Factors affecting the formation of alkaline phosphatase isozymes in Escherichia coli K-12.

Physiological and genetic factors affecting the formation of isozymes of alkaline phosphatase in Escherichia coli K-12 were studied. Our results are compatible with the hypothesis proposed by Schlesinger and his co-workers (Schlesinger et al., 1975) that the multiple forms of the enzyme are produced by converting a newly synthesized one (the least negatively charged one) into less negatively charged forms. Neither energy source nor de novo synthesis of the enzyme was necessary for the conversion. It is also confirmed that the conversion is effectively inhibited by externally added arginine (Piggott et al., 1972) but only partially by canavanine (arginine analog) or casamino acids. We isolated a mutant strain which did not form isozyme(s), if any, under the condition in which the wild type strain formed isozymes. The mutation(s) was proved to be mapped in the locus (or loci) other than alkaline phosphatase structure gene in the E. coli genetic map. We tentatively proposed to designate this the iap gene(s), an abbreviation for isozyme of alkaline phosphatase, which plays a role in isozyme formation.     

Alkaline phosphatase isozyme conversion by cell-free extract of Escherichia coli

Isozyme type 1 of alkaline phosphatase in Escherichia coli K-12 was converted to types 2 and 3 after incubation of type 1 isozyme with the supernatant of a sonicated cell-free extract prepared from the cells carrying the cloned iap+ gene on a multi-copy plasmid. By comparison, the lysate prepared from cells carrying the iap+ gene only on the chromosome showed much less isozyme-converting activity. The reaction was promoted by Mg2+ at concentrations of 10 to 50 mM. Protease inhibitors, antipain and leupeptin which inhibit the isozyme conversion in vivo, also inhibited the isozyme conversion in vitro. These results suggest that cells carrying the multiple copy iap+ plasmid overproduce a kind of proteolytic enzyme which removes the amino-terminal arginine residues from isozymes 1 and 2.     

Ribosomal Alterations Controlling Alkaline Phosphatase Isozymes in Escherichia coli

Different patterns of isozymes were obtained by starch-gel electrophoresis of alkaline phosphatase from Escherichia coli strains differing only by strA or ram mutations, or both, in the 30S ribosomal subunit. The isozyme spread was reduced in strA and increased in ram strains; this strictly parallels the restriction and enhancement of translational ambiguity produced by these mutations. Streptomycin present during growth had an effect similar to ram on both isozymes and ambiguity. The three isozymes analyzed have different N-terminal residues: aspartic acid, valine, and threonine. Different patterns of isozymes were also obtained in a wild-type strain through the specific action of exogenous arginine. A link between the mechanism of the effect of arginine and that of the ribosome is not obvious. The possibility is discussed that in both cases, although by different mechanisms, N-terminals are formed with different sensitivity to limited degradative attack.     

Isolation and characterization of mutant strains of Escherichia coli altered in H2 metabolism

A positive selection procedure is described for the isolation of hydrogenase-defective mutant strains of Escherichia coli. Mutant strains isolated by this procedure can be divided into two major classes. Class I mutants produced hydrogenase activity (determined by using a tritium-exchange assay) and formate hydrogenlyase activity but lacked the ability to reduce benzyl viologen or fumarate with H2 as the electron donor. Class II mutants failed to produce active hydrogenase and hydrogenase-dependent activities. All the mutant strains produced detectable levels of formate dehydrogenase-1 and -2 and fumarate reductase. The mutation in class I mutants mapped near 65 min of the E. coli chromosome, whereas the mutation in class II mutants mapped between srl and cys operons (58 and 59 min, respectively) in the genome. The class II Hyd mutants can be further subdivided into two groups (hydA and hydB) based on the cotransduction characteristics with cys and srl. These results indicate that there are two hyd operons and one hup operon in the E. coli chromosome. The two hyd operons are needed for the production of active hydrogenase, and all three are essential for hydrogen-dependent growth of the cell.     

Genetic analysis of the Escherichia coli K-12 srl region.

Specialized transducing lambda derivatives, deletion mapping, and Plkc transductional crosses have been used to analyze the genetic organization and regulation of the srl genes. Transducing phages obtained from a secondary site lambda insertion in srlA are of two types: lambdapsrlC1 and lambdaprecA are substituted in the b2 region of the lambda chromosome (galtype) and carry the srlC gene but not srlD; lambdapsrlD is substituted in the early region of the phage deoxyribonucleic acid (biotype) and carries the srlD gene but not srlC. The lambdapsrlC1 phage, which lysogenizes at attlambda, complements srlC mutants in trans, indicating that this gene codes for a diffusable positive regulatory element. The srl genes have been ordered relative to the cysC, recA, and alaS genes by two- and three-factor P1kc crosses. The order, cysC...srlD-srlA-srlC-recA-alaS, has been obtained. The srlA and srlD genes comprise an operon with srlD operator distal. From the secondary site lysogen, it has been possible to obtain deletion mutants of this region that are sensitive to ultraviolet light and are recombination deficient. Genetic evidence suggests that these deletions extend from srl into the recA gene.     

Initial-rate kinetics of human NMN-adenylyltransferases: substrate and metal ion specificity, inhibition by products and multisubstrate analogues, and isozyme contributions to NAD+ biosynthesis

Initial-rate and product inhibition studies revealed distinctive ordered ternary complex kinetic mechanisms, substrate specificities, and metal ion preferences for the three isozymes of human nicotinamide mononucleotide adenylyl-transferase (NMNAT, EC 2.7.7.1). ATP binds before NMN with nuclear isozyme NMNAT1 and Golgi apparatus NMNAT2, but the opposite order is observed with the mitochondrial isozyme NMNAT3. Only the latter utilizes ITP efficiently in place of ATP, and while NMNH conversion to NADH by NMNAT1 and NMNAT3 occurs at similar rates, conversion by NMNAT2 is much slower. These isozymes can also be discriminated by their action on tiazofurin monophosphate (TrMP), a metabolite of the antineoplastic prodrug tiazofurin. Our finding that TrMP is only a substrate with NMNAT1 and NMNAT3 reveals for the first time an organelle selectivity in the metabolism of this important drug. In search of additional ways to discriminate these isozymes, we synthesized and tested the P1-(nicotinamide/nicotinate-riboside-5')-Pn-(adenosine-5') dinucleotides Np3AD, Np4AD, and Nap4AD. In addition to being highly effective inhibitors, these multisubstrate geometric inhibitors gave inhibition patterns that are consistent with the aforementioned isozyme differences in substrate binding order. Distinctive differences in their substrate specificity and metal ion selectivity also permitted us to quantify individual isozyme contributions to NAD+ formation in human cell extracts.     

The origin of the high sensitivity of muscle fructose 1,6-bisphosphatase towards AMP

Adenosine 5'-monophosphate (AMP) inhibits muscle fructose 1,6-bisphosphatase (FBPase) about 44 times stronger than the liver isozyme. The key role in strong AMP binding to muscle isozyme play K20, T177 and Q179. Muscle FBPase which has been mutated towards the liver enzyme (K20E/T177M/Q179C) is inhibited by AMP about 26 times weaker than the wild-type muscle enzyme, but it binds the fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP), similarly to the wild-type liver enzyme. The reverse mutation of liver FBPase towards the muscle isozyme significantly increases the affinity of the mutant to TNP-AMP. High affinity to the inhibitor but low sensitivity to AMP of the liver triple mutant suggest differences between the isozymes in the mechanism of allosteric signal transmission.     

Genetic mapping of tomato cDNA clones encoding the chloroplastic and the cytosolic isozymes of superoxide dismutase

The isozyme pattern of superoxide dismutase (SOD) in tomato consists of two Cu,Zn isozymes located, respectively, in the chloroplast and in the cytosol, as well as additional isozymes of the Mn or Fe SOD type. We have shown that SOD-1 is the chloroplastic Cu,Zn SOD and is related to cDNA clone T10. Restriction fragment length polymorphism (RFLP) analysis was performed with two cDNA clones representing tomato Cu,Zn-superoxide dismutases. T10, coding for the chloroplast isozyme, was thus mapped to chromosome 11, between marker TG46 and TG108, while clone P31, coding for the cytosolic Cu,Zn SOD isozyme, was mapped to chromosome 1 between TG24 and TG81. SOD is associated with the response of plants to various environmental stresses; the mapping information presented here would permit the demonstration of this association by genetic analysis.     

Conformationally-restricted arginine analogues as alternative substrates and inhibitors of nitric oxide synthases.

Conformationally restricted arginine analogues (1-5) were synthesized and found to be alternative substrates or inhibitors of the three isozymes of nitric oxide synthase (NOS). A comparison of k(cat)/Km values shows that (E)-3,4-didehydro-D,L-arginine (1) is a much better substrate than the corresponding (Z)-isomer (2) and 3-guanidino-D,L-phenylglycine (3), although none is as good a substrate as is arginine; 5-keto-D,L-arginine (4) is not a substrate, but is an inhibitor of the three isozymes. Therefore, it appears that arginine binds to all of the NOS isozymes in an extended (E-like) conformation. None of the compounds exhibits time-dependent inhibition of NOS, but they are competitive reversible inhibitors. Based on the earlier report that N(omega)-propyl-L-arginine is a highly selective nNOS inhibitor (Zhang, H. Q.; Fast, W.; Marletta, M.; Martasek, P.; Silverman, R. B. J. Med. Chem. 1997, 40, 3869), (E)-N(omega)-propyl-3,4-didehydro-D,L-arginine (5) was synthesized, but it was shown to be weakly potent and only a mildly selective inhibitor of NOS. Imposing conformational rigidity on an arginine backbone does not appear to be a favorable approach for selective NOS inhibition.     

Inhibitory effect of adenosine 3'',5''-phosphate on cell division of Escherichia coli K-12 mutant derivatives

Cell division of Escherichia coli K-12 strain PA3092 was inhibited by the addition of adenosine 3',5'-phosphate (cAMP), and the cellular morphology was changed from rods into filaments. Nucleoids in the filaments were regularly distributed and septum formation was perfectly inhibited. This inhibition of cell division by cAMP was reversed by the addition of guanosine 3',5'-monophosphate. To examine whether the inhibitory effect of cAMP on cell division in E. coli PA3092 was specific, its effect in several parental strains was investigated. Induction of cell filamentation by cAMP was observed in E. coli PA309 and P678, but not in E. coli W505, W1, Y10, or the wild-type strain. This result suggests that filamentation by cAMP in E. coli PA3092, PA309, and P678 was due to the mutagenesis by which E. coli P678 was derived from E. coli W595.     

AMP deaminase isozymes in human tissues

In human, there are four AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) isozymes: E1, E2, M and L. Chromatographic, electrophoretic and immunological studies showed the existence of isozymes E1 and E2 in erythrocytes, isozyme M in muscle and isozyme L in liver and brain. The tissues such as heart, kidney and spleen contained isozymes E1, E2 and L. Isozymes E1, M and L were isolated as apparently homogeneous preparations. The three isozymes were all tetramers composed of identical subunits, but differing slightly in molecular weight; isozyme E1 showed a subunit molecular weight of 80 000, isozyme M 72 000 and isozyme L 68 000. They were immunologically different from one another. The antisera precipitated only the corresponding enzyme and did not precipitate any other isozyme. The three isozymes were also different in kinetic and regulatory properties. Isozyme E2 was very similar to isozyme E1 in immunological and kinetic properties, although isozyme E2 could be separated from isozyme E1 by phosphocellulose chromatography, and zonal electrophoresis.     

Substrate specificity of collagenolytic proteases from the hepatopancreas of the of the Kamchatka crab]

The substrate specificity of two isozymes of collagenolytic protease of the crab (Paralithodes camtschatica) was studied. It was found that both proteases can effectively hydrolyze type I and III collagens, as well as gelatin, the set of products yielded by enzymatic hydrolysis being different for isozymes A and C. Hydrolysis of some well-known peptides revealed that isozyme A predominantly cleaves the peptide bonds containing arginine and lysine residues, whereas isozyme C predominantly hydrolyzes bonds containing hydrophobic amino acids. The catalytic constants for the hydrolysis of several low molecular weight substrates in the presence of P. camtschatica proteases were determined, which allowed to attribute isozyme A to trypsin-like, and isozyme C to chymotrypsin-like proteinases. The peptide substrates of collagenase, Pz-Pro-Leu-Gly-Pro-D-Arg and Z-Gly-Pro-Ala-Gly-Pro-Ala are not hydrolyzed isozymes of crab collagenolytic protease.     

Sodium dodecyl sulfate hypersensitivity of clpP and clpB mutants of Escherichia coli

We studied the hypersensitivity of clpP and clpB mutants of Escherichia coli to sodium dodecyl sulfate (SDS). Both wild-type E. coli MC4100 and lon mutants grew in the presence of 10% SDS, whereas isogenic clpP and clpB single mutants could not grow above 0.5% SDS and clpA and clpX single mutants could not grow above 5.0% SDS. For wild-type E. coli, cellular ClpP levels as determined by Western immunoblot analysis increased ca. sixfold as the levels of added SDS increased from 0 to 2%. Capsular colanic acid, measured as uronic acid, increased ca. sixfold as the levels of added SDS increased from 2 to 10%. Based on these findings, 3 of the 19 previously identified SDS shock proteins (M. Adamowicz, P. M. Kelley, and K. W. Nickerson, J. Bacteriol. 173:229-233, 1991) are tentatively identified as ClpP, ClpX, and ClpB.     

The DNA sequence of the sulfate activation locus from Escherichia coli K-12.

The DNA sequence of the sulfate activation locus from Escherichia coli K-12 has been determined. The sequence includes the structural genes encoding the enzymes ATP sulfurylase (cysD and cysN) and APS kinase (cysC) which catalyze the synthesis of activated sulfate. These are the only genes known to reside in the sulfate activation operon. Consensus elements of the operon promoter were identified, and the start codons and open reading frames of the Cys polypeptides were determined. During this work, another gene, iap, was partially sequenced and mapped. The activity of ATP sulfurylase is stimulated by an intrinsic GTPase. Comparison of the primary sequences of CysN and Ef-Tu revealed that CysN has conserved many of the residues integral to the three-dimensional structure important for guanine nucleotide binding in Ef-Tu and RAS. nodP and nodQ, from Rhizobium meliloti, are essential for nodulation in leguminous plants. The Cys and Nod proteins are remarkably similar. NodP appears to be the smaller subunit of ATP sulfurylase. NodQ encodes homologues of both CysN and CysC; thus, these enzymes may be covalently associated in R. meliloti. The consensus GTP-binding sequences of NodQ and CysN are identical suggesting that NodQ encodes a regulatory GTPase.     

Isolation, characterization, and mapping of Escherichia coli mutants blocked in the synthesis of ornithine decarboxylase.

Several Escherichia coli K-12 mutants blocked in the synthesis of ornithine decarboxylase (OD) were isolated after transduction for serA+ in a strain (MA197) blocked in agmatine ureohydrolase (AUH) with a mutagenized phage lysate of P1. The new double-polyamine mutants were characterized by an unconditional polyamine dependence; either putrescine or spermidine was required for normal growth. The mutational block was varified by the demonstration of a virtual absence of OD activity in cellular extracts. The mutation, designated speC, was mapped by P1 transduction in several strains and was shown to have a cotransduction frequency of 17.2% with serA. Map order was established as serA speB speC metK. A derivative of one of the OD mutants having wild-type levels of AUH and blocked in OD was utilized along with an OD AUH mutant and an OD+ AUH strain to explore the phenomenon of "pathway selection" using growth rate as a parameter. Polyamine pool studies were carried out simultaneously. The results presented here support the hypothesis of pathway selection, implying a preferential utilization of exogenous arginine rather than endogenously produced arginine in polyamine biosynthesis.     

Enhancement of the catalytic properties of human carbonic anhydrase III by site-directed mutagenesis

Among the seven known isozymes of carbonic anhydrase in higher vertebrates, isozyme III is the least efficient in catalytic hydration of CO2 and the least susceptible to inhibition by sulfonamides. We have investigated the role of two basic residues near the active site of human carbonic anhydrase III (HCA III), lysine 64 and arginine 67, to determine whether they can account for some of the unique properties of this isozyme. Site-directed mutagenesis was used to replace these residues with histidine 64 and asparagine 67, the amino acids present at the corresponding positions of HCA II, the most efficient of the carbonic anhydrase isozymes. Catalysis by wild-type HCA III and mutants was determined from the initial velocity of hydration of CO2 at steady state by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and water at chemical equilibrium by mass spectrometry. We have shown that histidine 64 functions as a proton shuttle in carbonic anhydrase by substituting histidine for lysine 64 in HCA III. The enhanced CO2 hydration activity and pH profile of the resulting mutant support this role for histidine 64 in the catalytic mechanism and suggest an approach that may be useful in investigating the mechanistic roles of active-site residues in other isozyme groups. Replacing arginine 67 in HCA III by asparagine enhanced catalysis of CO2 hydration 3-fold compared with that of wild-type HCA III, and the pH profile of the resulting mutant was consistent with a proton transfer role for lysine 64. Neither replacement enhanced the weak inhibition of HCA III by acetazolamide or the catalytic hydrolysis of 4-nitrophenyl acetate.     

Human T lymphocyte cAMP-dependent protein kinase: subcellular distributions and activity ranges of type I and type II isozymes.

The role of the type I and type II protein kinase A isozymes in the regulation of human T lymphocyte immune effector functions has not been ascertained. To approach this question, we first characterized the distribution and enzyme activities of the type I and type II protein kinase A (PKA) isozymes in normal, human T lymphocytes. T cells possess both type I and type II isozymes with an activity ratio of 5.0:1 +/- 0.71 (mean +/- SD). The type I isozyme associates predominately with the plasma membrane whereas the type II isozyme localizes primarily to the cytosol. Analyses of isozyme activities demonstrated that T cells from approximately one-third of 16 healthy donors exhibited significantly higher type II isozyme activities (higher type II, type IIH) than the remaining donors (lower type II, type IIL) (mean = 605 +/- 75 pmol.min-1.mg protein-1, P less than 0.001). Scatchard analyses of [3H]cAMP binding in the cytosolic fraction demonstrated similar Kd values (type IIH, 1.1 x 10(-7) M; type IIL, 9.0 x 10(-8) M); however, the Bmax (maximal binding) of the type IIH was 400 fmol/mg protein compared to the Bmax of the type IIL of 126 fmol/mg protein. Scatchard analysis of [3H]cAMP binding to the type I isozyme associated with membrane fragments had a Kd of 5.6 x 10(-8) M and a Bmax of 283 fmol/mg protein. Eadie-Hofstee plots of type IIH and type IIL gave a Km and Vmax of 2.3 mg/ml and 1.5 nmol.mg-1.min-1, and 2.1 mg/ml and 1.6 nmol.mg-1.min-1, respectively. The 3.2-fold higher maximal binding of the type II isozyme in one-third of healthy donors may reflect a greater amount of isozyme protein. The compartmentalization of type I PKA isozyme to the plasma membrane and type II PKA isozyme to the cytosol may serve to localize the isozymes to their respective substrates in T lymphocytes.     

Purification and properties of neuraminidase isozymes in Arthrobacter ureafaciens mutant

An Arthrobacter ureafaciens mutant (M1057) capable of producing neuraminidase constitutively was isolated by NTG mutagenesis from A. ureafaciens KMS 3663. Four molecular species (L, M1, M2, and S) of neuraminidase isozymes were homogeneously purified from the mutant and parent strains by means of DEAE-cellulose, affinity chromatography, ammonium sulfate precipitation, chromatofocusing, and Ultrogel AcA44 gel filtration. The molecular weights of L, M1, M2, and S isozymes were shown to be approximately 88,000, 66,000, 66,000, and 52,000, respectively. The optimal pHs and Km values of these isozymes for N-acetylneuraminosyl-alpha,(2-6)-lactose were 4.5-5.5 and 0.6-0.8 mM. Neuraminidase L, M1, M2, and S were able to hydrolyze oligosaccharides, glycoproteins and gangliosides containing alpha,(2-3)-, alpha,(2-6)-, and alpha,(2-8)-linked N-acetylneuraminic acid. Among these isozymes isolated, isozyme S was most active on colominic acid.     

Acetohydroxy acid synthase activity from a mutation at ilvF in Escherichia coli K-12.

Examination of the ilvF locus at 54 min on the Escherichia coli K-12 chromosome revealed that it is a cryptic gene for expression of a valine-resistant acetohydroxy acid synthase (acetolactate synthase; EC 4.1.3.18) distinct from previously reported isozymes. A spontaneous mutation, ilvF663, yielded IlvF+ enzyme activity that was multivalently repressed by all three branched-chain amino acids, was completely insensitive to feedback inhibition, was highly stable at elevated temperatures, and expressed optimal activity at 50 degrees C. The IlvF+ enzyme activity was expressed in strains in which isozyme II was inactive because of the ilvG frameshift in the wild-type strain K-12 and isozymes I and III were inactivated by point mutations or deletions. Tn5 insertional mutagenesis yielded two IlvF- mutants, with the insertion in ilvF663 in each case. These observations suggest that the ilvF663 locus may be a coding region for a unique acetohydroxy acid synthase activity.