Cephalosporinase and penicillinase activities of a β-lactamase from Pseudomonas pyocyanea

1. Pseudomonas pyocyanea N.C.T.C. 8203 produces a beta-lactamase that is inducible by high concentrations of benzylpenicillin or cephalosporin C. Methicillin appeared to be a relatively poor inducer, but this could be attributed in part to its ability to mask the enzyme produced. Much of the enzyme is normally cell-bound. 2. No evidence was obtained that the crude enzyme preparation consisted of more than one beta-lactamase and the preparation appeared to contain no significant amount of benzylpenicillin amidase or of an acetyl esterase. 3. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by the crude enzyme was more than five times that of benzylpenicillin. Methicillin, cloxacillin, 6-aminopenicillanic acid and 7-aminocephalosporanic acid were resistant to hydrolysis, and methicillin and cloxacillin were powerful competitive inhibitors of the action of the enzyme on easily hydrolysable substrates. 4. Cephalosporin C, cephalothin and cephaloridine yielded 2 equiv. of acid/mole on enzymic hydrolysis, and deacetylcephalorsporin C yielded 1 equiv./mole. Evidence was obtained that the opening of the beta-lactam ring of cephalosporin C and cephalothin is accompanied by the spontaneous expulsion of an acetoxy group and that of cephaloridine by the expulsion of pyridine. 5. A marked decrease in the minimum inhibitory concentration of benzylpenicillin and several hydrolysable derivatives of 7-aminocephalosporanic acid was observed when the size of the inoculum was decreased. This suggested that the production of a beta-lactamase contributed to the factors responsible for the very high resistance of Ps. pyocyanea to these substances. It was therefore concluded that the latter might show synergism with the enzyme inhibitors, methicillin and cloxacillin, against this organism.     

Inhibition of Aerobacter Cephalosporin β-Lactamase by Penicillins

Cephalosporinase (beta-lactamase) was obtained from cell washings of Aerobacter (Enterobacter) cloacae as a highly active preparation. An alkalimetric method was used to determine the enzyme activity and to estimate its inhibition by 6-amino-penicillanic acid derivatives. Their order of decreasing inhibitory effect was as follows: cloxacillin, oxacillin, methicillin, ampicillin, and penicillin G. We found that 2 to 3 ng of cloxacillin per ml was sufficient to decrease the enzyme activity by 50% in the presence of 400 mug of cephalosporin C per ml. Cloxacillin exerted a potentiating effect on the inhibition of the E. cloacae organisms by cephalosporin C.     

Studies of triose phosphate isomerase by hydrogen exchange

The (3)H-H exchange of chicken muscle and rabbit muscle triose phosphate isomerases was studied. Their behaviour was mostly very similar. ;Exchange-in' (acquisition of radioactivity when protein was incubated in (3)H(2)O) was measured at 37 degrees C and at pH7.5, and the rates of exchange of the native and liganded enzymes were compared. Inhibitors and substrates retarded exchange, substrates showing the most marked effect; structural rearrangements in the enzyme may thus play some part in catalysis. The inhibitor phosphoglycollate affected the rabbit enzyme, but had little or no effect on the chicken enzyme. ;Exchange-out' (loss of radioactivity from protein previously labelled by incubation in (3)H(2)O) was measured by hollow-fibre dialysis. When ligand was removed during the course of dialysis (by replacing buffer that contained ligand with buffer that lacked ligand) there was a prompt decrease in the number of labelled H atoms of the protein. Analysis of the curves provides some information about the number and half-lives of the responsive H atoms. Ligands decrease the motility of the protein and affect about one-fifth of the chain. Low concentrations of glycerol 3-phosphate have an effect that is greater than expected.     

Human-liver alanine aminopeptidase. A kinin-converting enzyme sensitive to beta-lactam antibiotics

Human liver alanine aminopeptidase (EC 3.4.11.14; L-alpha-aminoacyl-peptide hydrolase) catalyzes the stepwise hydrolysis of methionyl-lysyl-bradykinin to yield methionine, lysine, and the limit nonapeptide, bradykinin which is resistant to further hydrolytic cleavage by this enzyme. Alanine aminopeptidase also catalyzes the hydrolysis of various neutral amino acid beta-naphthylamides. This enzyme cleaves N-terminal arginyl residues unless the adjacent penultimate residue is proline as is the case for bradykinin. The properties are consistent with the requirements of a kinin converting enzyme. Human alanine aminopeptidase activity is reduced by several beta-lactam antibiotics, with the cloxacillin, oxacillin, and methicillin Ki values being 0.51 mM, 1.6 mM, and 2.4 mM respectively. Our experiments with radioactively labelled penicillin indicate that two moles of antibiotic are bound per mole of enzyme. Neither chromatography of the penicillin-treated enzyme on G-25 Sephadex, treatment of penicillin-G-treated enzyme with penicillinase, nor extensive dilution of cloxacillin-treated enzyme diminished the degree of inactivation produced. Inhibition was obtained with 6-aminopenicillanic acid, which indicated that the penicillin nucleus itself was being bound, but substitutions, as in cloxacillin, could enhance the binding.     

Kinetic and structural characterization of reversibly inactivated beta-lactamase

The reversible inhibition of beta-lactamase I from Bacillus cereus by cloxacillin, methicillin, and nafcillin has been systematically investigated. For these substrates the enzymatic reaction involves partitioning of the substrate between turnover and inhibition. Typically, concentrations of several hundred millimolar are necessary for complete inactivation. The completely inactivated enzyme could be formed by incubation at temperatures above 20 degrees C, where inhibition competes more effectively with turnover, and then stabilized by dropping the temperature to 0 degrees C or lower. The inactivated enzyme was rapidly separated from unreacted substrate and product at low temperature by centrifugal gel filtration or ion exchange and examined by far-UV circular dichroism for evidence of a conformational change. At pH 7 the inactivated enzyme had a secondary structure essentially identical with that of the native enzyme. The fluorescence emission spectrum of the inactivated enzyme (at pH 7) was also identical with that of the native enzyme. However, the inactivated enzyme was found to be considerably more sensitive to thermal denaturation, to acid-induced conformational isomerization, and to trypsinolysis than the native enzyme. We conclude from the circular dichroism results that the structure of the reversibly inactivated enzyme cannot be significantly different from that of the native enzyme. Therefore, previous findings that have been interpreted as indicating a major conformational change must be reevaluated. From examination of the low-resolution crystallographic structure of the enzyme we propose that the most likely cause of the inactivation is an alternate conformational state of the acyl-enzyme intermediate involving movement of one or more of the alpha-helices forming part of the active site. Such a structural effect could leave the secondary structure unchanged but have significant effects on the tertiary structure, catalysis, mobility, and susceptibility to trypsin and denaturation. We propose that the underlying physical reason why certain beta-lactam substrates bring about this "substrate-induced deactivation", or suicide inactivation, of the enzyme is due to the presence of the alternative acyl-enzyme conformation of similar free energy to the productive one, in which one (or more) essential catalytic group is no longer optimally oriented for catalyzing deacylation. Thus for substrates with a slow rate of deacylation (less than or equal to 100 s-1) the conformational transition can compete effectively on the time scale of the turnover reaction.     

Comparison of the Substrate Specificities of the β-Lactamases from Klebsiella aerogenes 1082E and Enterobacter cloacae P99

A potent beta-lactamase (EC 3.5.2.6) produced by a strain of Klebsiella aerogenes (K. pneumoniae), 1082E, isolated from a hospital patient, has been examined. Its properties were different from those of most gram-negative beta-lactamases previously reported. The enzyme has been partly purified, and its activity against a range of substrates has been compared with that of the enzyme from Enterobacter cloacae (Aerobacter cloacae) P99. The K. aerogenes enzyme, although predominantly a penicillinase, had a wide range of specificity. In addition to hydrolyzing the cephalosporins, it attacked the normally beta-lactamaseresistant compounds methicillin and cloxacillin as well as cephalosporin analogues with the same acyl substituents. The results obtained with the E. cloacae enzyme confirmed its cephalosporinase activity and showed that, unlike the enzyme from K. aerogenes, it was relatively inactive against the penicillins.     

Phenylalanyl-tRNA synthetase from E. coli MRE-600. Effect of chemical modification of lysine residues on the enzyme interaction with substrates

The effect of modification of Phe-RSase from E. coli MRE-600 by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP and L-phenylalanynyl-5'-adenylate obtained by periodate oxidation on the enzyme interaction with substrates was investigated. It was shown that modification of Phe-RSase by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP leads to a decrease of the aminoacylation rate without changing the rate of the ATP-[32P]-pyrophosphate exchange reaction. The substrate analogs L-phenylalanynol and L-phenyl-alanynyladenylate increase the degree of Phe-RSase inactivation in the aminoacylation reaction. tRNAphe strongly protects the enzyme against inactivation. ATP, both in the absence (in case of modification with pyridoxal-5'-phosphate) and in- the presence of Mg2+ and phenylalanine (in case of modification with o-ATP) exhibits a pronounced protective effect. L-Phe does not protect the enzyme against the inactivation by pyridoxal-5'-phosphate or o-ATP. The dissociation constant of the Phe-RSase[14C]-Phe-tRNAphe complex increases 2.5 -- 5-fold after the enzyme modification by pyridoxal-5'-phosphate, while the Km value for tRNAphe decreases approximately two times in the aminoacylation reaction. There are no changes in the Km values for amino acid and ATP and the Hill coefficients for all substrates tested. Modification of Phe-RSase by pyridoxal-5'-phosphate leads to a decrease of stability of the aminoacyladenylate -- enzyme complex. Oxidized L-phenylalanynyladenylate does not produce enzyme inactivation either by aminoacylation or in the isotropic ATP-PP iota exchange reaction. It is assumed that Phe-RSase from E. coli MRE-600 contains some lysine residues essential for binding and aminoacylation of tRNA, which do not occur in the ATP-binding subsite and aminoacyladenylate formation center.     

A survey of the kinetic parameters of class C beta-lactamases. Penicillins.

The interaction between six class C beta-lactamases and various penicillins has been studied. All the enzymes behaved in a very uniform manner. Benzylpenicillin exhibited relatively low kcat. values (14-75 s-1) but low values of Km resulted in high catalytic efficiencies [kcat./Km = 10 X 10(6)-75 X 10(6) M-1.s-1]. The kcat. values for ampicillin were 10-100-fold lower. Carbenicillin, oxacillin cloxacillin and methicillin were very poor substrates, exhibiting kcat. values between 1 x 10(-3) and 0.1 s-1. The Km values were correspondingly small. It could safely be hypothesized that, with all the tested substrates, deacylation was rate-limiting, resulting in acyl-enzyme accumulation.     

beta-lactamase from Streptomyces cacaoi. Purification and properties

A beta-lactamase was purified to an apparently homogeneous state from Streptomyces cacaoi. The molecular weight calculated from the mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis was 34,000. pI was 4.7 and the optimal pH was 6.5. The optimum temperature was found to be between 40 degrees C and 45 degrees C, but the enzyme lost activity above 50 degrees C. N-Bromosuccinimide was the strongest inhibitor among the reagents tested, followed by iodine. p-Chloromercuribenzoate showed a weak inhibitory effect. Diisopropylfluorophosphate and sodium chloride did not show any inhibitory effect on the enzyme. The beta-lactamase catalyzed the hydrolysis of methicillin and cloxacillin at two-thirds to one-third the rate of benzylpenicillin. On the other hand, the enzyme hydrolyzed cephalosporins and 7-methoxycephalosporin only slowly. With benzylpenicillin as a substrate, the Km increased sharply with decreasing pH and the pK alpha estimated from the Km versus pH curve was 6.5 to 7.0. In contrast, with cloxacillin as a substrate, the Km showed a minimum at pH 7.5. The Vmax changed with pH in a bell-shaped curve in the case of benzylpenicillin, but the Vmax for cloxacillin changed only within a small range. In addition, the ratio of the hydrolysis rate of benzylpenicillin and cloxacillin at 30 degrees C and 20 degrees C (V30 degrees/V20 degrees) was found to be 1.23 and 1.55, respectively. These results indicate that the S. cacaoi beta-lactamase behaves differently toward benzylpenicillin and cloxacillin, although both are penicillins. S. cacaoi seems to release beta-lactamase into the culture medium soon after its biosynthesis without retaining it in the membrane and the soluble fraction. The possible relationships between beta-lactamases from Streptomyces and those from pathogenic bacteria are discussed.     

Stereochemical studies of hydrogen incorporation from nucleotides with fatty acid synthetase from Brevibacterium ammoniagenes.

The biosynthesis of fatty acids from malonyl-CoA and acetyl-CoA was investigated with an enzyme preparation which was purified 100-fold from Brevibacterium ammoniagenes. Fatty acids synthesized in the presence of D2O and stereospecifically deuterated NADPH and NADH were isolated and analyzed by mass chromatography to examine the localization of deuterium in the molecule. The following results were obtained: 1) HB hydrogen of NADPH was used for beta-ketoacyl reductase. 2) HB hydrogen of NADH was used for enoyl reductase. 3) Hydrogen atoms from water were found on the even-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom) and some were also found on the odd-numbered methylene carbon atoms. 4) Hydrogen atoms from NADPH was found on the odd-numbered methylene carbon atoms (1 hydrogen per carbon). 5) Hydrogen atoms from NADH was also found on the odd-numbered methylene carbon atoms, but the number of incorporated hydrogen atoms was less than expected. The exchange of HB hydrogen of NADH with water catalyzed by enoyl reductase was suspected. 6) The exchange of methylene hydrogen atoms of malonyl-CoA with protons of water was suggested by 13C NMR analysis.     

Investigation of the mechanism and steric course of the reaction catalyzed by 6-methylsalicylic acid synthase from Penicillium patulum using (R)-[1-13C;2-2H]- and (S)-[1-13C;2-2H]malonates.

Chiral malonyl-CoA derivatives, enzymically synthesized from (R)- and (S)-[1-13C;2-2H]malonates using succinyl-CoA transferase, were incorporated into 6-methylsalicylic acid with homogeneous 6-methylsalicylic acid synthase isolated from Penicillium patulum. Analysis of the 6-methylsalicylic acid formed established that the hydrogen atoms at the 3- and 5-positions are derived from opposite absolute configurations in malonyl-CoA. When acetoacetyl-CoA was used as the starter molecule, a single hydrogen atom is incorporated from the chiral malonates into the 3-position of the 6-methylsalicylic acid. Mass spectrometric analysis of the 6-methylsalicylic acid indicates that this hydrogen atom originates from HRe of malonyl-CoA or HSi in the polyketide intermediate. It is thus concluded that the hydrogen atom at the 5-position of 6-methylsalicylic acid originates from HSi of malonyl-CoA or HRe in the polyketide intermediate. During the reaction the enzyme also catalyzes the stereospecific exchange of hydrogen atoms in the polyketide intermediates. The implications of the stereochemical information from these experiments are discussed in relation to the mechanism of the 6-methylsalicylic acid synthase reaction.     

Isotopic hydrogen exchange in reactions catalysed by cysteine lyase and serine sulphhydrase.

Serine sulphhydrase from chicken liver and cysteine lyase from chicken-embryo yolk sac catalyse the exchange of alpha-H atoms of the amino acid substrate with 3-H-2O. The degree of labelling of the unreacted substrate approaches a maximum of one atom per mol of amino acid. In the absence of replacing agent there is practically no H-exchange in the substrate. The alpha-H of the accumulating beta-substitution product is completely replaced by the labelled hydrogen of the solvent water, irrespective of the duration of incubation. The amount of labelled alpha-hydrogen incorporated into excess (unreacted) amino acids substrate within 3.5-h incubation is somewhat less than the amount incorporated into the product of the complete enzymic beta-replacement reaction. Within the sensitivity limits of detection, the enzymes do not induce any isotopic exchange either of b-H atoms in the amino substrate or of 18-O-labelled beta-HO groups, in the case of L-serine. Neither serine sulphhydrase nor cysteine lyase will catalyse alpha-hydrogen exchange in close structural analogues of their substrates, e.g. L-alanine, D-serine, threonin, 3-phosphoserine. A special case is the interaction of cysteine lyase with the competitive inhibitor, L-serine (whose inhibitor constant, K-i, is equal to the Michaelis constant, K-m, of L-cysteine): the lyase catalyses, only in presence of a cosubstrate thiol, alpha-H exchange in L-serine at approximately the same rate as in L-cysteine. The present data concerning isotopic alpha-H exchange in substrate amino acids, and evidence published earlier, suggest that the catalytic mechanism of replacement-specific beta-lyases may significantly differ from that of the eliminating or ambivalent (mixed-function) lyases. Formation of alpha, beta-unsaturated pyridoxylidene aldimines as real reaction intermediates is unlikely in the case of lyases specifically catalysing beta-replacement reactions; these may proceed by some alternative mechanism of the type suggested in this paper.     

Origin of hydrogen atoms in the fatty acids synthesized with yeast fatty acid synthetase.

The mechanism of hydrogen incorporation into fatty acids was investigated with an enzyme preparation from baker's yeast. Fatty acids synthesized from malonyl-CoA and acetyl-CoA in the presence of D2O or stereospecifically deuterium-labeled NADPH were isolated and analyzed by mass chromatography to examine the localization of deuterium atoms in the molecule. The following results were obtained: 1. Hydrogen atoms from water were found on the even-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom). The second hydrogen atom was incorporated as the result of hydrogen exchange phenomenon between the methylene group of malonyl CoA and water. 2. HB hydrogen of NADPH was used for beta-ketoacyl reductase. 3. HB hydrogen of NADPH was also used for enoyl reductase. 4. Hydrogen atoms from HB position of NADPH were found on the odd-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom).     

Purification and characterization of an extracellular beta-lactamase produced by Acinetobacter calcoaceticus.

A beta-lactamase was purified 430-fold from the culture supernatant of Acinetobacter calcoaceticus by ion exchange chromatography on CM-Sephadex and affinity chromatography on phenylboronic-acid-agarose. The purified enzyme was homogeneous as judged by SDS-PAGE, and was characterized with respect to molecular mass (38 and 41 kDa by gel filtration on Sephadex G-75 and SDS-PAGE, respectively), pH optimum (pH 7.0), temperature optimum (45 degrees C) and isoelectric point (9.3). The beta-lactamase showed mainly cephalosporinase activity. It was inhibited by cloxacillin, carbenicillin, penicillanic acid sulphone (sulbactam) and aztreonam. It was not inhibited by clavulanic acid up to a concentration of 0.25 mM. Neither EDTA nor p-chlormercuribenzoate, up to concentrations of 1 or 100 mM, respectively, affected activity. According to these characteristics, it is a typical CEP-N cephalosporinase.     

Structural and catalytic properties of hydrogenase from Chromatium.

The enzyme hydrogenase, from the photosynthetic bacterium Chromatium, was purified to homogeneity after solubilization of the particulate enzyme with deoxycholate. The purification procedure included ammonium sulfate fractionation, treatment with manganous phosphate gel, heating at 63 degrees, DEAE-cellulose chromatography, and isoelectric focusing. The last step gave two active enzyme fractions with isoelectric points of 4.2 and 4.4. It was shown that the two fractions were different forms of the same protein. The enzyme was obtained in 23% yield and was purified 1700-fold. The enzyme had a molecular weight of 98,000, a sedimentation coefficient of 5.16 S and gave a single protein and activity band on disc gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis gave a single band of mol wt 50,000, suggesting that the active enzyme was composed of two subunits of the same molecular weight. The pure hydrogenase contained four atoms of iron and four atoms of acid-labile sulfide, and had a visible absorption peak at 410 nm. Electron paramagnetic resonance (EPR) spectroscopy at 10--15 K showed a free-radical signal at g' = 2.003 in the oxidized enzyme and signals at g' = 2.2 and 2.06 in the reduced enzyme. These findings suggest that Chromatium hydrogenase is an iron-sulfur protein. The pure hydrogenase catalyzed the exchange reaction between H2 and HDO or HTO, the reduction of Benzyl Viologen and Methylene Blue, and the evolution of hydrogen from reduced Methyl Viologen. The mechanism of hydrogen activation was shown to be heterolytic cleavage to an enzyme hydride and a proton. Hydrogenase could not catalyze reduction of pyridine nucleotides or ferredoxin with H2. The effect of pH and various inhibitors on the enzymatic activity has been studied.     

Enzymes of vitamin B6 degradation. Purification and properties of 5-pyridoxic-acid oxygenase from Arthrobacter sp

5-Pyridoxic-acid oxygenase, a cytoplasmic enzyme formed when Arthrobacter Cr-7 is grown with pyridoxine as a sole source of carbon and nitrogen, was purified about 190-fold to homogeneity from fully induced cells. The enzyme catalyzes Reaction a, (Formula: see text) the essential ring-opening step in the degradation of pyridoxine, and provides a second example of an FAD-dependent oxygenase that adds both two hydrogen and two oxygen atoms to its substrate. 5-Pyridoxic-acid oxygenase has an isoelectric point of 4.6, functions optimally between pH 7 and 8, appears to contain a single subunit of Mr = 51,000 and one FAD (but no iron) per subunit, and is readily resolved by precipitation with ammonium sulfate at pH 3.0. FMN and riboflavin do not replace FAD as coenzyme, but their presence enhances a normally minor side reaction (Reaction b) NAD(P)H + H+ + O2----NAD(P)+ + H2O2 (b) catalyzed by the holoenzyme. Reaction b also is enhanced when the poorly utilized analogues, 3-hydroxy-2-methylpyridine-5-carboxylic acid or NADH, replace 5-pyridoxic acid or NADPH, respectively, as substrates in Reaction a. Each of the enzymes required in two different pathways for degradation of pyridoxine to anabolic intermediates has now been studied. A comparison of these two pathways and their enzymes is provided.     

Identification of uronic acid oxidase in plant peroxidase preparations

Commercial plant peroxidase preparations contained a uronic acid oxidase, separable from the peroxidase activity by ion exchange chromatography. The partially purified enzyme, devoid of peroxidase, oxidized hexuronic acids, with the greatest activity for D-glucuronic acid, whereas other aldoses were not substrates. The immediate products of reaction of D-glucuronic acid with oxygen were hydrogen peroxide and a D-glucarolactone, which was a very strong inhibitor of β-glucuronidase and believed to be the 1,5-lactone. The sensitivity to sulphite inhibition suggests that the enzyme is a flavoprotein.     

Influence of chloride on modification of unsaturated phosphatidylcholines by the myeloperoxidase/hydrogen peroxide/bromide system

The leukocyte enzyme myeloperoxidase (MPO) is capable of catalyzing the oxidation of chloride and bromide ions, at physiological concentrations of these substrates, by hydrogen peroxide, generating hypochlorous acid (HOCl) and hypobromous acid (HOBr), respectively. Our previous results showed that the hypohalous acids formed react with double bonds in phosphatidylcholines (PCs) to produce chloro- and bromohydrins. Lysophosphatidylcholine (lyso-PC) is additionally formed in PCs with two or more double bonds. This study was conducted to determine the effect physiological chloride concentration (140 mM) has on the formation of bromohydrins and lyso-PC from unsaturated PC upon treatment with the myeloperoxidase/hydrogen peroxide/bromide (MPO/H2O2/Br-) system using physiological bromide concentrations (20-100 microM). The composition of reaction products was analyzed by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). With monounsaturated PC, we demonstrated that the rate and extent of mono-bromohydrin formation were higher in the samples with 140 mM chloride compared to those with no added chloride. Moreover, mono-bromohydrin came to be the major product and no mono-chlorohydrin was observed already at 60 microM bromide. We attributed these effects to the involvement of HOBr arising from the reaction of MPO-derived HOCl with bromide rather than to the exchange of bromide with chlorine atoms of chlorohydrins or direct formation of HOBr by MPO. The presence of chloride shifted the pH optimum for mono-bromohydrin formation (pH 5.0) toward neutral values, and a significant yield of mono-bromohydrin was detected at physiological pH values (7.0-7.4). For polyunsaturated PC, chloride enhanced also lyso-PC production, the effect being pronounced at bromide concentrations below 40 microM. The results indicate that at physiological levels of chloride and bromide, chloride promotes MPO-mediated formation of bromohydrins and lyso-PC in unsaturated phospholipids.     

Susceptibility of Coagulase-negative Staphylococci to Lysostaphin and Other Antibiotics

In general, coagulase-negative staphylococci were found to be relatively less susceptible to the lytic action of lysostaphin than coagulase-positive staphylococci. To achieve, arbitrarily, a lysis greater than 75%, it was necessary to use an increased concentration of enzyme or a longer incubation period than that usually required with coagulase-positive strains. For the most part, the cultures studied were sensitive to oxacillin, cloxacillin, dicloxacillin, nafcillin, ancillin, cephalothin, cephaloridine, fusidic acid, lincomycin, novobiocin, and neomycin [median minimal inhibitory concentrations (MIC) of 1.56 mug/ml or less]. Some degree of resistance (median MIC values of 12.5 mug/ml or greater) to benzylpenicillin, ampicillin, methicillin, tetracycline, chloretetracycline, erythromycin, ristocetin, and lysostaphin was found. Ten methicillin-resistant, coagulase-negative staphylococal strains were found to be cross-resistant to all nine of the penicillins tested, but much less resistant to the two cephalosporin analogues. In several instances, some of these strains seemed to be more sensitive to benzylpenicillin and to certain of the semisynthetic penicillins than to methicillin. Of the 18 antibiotics tested with the viable plate count method, the methicillin-resistant strains were found to be the most sensitive to lincomycin and novobiocin.     

The internal cavities of pike alpha-parvalbumin probably contain water

The kinetics of hydrogen exchange of pike a-parvalbumin was investigated using the method of infrared spectroscopy (sensitive to the amide hydrogen atoms in the peptide) and radioisotope method (sensitive to all labile hydrogen atoms). Ultraslow exchangeable hydrogen atoms were found to be substantially less in the first case than in the second one. Taking into account that the internal cavities in the parvalbumin are formed by hydrophobic amino acid residues, devoid of labile hydrogen atoms, it is possible to make the most appropriate assumption, namely, these cavities contain water molecules, hydrogen atoms of which are ultraslow exchangeable.