production of the peak I form, which implicated protein phosphorylation in the interconversion of the various PKC forms.

Isolation and characterization of the calcium- and phospholipid-dependent protein kinase (protein kinase C) subtypes from bovine heart

Protein kinase C was isolated from bovine heart by chromatography on DEAE-Sephacel, phenyl-Sepharose, poly(L-lysine) agarose, and hydroxylapatite. Estimates based upon enzyme recovery indicate 10-20 nmol/min of protein kinase C activity per gram of bovine ventricular myocardium. Hydroxylapatite column chromatography resolved the preparation into two peaks of calcium- and phospholipid-dependent protein kinase activity. By Western blot analysis, peaks 1 and 2 contained subtypes II (beta 2) and III (alpha), respectively. No cross-reactivity was observed, indicating that separation was complete. Type III, the major subtype detected, was subsequently purified to apparent homogeneity by chromatography on phosphatidylserine (PS) acrylamide. Type II activity could not be recovered following phosphatidylserine affinity chromatography. Phospho amino acid analysis showed that type III autophosphorylated at serine residues, whereas type II autophosphorylated at both serine and threonine residues. Among the various phospholipids tested for activity, PS was the most effective. Both subtypes were activated by 1-stearoyl-2-arachidonylglycerol (SAG) in the presence of phosphatidylserine and calcium. Activation of both subtypes occurred at calcium concentrations of less than 1 microM. In addition to several similarities, these two subtypes showed differences in activation and kinetic properties: type II was activated by cardiolipin, 1,2-and 1,3-dioleoylglycerol, and both cis- and trans-unsaturated fatty acids. Type III was activated to a lesser degree by cardiolipin and showed no response to 1,3-dioleoylglycerol. Type III was activated to a greater extent by 1,2-diacylglycerols and by cis-unsaturated fatty acids. In the presence of PS and SAG, type II exhibited substantial activity in the presence of 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) without added calcium. Activation of types II and III by unsaturated fatty acids was independent of phospholipid and showed a lower apparent calcium affinity than that observed for activation by phosphatidylserine. These results show that cardiac protein kinase C subtypes II and III were functionally distinguishable and may play unique roles in the regulation of cardiac function.

Purification and properties of human serum carnosinase

Carnosinase from human plasma was purified 18000-fold to apparent homogeneity in a four step procedure. The dipeptidase was partially inactivated during DEAE-cellulose chromatography; however, it reactivated slowly when concentrated and stored at 4°C. In the second purification step, hydroxylapatite column chromatography, two forms of the enzyme were separated from one another. Human serum carnosinase was found to be a glycoprotein with a p I of 4.4 and a subunit M r of 75 000; the active enzyme was a dimer, the two subunits being connected by one or more disulfide bonds. The enzyme was especially active in hydrolyzing carnosine and anserine, preferring dipeptides with histidine in the C-terminal position. In most human tissues, the concentration of serum carnosinase was proportional to the percentage of trapped blood in the sample. However, the brain contained about 9 times more enzyme than expected, based on the amount of trapped blood present. The physiological function of this enzyme seems to be the hydrolysis of homocarnosine in the brain and the splitting of carnosine and anserine in the blood stream. Six higher primates were found to have serum carnosinase. Twelve nonprimate mammals were tested; all were lacking the serum enzyme except for the Golden hamster, which had very high concentrations of a carnosinase having somewhat different properties than the higher primate enzyme.

20] Purification and analysis of protein kinase C isozymes

Protein kinase C (PKC) as a collection of Ca 2+ /phosphatidylserine (PS)/diacylglycerol (DAG)-stimulated kinases has been purified from various sources. The three Ca 2+ /PS/DAG-stimulated kinases with corresponding Ca 2+ /PS-dependent phorbol ester-binding activity peaks are designated a PKC I, II, and III. PKC I, II, and III are structurally homologous and behave similarly during purification by ion-exchange, gel-filtration, hydrophobic, and affinity column chromatography. Thus, by using hydroxylapatite column chromatography as a last step of purification, these isozymes are separated and each purified in milligram quantity. For the purification process, fresh rat brains (120 g wet wt) from 80 male Sprague-Dawley rats (200–250 g) are homogenized in 600 ml of ice-cold homogenizing buffer using a Polytron at setting 5 with four 15- sec bursts. The homogenate is centrifuged at 34,000 rpm at 4° for one hr using Beckman 35 rotors. The supernatant fluid is decanted carefully to avoid the turbid fluffy layer and the combined fluffy layer and the pellet are extracted once again with 400 ml of the homogenizing buffer. The combined high-speed supernatant fluid is adjusted to pH 7.5 by adding solid Tris and applied to a DEAE-cellulose (DE-52) column (4.0 × 16 cm) equilibrated with buffer A. The surface of the column is periodically stirred up gently with a glass rod to avoid clotting of the DEAE-cellulose.