Effect of calcium on the endogenous phosphorylation of mouse brain microsomes in vitro

1. The endogenous phosphorylation of mouse brain microsomes was studied using the technique of acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). 2. It was found that specific proteins and lipids in brain microsomes were phosphorylated by the terminal phosphate of ATP under appropriate conditions. Six peaks of radioactivity were observed on SDS-polyacrylamide gel electrophoresis of 32Pi-labelled brain microsomes. The peaks were designated as P-I, P-II, P-III, P-IV, P-V, and P-VI. The peaks from P-I to P-V, which consist of phosphoproteins, underwent rapid dephosphorylation. On the other hand, P-VI, which consists of phospholipids, remained unaffected even after the complete hydrolysis of added ATP. 3. With the addition of 100 muM CaCl2 to the assay medium, the phosphorylation of brain microsomal proteins was stimulated; in the regions of P-I, P-II, and P-III, the amounts of 32Pi incorporation were approximately twice the 32Pi incorporation in the absence of Ca2+. On the other hand, 32Pi incorporation into P-VI was unaffected irrespective of the presence or absence of 100 muM CaCl2. In the presence of higher concentrations of Ca2+ (1-10 mM), the phosphorylation of all components was inhibited.     

Evidence for two α-amanitin-resistant RNA polymerases in vegetative amoebae of Dictyostelium discoideum

Three DNA-dependent RNA polymerases (EC 2.7.7.6), P-I, P-II and P-III, have been isolated from the sonicated nuclear extract of vegetative amoebae of $\text{Dictyostelium discoideum}$ by phosphocellulose chromatography. P-I was inhibited by α-amanitin, while P-II and P-III were not. Rifampicin did not prevent all the polymerase activities. These polymerases were more active in the presence of Mg²⁺ than Mn²⁺. P-III was reduced in the enzyme activity by being passed through DEAE-Sephadex column and not obtained from the nuclear extract of amoebae at the culmination stage during morphogenetic development.     

Cell wall proteins from sugar beet cells in suspension culture

Several proteins were extracted from the purified cell walls of suspension-cultured sugar beet cells with 0.5% EDTA (pH 6.8) after prior extraction of the walls with 0.5% deoxycholate and then with 2 molar NaCl. Two abundant proteins (P-I and P-II protein) were separately purified to homogeneity by procedures that included fractionation with ammonium sulfate, column chromatography on DEAE-cellulose and butyl Toyopearl, and preparative polyacrylamide electrophoresis. P-I exists as a dimer of identical subunits, and P-II is composed of four different subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that quite different polypeptides are present in the culture medium and in the NaCl and EDTA extracts of the wall.     

Isolation of Alkaline and Neutral Proteases from Aspergillus flavus var. columnaris, a Soy Sauce Koji Mold

Two different extracellular proteases, protease I (P-I), an alkaline protease, and protease II (P-II) a neutral protease, from Aspergillus flavus var. columnaris were partially purified by using (NH(4))(2)SO(4) precipitation, diethylaminoethyl-Sephadex A-50 chromatography, carboxymethylcellulose CM-52 chromatography, and Sephadex G-100 gel filtration. The degree of purity was followed using polyacrylamide gel electrophoresis. The activity of P-I was completely inhibited by 0.1 mM phenylmethylsulfonyl fluoride, and that of P-II was completely inhibited by 1 mM ethylenediaminetetraacetate. By using these inhibitors with extracts of wheat bran koji, the proportions of total activity that could be assigned to P-I and P-II were 80 and 20%, respectively. This compared favorably with activities estimated by using polyacrylamide gel electrophoresis slices (82 and 18%, respectively). Extracts from factory-run soybean koji gave comparable results. Both enzymes demonstrated maximum activity at 50 to 55 degrees C and only small changes in activity between pH 6 and 11. For P-I, activity was somewhat higher from pH 8.0 to 11.0, whereas for P-II it was somewhat higher from pH 6 to 9. In the presence of 18% NaCl, the activities of both P-I and P-II dropped by approximately 90 and 85%, respectively. P-I was inferred to possess aminopeptidase activity since it could hydrolyze l-leucyl-p-nitroanilide hydrochloride. P-II was devoid of such activity. The ramifications of the results for factory-produced soy sauce koji are discussed.     

Purification and Some Properties of Melanoidin Decolorizing Enzymes,P-III and P-IV,from Mycelia of Coriolus versicolor Ps4a

Melanoidin decolorizing enzymes (MDE) were extracted from mycelia of Coriolus versicolor Ps4a and purified by DEAE-Sephadex, DEAE-Sephacel and Sephadex G-200 column chromatographies. MDE of this strain consisted of a main fraction, P-fraction, and a minor fraction, E-fraction, and the P-fraction was composed of at least five enzymes. P-III and P-IV in the P-fraction were picked as typical enzymes of this strain, and their enzymatic properties were investigated. P-III had a molecular weight of 48,400 ~ 50,000, an optimum pH of 5.5 and an optimum temperature of 30~35°C. P-III required glucose and 02 for the appearance of the activity, and was inhibited by p-CMB, N-BSI, Ag⁺ and o-phenanthroline.

On the other hand, P-IV had a molecular weight of 43,800 ~ 45,000, an optimum pH of 4.0~4.5 and an optimum temperature of 30~35°C. P-IV could decolorize melanoidin in the absence of glucose and O₂, and was inhibited weakly by Ag⁺, p-CMB and N-BSI. P-IV is the enzyme that attacks the melanoidin directly in comparison with P-III which attacks melanoidin indirectly as in the sub-reaction of sugar oxidase.

Incidentally, a multiplicative effect between P-III and P-IV for decolorization was observed.     

Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult

Proteolytic activity of 0-12 day old eggs, miracidium and adult worm of Fasciola gigantica was assessed and proteases were partially purified by DEAE-Sepharose and CM-cellulose columns. Four forms of protease were separated, PIa, PIb, PIc and PII. Purifications were completed for PIc and PII using Sephacryl S-200 chromatography. A number of natural and synthetic proteins were tested as substrates for F. gigantica PIc and PII. The two proteases had moderate activity levels toward azoalbumin and casein compared to azocasein, while gelatin, hemoglobin, albumin and fibrin had very low affinity toward the two enzymes. Amidolytic substrates are more specific to protease activity. PIc had higher affinity toward BAPNA-HCl (N-benzoyl-arginine-p-nitroanilide-HCl) and BTPNA-HCl (N-benzoyl-tyrosine-p-nitroanilide-HCl) at pH 8.0 indicating that the enzyme was a serine protease. However, PII had higher affinity toward BAPNA at pH 6.5 in the presence of sulfhydryl groups (beta-mercaptoethanol) indicating that the enzyme was a cysteine protease. The effect of specific protease inhibitors on these enzymes was studied. The results confirmed that proteases PIc and PII could be serine and cysteine proteases, respectively. The molecular weights of F. gigantica PIc and PII were 60,000 and 25,000, respectively. F. gigantica PIc and PII had pH optima at 7.5 and 5.5 and K(M) of 2 and 5 mg azocasein/mL, respectively. For amidolytic substrates, PIc had K(M) of 0.3 mM BAPNA/mL and 0.5 mM BTPNA/mL at pH 8.0 and PII had K(M) of 0.6 mM BAPNA/mL at pH 6.5 with reducing agent. F. gigantica PIc and PII had the same optimum temperature at 50 degrees C and were stable up to 40 degrees C. All examined metal cations tested had inhibitory effects toward the two enzymes. From substrate specificity and protease inhibitor studies, PIc and PII could be designated as serine PIc and cysteine PII, respectively.     

Isolation and partial characterization of chromoprotein from the larval hemolymph of the Japanese oak silkworm (Antheraea yamamai)

A total of two different hemolymph proteins (designated P-I and P-II) of the Japanese oak silkworm, Antheraea yamamai, were purified from the hemolymph of the fifth instar larvae using four chromatographic steps: (a) hydrophobic interaction chromatography; (b) ion exchange chromatography; (c) gel-filtration; and (d) reverse-phase high performance liquid chromatography (HPLC). These two proteins were separated by TSKgel Phenyl-5PW RP column chromatography. P-I has an apparent molecular weight of 31 000 or 35 000, as determined by gel-filtration and SDS-PAGE, respectively. P-II shows a molecular weight of 22 000 or 25 000, by gel-filtration and SDS-PAGE, respectively. The molecular weight of P-I and P-II were determined to be 31 076 and 21 500 by MALDI-TOF MS, respectively. These results suggest that both P-I and P-II are monomers. The N-terminal sequence analysis suggests that P-I is closely related to the ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta, with 40% identity in the first 30 residues, while P-II is similar to the biliproteins (BPs) from other lepidopteran insects (50% identity). Spectroscopic analysis shows that the blue chromophore of A. yamamai BP is not biliverdin IX, which is present in the biliproteins of most insects.     

Immunotyping of Chlamydia psittaci by indirect immunofluorescence antibody test with monoclonal antibodies

Monoclonal antibodies against pigeon and budgerigar strains of Chlamydia psittaci were used to classify the immunotypes of C. psittaci strains by an indirect immunofluorescence antibody (IFA) test. Thirty-three C. psittaci strains from pigeons and 24 from budgerigars were divided into three immunotypes (P-I, P-II, and P-III) and (B-I, B-II, and B-III), respectively. Two strains from human psittacosis patients were identified as P-III and B-I, coinciding with the epidemiological evidence of each human infection. Two strains from psittacine birds, a parrot and a parakeet, were identical to the B-II immunotype. Other mammalian strains were quite distinct from avian strains in their IFA reaction with the monoclonal antibodies.     

Population genetic structure of Legionella pneumophila inferred from RNA polymerase gene (rpoB) and DotA gene (dotA) sequences

The population structure of Legionella pneumophila was studied by using partial RNA polymerase gene (rpoB) and DotA gene (dotA) sequences. Trees inferred from rpoB sequences showed that two subspecies of L. pneumophila, Legionella pneumophila subsp. pneumophila and Legionella pneumophila subsp. fraseri, were clearly separated genetically. In both rpoB and dotA trees, 79 Korean isolates used in this study constituted six clonal populations, four of which (designated subgroups P-I to P-IV) were identified in L. pneumophila subsp. pneumophila and two of which (designated subgroups F-I and F-II) were identified in L. pneumophila subsp. fraseri. Although the relationships among subgroups were not identical, such subgrouping was congruent between the rpoB and dotA trees. Type strains of several serogroups did not belong to any subgroup, presumably because isolates similar to these strains were not present among our local sample of the population. There was evidence that horizontal gene transfer or recombination had occurred within L. pneumophila. Contrary to the phylogeny from rpoB and the taxonomic context, subgroups P-III and P-IV of L. pneumophila subsp. pneumophila proved to be closely related to those of L. pneumophila subsp. fraseri or showed a distinct clustering in the dotA tree. It can be inferred that dotA of subgroups P-III and P-IV has been transferred horizontally from other subspecies. The diverse distribution of serogroup 1 strains through the gene trees suggests that surface antigen-coding genes that determine serogroup can be exchanged. Thus, it can be inferred that genetic recombination has been important in the evolution of L. pneumophila.     

Heat-stable proteases from psychrotrophic pseudomonads: comparison of immunological properties.

A heat-stable extracellular protease from Pseudomonas fluorescens was purified by chromatography on a DEAE-cellulose column and gel filtration on a Sephadex G100 column. The homogeneous enzyme preparation was used to prepare antiserum in rabbits. The rabbit antiserum was used to study the antigenic relatedness of proteases from 19 psychrotrophic pseudomonads isolated from raw milk. The inhibition of the proteases by the antiserum and the gel precipitin reactions revealed similar antigenic determinants in proteases from different isolates. Rabbit antiserum to the purified protease gave precipitin bands with antigens (proteases) from 10 different isolates. However, the same antiserum did not inhibit the protease activity in cell extracts of isolates T10, T13, and T24. By determining serological cross-reactions, proteases from psychrotrophic pseudomonads were shown to be different from one another.     

Heat-stable proteases from psychrotrophic pseudomonads: comparison of immunological properties

A heat-stable extracellular protease from Pseudomonas fluorescens was purified by chromatography on a DEAE-cellulose column and gel filtration on a Sephadex G100 column. The homogeneous enzyme preparation was used to prepare antiserum in rabbits. The rabbit antiserum was used to study the antigenic relatedness of proteases from 19 psychrotrophic pseudomonads isolated from raw milk. The inhibition of the proteases by the antiserum and the gel precipitin reactions revealed similar antigenic determinants in proteases from different isolates. Rabbit antiserum to the purified protease gave precipitin bands with antigens (proteases) from 10 different isolates. However, the same antiserum did not inhibit the protease activity in cell extracts of isolates T10, T13, and T24. By determining serological cross-reactions, proteases from psychrotrophic pseudomonads were shown to be different from one another.     

The proteases in the gut of the locust, Locusta migratoria

The intestinal fluid of Locusta migratoria was purified by ionexchange chromatography on a DEAE-cellulose column. Four fractions (P_I–P_IV) with endopeptidase activity have been obtained and characterized in further studies. All proteolytic fractions were found to react with PMSF. Therefore, they seem to be typical serine proteases. Two of them, P_I and P_IV, resemble bovine trypsin and bovine chymotrypsin, respectively. These proteases hydrolyse the B-chain of oxidized insulin and the synthetic substrates BTEE,² APNE and BAEE, BANA with a specificity very similar to the bovine enzymes. Moreover, they show similar inhibition characteristics and pH activity profiles. Their molecular weights were found to be 17,000 and 18,200, respectively, according to gel filtration. Fraction P_III did not hydrolyse any of the applied synthetic substrates, P_II was active only with GluPNA. The pH optima of these enzymes lay near neutrality. Their molecular weights were found to be 27,000 and 32,000, respectively. Probably they belong to a type of proteases hitherto scarcely described and not to be found in vertebrates.     

Purification and serological comparison of the yeast hexokinases P-I and P-II

An improved procedure for purification of the hexokinases P-I and P-II from baker's yeast is described. Yields, reproducibility, and purity are improved over those found by the methods used previously in this laboratory. The growth of large crystals of form P-I is described.Antisera prepared against the two purified hexokinases show only slight cross reaction by microcomplement fixation. The anti-sera have been used to demonstrate the presence of both P-I and P-II in crude extracts of various yeasts, including two haploid strains, and their absence in a yeast which contains glucokinase but no hexokinases.     

Identification of an ascorbate-dependent cytochrome<Emphasis Type="Italic"> b</Emphasis> of the tonoplast membrane sharing biochemical features with members of the cytochrome<Emphasis Type="Italic"> b</Emphasis>561 family

Two membrane-bound, ascorbate-dependent b-type cytochromes were identified in etiolated bean (Phaseolus vulgaris L.) hypocotyls. Following solubilization of microsomal membranes and anion-exchange chromatography at pH 8.0, two major cytochrome peaks (P-I and P-II) were separated. Both cytochromes were reduced by ascorbate and re-oxidized by monodehydroascorbate, but P-I reduction by ascorbate was higher and saturated at far lower concentrations of ascorbate with respect to P-II. The -band was symmetrically centered at 561 nm in P-I, but it was asymmetric in P-II with a maximum at 562 nm and shoulder at 557 nm. Ascorbate reduction of P-II, but not P-I, was inhibited by diethyl pyrocarbonate. Reduced P-II but not P-I was readily oxidized by certain ferric chelates, including FeEDTA and Fe-nitrilotriacetic acid. Purified P-I, associated with the plasma membrane, showed up as a 63-kDa glycosylated protein during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and behaved as a monomer of about 70 kDa during size-exclusion chromatography. P-I identified with a previously purified ascorbate-dependent b-type cytochrome of bean hypocotyl plasma membranes [P. Trost et al. (2000) Biochim Biophys Acta 1468:1–5]. Partially purified P-II, on the other hand, correlated with a heme-protein of 27 kDa in SDS–PAGE gels, was dimeric (60 kDa) during size-exclusion chromatography, and was associated with the tonoplast marker V-ATPase in sucrose gradients. The sequence of a peptide of 11 residues obtained by tryptic digestion of P-II was found to be identical to a segment of a putative cytochrome b561 of Zea mays and highly conserved in other related plant sequences, including that of Arabidopsis thaliana cytochrome b561-1 (CAA18169). The biochemical features fully support the assignment of P-II cytochrome to the family of cytochrome b561, ascorbate-dependent (CYBASC) cytochromes, which also includes cytochrome b561 of animal chromaffin granules. The presence of a cytochrome reducing ferric chelates on the tonoplast is consistent with the role of plant vacuoles in iron homeostasis.     

Gastric proteases of the Greenland cod Gadus ogac. I. Isolation and kinetic properties

The zymogens of three gastric proteases of the Greenland cod (Gadus ogac) were isolated by exclusion chromatography and chromatofocusing. The cod zymogens were activated more rapidly at lower temperatures than porcine pepsinogen and, after activation, were further purified by exclusion chromatography. The cod proteases had more alkaline pH optima and were active over a wider range of pH than porcine pepsin. The specific activity of porcine pepsin on protein substrates was greater than that of the individual cod proteases. However, the cod proteases had cumulative activity on protein substrates that was greater than the sum of their individual activities. Cod protease 1 was active on pepsin-specific substrates, and cod proteases 2 and 3 were active as gastricsin-specific substrates. All three cod proteases had greater milk-clotting activity and hydrolysed hemoglobin to a greater extent than porcine pepsin. The Vmax and Km,app of the cod proteases were dependent upon the substrate, and Vmax/Km,app values of the cod proteases were generally lower than porcine pepsin. It is suggested that the cod proteases together exhibit broad substrate specificity and maintain activity over a wide range of conditions to enhance protein digestion in the cod stomach.     

Intracellular serine protease-4, a new intracellular serine protease activity from Bacillus subtilis

A previously undiscovered intracellular serine protease activity, which we have called intracellular serine protease-4, was identified in extracts of stationary Bacillus subtilis cells, purified 260 fold from the cytoplasmic fraction, and characterized. The new protease was stable and active in the absence of Ca²⁺ ions and hydrolyzed azocasein and the chromogenic substrate carbobenzoxy-carbonyl-alanyl-alanyl-leucyl-p-nitroanilide, but not azocollagen or a variety of other chromogenic substrates. The protease was strongly inhibited by phenylmethylsulfonylfluoride, chymostatin and antipain, but not by chelators, sulfhydryl-reactive agents or trypsin inhibitors. Its activity was stimulated by Ca²⁺ ions and gramicidin S; its pH and temperature optima were 9.0 and 37°C, respectively. Although intracellular serine protease-4 was immunochemically distinct from intracellular serine protease-1, it was absent from a mutant in which the gene encoding the latter was disrupted.     

The activation of expressed cGMP-dependent protein kinase isozymes I alpha and I beta is determined by the different amino-termini.

cDNA of bovine cGMP-dependent protein kinase (cGMP kinase) isozymes I alpha and I beta differ only in their amino-terminal domains (amino acids 1-89 and 1-104, respectively). Each recombinant isozyme (rI alpha and rI beta) was transiently expressed in COS-7 cells and its properties were compared with the cGMP kinase isozymes P-I and P-II purified from bovine trachea. The subunit of P-I, P-II, rI alpha and rI beta had a molecular mass of about 75 kDa. rI alpha and rI beta had S20,W values of 7.6 and 7.2, respectively, indicating that they were present as dimeric holoenzymes. Immunostaining with specific antibodies showed that P-I and rI alpha, and P-II and rI beta, were immunologically indistinguishable. P-I, P-II, rI alpha and rI beta had the same catalytic activity. However, rI alpha and rI beta were half-maximally activated at 0.1 microM and 1.3 microM cGMP, and 0.3 microM and 12 microM 8-bromoguanosine 3',5'-(cyclic)phosphate (Br8-cGMP), respectively. P-I and P-II had a similar shift in their apparent KA values. P-I and rI alpha bound 2 mol cGMP/mol subunit to high-affinity (site 1) and low-affinity (site 2) cGMP-binding sites. The exchange rates were 0.005-0.009 min-1 for site 1 and 3.7 min-1 for site 2. In contrast, P-II and rI beta bound and rI beta bound 2 mol cGMP/mol enzyme subunit at only two low-affinity binding sites (site 2) with k-1 values of 0.92 min-1 and 4.8 min-1. These results suggest that a change from the I alpha amino-terminal domain to that of I beta increases the apparent KA value for cGMP 10-fold by altering the binding properties of binding site 1. The differential expression of the cGMP kinase isozymes could be an important mechanism in vivo to dampen the effect of long-term elevation of cGMP level.     

A preliminary study of Cu-, Cd- and Zn-binding components in the hepatopancreas of Palaemon elegans (Crustacea: Decapoda)

Three low molecular weight, metal-binding components have been isolated from the hepatopancreas of the shrimp Palaemon elegans. The two larger components P-I (11-15,000) and P-II (4-6000) were associated with large amounts of copper in field-collected and copper-exposed shrimps and with cadmium in shrimps exposed to elevated Cd concentrations. A smaller component, P-III was associated with zinc. The recovery of these components was dependent upon the use of a protease inhibitor and the reducing agent mercaptoethanol (2-M) during the separation procedure. Copper, zinc and cadmium are not evenly distributed between individual tissues of P. elegans, the highest concentrations occurring in the hepatopancreas, gills and eyes.     

Thyroid hormone induces a 52 kDa soluble protein in goat testis Leydig cell which stimulates androgen release

Incubation of goat testicular Leydig cells with 3,5,3′-triiodothyronine(T₃) induces the generation of a proteinaceous factor (factors) which was located in the soluble supernatant fraction (100 000 × g supernatant, 100k sup) of sonicated Leydig cells. Addition of this factor to Leydig cell incubation greatly stimulated androgen release. This factor(s) was purified based on its biological properties, i.e., its addition to Leydig cell incubation augmented the release of androgen. This was designated as TIP (T₃-induced protein) activity. 100 k sup prepared from Leydig cells incubated in the absence (control) or presence of T₃ was gel filtrated through Sephadex G-100. 100 k sup from T₃ incubate gave two protein peaks, P-I and P-II, control 100 k sup had similar nature of P-I, but P-II was not well marked. Incubation in the presence of [¹⁴C]leucine clearly showed TCA precipitable radioactivity only in the P-II region of T₃ incubate. 5 μg of P-II protein stimulated androgen release from Leydig cells (1 · 10⁶ cells/well) to more than 5-fold as compared to control. P-II protein was further purifies by FPLC Mono-Q column chromatography where one unadsorbed (MQ-I) and two adsorbed(MQ-II and MQ-III) protein peaks could be detected. MQ-II, which was eluted with 0.20 M NaCl gradient, demonstrated strong TIP activity (2 μg protein released 4.8-fold more androgen as compared to control). MQ-II was passed through FPLC Superose-6 column where it gave two peaks and Peak-I(SP-I) showed strong TIP activity (2 μg protein stimulated a 6-fold increase in androgen release as compared to control). Polyacrylamide gel electrophoresis (PAGE) indicated SP-I to be a homogeneous protein and SDS-PAGE demonstrated it to be a 52 kDa monomer protein. Results show that T₃ induces the synthesis of a 52 kDa protein in testicular Leydig cells which in turn causes stimulation of androgen release suggesting this protein to be a novel mediator of T₃ function in Leydig cells.