Specific Activity In Extracts Research Articles

Biochemical analysis of patients with mutations in MTHFD1 and a diagnosis of methylenetetrahydrofolate dehydrogenase 1 deficiency

MTHFD1 is a trifunctional protein containing 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylenetetrahydrofolate dehydrogenase activities. It is encoded by MTHFD1 and functions in the cytoplasmic folate cycle where it is involved in de novo purine synthesis, synthesis of thymidylate and remethylation of homocysteine to methionine. Since the first reported case of severe combined immunodeficiency resulting from MTHFD1 mutations, seven additional patients ascertained through molecular analysis have been reported with variable phenotypes, including megaloblastic anemia, atypical hemolytic uremic syndrome, hyperhomocysteinemia, microangiopathy, infections and autoimmune diseases. We determined the level of MTHFD1 expression and dehydrogenase specific activity in cell extracts from cultured fibroblasts of three previously reported patients, as well as a patient with megaloblastic anemia and recurrent infections with compound heterozygous MTHFD1 variants that were predicted to be deleterious. MTHFD1 protein expression determined by Western blotting in fibroblast extracts from three of the patients was markedly decreased compared to expression in wild type cells (between 4.8 and 14.3% of mean control values). MTHFD1 expression in the fourth patient was approximately 44% of mean control values. There was no detectable methylenetetrahydrofolate dehydrogenase specific activity in extracts from any of the four patients. This is the first measurement of MTHFD1 function in MTHFD1 deficient patients and confirms the previous molecular diagnoses.

Identification and function of the pdxY gene, which encodes a novel pyridoxal kinase involved in the salvage pathway of pyridoxal 5'-phosphate biosynthesis in Escherichia coli K-12.

pdrK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5'-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY+ overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette into pdxY and crossed the resulting pdxY::omegaKan(r) mutation into the bacterial chromosome of a pdrB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts of pdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6 vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show that pdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNA(Tyr) synthetase) in a multifunctional operon. pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in the tyrS-pdxY intercistronic region.

Glucosamine 6‐phosphate deaminase in normal human erythrocytes

In the course of an investigation of hexosamine catabolism in the human malaria parasite, Plasmodium falciparum, it became apparent that a basic understanding of the relevant enzymatic reactions in the host erythrocyte is lacking. To acquire the necessary basic knowledge, we have determined the activities of several enzymes involved in hexosamine metabolism in normal human red blood cells. In the present communication we report the results of studies of glucosamine 6-phosphate deaminase (GlcN6-P) using a newly developed sensitive radiometric assay. The mean specific activity in extracts of fresh erythrocytes assayed within 4h of collection was 14.7 nmol/h/mg protein, whereas preparations from older erythrocytes that had been stored at 4 degrees C for up to 4 weeks had a mean specific activity of 6.2 nmol/h/mg. Characterization of the deaminase by chromatofocusing gave a pI of 8.55. The enzyme was optimally active at pH 9.0 and had a Km of 41 microM. The metal chelators EDTA and EGTA were non-inhibitory; however, inhibition was observed in the presence of metal ions, especially Cu2+, Ni2+ and Zn2+. In addition, the deaminase was also inhibited by several sugar phosphates including the reaction product, fructose 6-phosphate.

Inorganic pyrophosphatase from bovine retinal rod outer segments.

Rod outer segments from bovine retina contain a higher level of intracellular inorganic pyrophosphatase (EC 3.6.1.1) activity than has been found in any other mammalian tissue; the specific activity in extracts of soluble outer segment proteins is more than 6-fold higher than in extracts from bovine liver and more than 24-fold higher than in skeletal muscle extracts. This high activity may be necessary to keep inorganic pyrophosphate concentrations low in the face of the high rates of pyrophosphate production that accompany the cGMP flux driving phototransduction. We have begun to explore the role of inorganic pyrophosphatase in photoreceptor cGMP metabolism by 1) studying the kinetic properties of this enzyme and its interactions with divalent metal ions and anionic inhibitors, 2) purifying it and studying its size and subunit composition, and 3) examining the effects of pyrophosphate on rod outer segment guanylyl cyclase. Km for magnesium pyrophosphate was 0.9-1.5 microM, and the purified enzyme hydrolyzed > 885 mumol of PPi min-1 mg-1. The enzyme appears to be a homodimer of 36-kilodalton subunits when analyzed by gel electrophoresis and density gradient centrifugation, implying that kcat = 10(3) s-1, and kcat/Km = 0.7-1 x 10(9) M-1 s-1. The enzyme was inhibited by Ca2+ at submicromolar levels: 28% inhibition was observed at 138 nM [Ca2+], and 53% inhibition at 700 nM [Ca2+]. Imidodiphosphate acted as a competitive inhibitor, with Ki = 1.2 microM, and fluoride inhibited half-maximally approximately 20 microM. Inhibition studies on rod outer segment guanylyl cyclase confirmed previous reports that pyrophosphate inhibits guanylyl cyclase, suggesting an essential role for inorganic pyrophosphatase in maintaining cGMP metabolism.

UV-B-Inducible and Temperature-Sensitive Photoreactivation of Cyclobutane Pyrimidine Dimers in Arabidopsis thaliana

Removal of cyclobutane pyrimidine dimers (CBPDs) in vivo from the DNA of UV-irradiated eight-leaf seedlings of Arabidopsis thaliana was rapid in the presence of visible light (half-life about 1 hour); removal of CBPDs in the dark, presumably via excision repair, was an order of magnitude slower. Extracts of plants contained significant photolyase in vitro, as assayed by restoration of transforming activity to UV-irradiated Escherichia coli plasmids; activity was maximal from four-leaf to 12-leaf stages. UV-B treatment of seedlings for 6 hours increased photolyase specific activity in extracts twofold. Arabidopsis photolyase was markedly temperature-sensitive, both in vitro (half-life at 30 degrees C about 12 minutes) and in vivo (half-life at 30 degrees C, 30 to 45 minutes). The wavelength dependency of the photoreactivation cross-section showed a broad peak at 375 to 400 nm, and is thus similar to that for maize pollen; it overlaps bacterial and yeast photolyase action spectra.

Functional methionine synthase deficiency (cblE and cblG): clinical and biochemical heterogeneity.

Functional methionine synthase deficiency is generally characterized by homocystinuria and hypomethioninemia in the absence of methylmalonic aciduria. Patients are divided into two classes, cblE and cblG, on the basis of complementation analysis. Presentation has usually been in the first 2 years of life, but one patient came to medical attention at age 21 years with symptoms initially diagnosed as multiple sclerosis. Common findings among 11 patients (4 with cblE and 7 with cblG) have included megaloblastic anemia (all patients) and various neurological deficits including developmental retardation (10 patients), cerebral atrophy (8 patients), hypotonia (7 patients), EEG abnormalities (6 patients), and nystagmus (5 patients). Hypertonia, seizures, blindness, and ataxia were less frequent. All patients have responded to therapy with cobalamin with resolution of anemia and biochemical abnormalities; neurological deficits resolved more slowly and in some cases incompletely. Hydroxycobalamin has been more effective than cyanocobalamin. Fibroblasts from patients with cblE (5 patients) and cblG (6 patients) all showed decreased intracellular levels of methylcobalamin (MeCbl) and decreased incorporation of label from 5-methyltetrahydrofolate into macromolecules, suggesting decreased activity of the MeCbl-dependent enzyme methionine synthase. Methionine synthase specific activity in extracts of all cblE fibroblasts was normal or near-normal under standard reducing conditions; synthase specific activity in extracts of 5 cblG patients was low but was high in a 6th patient measured in another laboratory. Thus, there is heterogeneity among patients with functional methionine synthase deficiency both in clinical presentation and in the results of biochemical studies of cultured cells.

Extracellular localization of chitinase in cucumber

The localization of systemically induced chitinase activity was examined in the second leaves of cucumber plants that had been infected on the first leaf with tobacco necrosis virus. Leaf pieces were infiltrated with a buffered sodium chloride solution, and the intercellular fluid was subsequently extracted by centrifugation. Chitinase had a much higher specific activity in extracts of the intercellular fluid than in leaf homogenates. The specific activity of chitinase in protoplasts obtained from the leaves was only 5% of that in tissue homogenates. These data indicate that most of the chitinase activity is located in the extracellular space.

Two chitin synthases in Saccharomyces cerevisiae.

Disruption of the yeast CHS1 gene, which encodes trypsin-activable chitin synthase I, yielded strains that apparently lacked chitin synthase activity in vitro, yet contained normal levels of chitin (Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L., and Robbins, P. W. (1986) Cell 46, 213-225). It is shown here that disrupted (chs1 :: URA3) strains have a particulate chitin synthetic activity, chitin synthase II, and that wild type strains, in addition to chitin synthase I, have this second activity. Chitin synthase II is measured in wild type strains without preincubation with trypsin, the condition under which highest chitin synthase II activities are obtained in extracts from the chs1 :: URA3 strain. Chitin synthase II, like chitin synthase I, uses UDP-GlcNAc as substrate and synthesizes alkali-insoluble chitin (with a chain length of about 170 residues). The enzymes are equally sensitive to the competitive inhibitor Polyoxin D. The two chitin synthases are distinct in their pH and temperature optima, and in their responses to trypsin, digitonin, N-acetyl-D-glucosamine, and Co2+. In contrast to the report by Sburlati and Cabib (Sburlati, A., and Cabib, E. (1986) Fed. Proc. 45, 1909), chitin synthase II activity in vitro is usually lowered on treatment with trypsin, indicating that chitin synthase II is not activated by proteolysis. Chitin synthase II shows highest specific activities in extracts from logarithmically growing cultures, whereas chitin synthase I, whether from growing or stationary phase cultures, is only measurable after trypsin treatment, and levels of the zymogen do not change. Chitin synthase I is not required for alpha-mating pheromone-induced chitin synthesis in MATa cells, yet levels of chitin synthase I zymogen double in alpha factor-treated cultures. Specific chitin synthase II activities do not change in pheromone-treated cultures. It is proposed that of yeast's two chitin synthases, chitin synthase II is responsible for chitin synthesis in vivo, whereas nonessential chitin synthase I, detectable in vitro only after trypsin treatment, may not normally be active in vivo.

Interaction of saccharin with hexitol metabolism by Streptococcus mutans.

Saccharin markedly decreased the sorbitol- and mannitol-dependent growth rate and growth yield of Streptococcus mutans NCTC 10449 and also decreased the amount of acid produced from these hexitols by the organism. In addition, saccharin increased both the lag phase when glucose-grown cells were placed in a medium which contained sorbitol or mannitol as the primary energy source and to also increase the diauxie portion of the growth-response curve when glucose-grown cells were placed in a medium which contained a mixture of glucose and sorbitol or mannitol as fermentable energy sources. One mechanism by which saccharin may exert its negative effect upon hexitol catabolism by S. mutans is through inhibiting the activity of two inducible NAD-dependent oxidoreductases, sorbitol-6-phosphate dehydrogenase and mannitol-1-phosphate dehydrogenase which are found at high specific activities in extracts obtained from sorbitol- or mannitol-adapted cells respectively. Saccharin exerted a negative effect upon the enzymes by competitively inhibiting their interaction with both their oxidized and reduced coenzymes, NAD and NADH. In contrast, the inhibitor had no effect on the interaction of the enzymes with their respective substrates, mannitol-1-phosphate and sorbitol-6-phosphate.

Ferric iron reductase of Rhodopseudomonas sphaeroides.

Ferric iron reductase activity was examined in the facultative photosynthetic bacterium Rhodopseudomonas sphaeroides. The specific activities of extracts from cells grown under phototrophic and aerobic conditions were similar and not affected by the concentration of iron in the growth media. The activity was resolved by ion-exchange column chromatography into two fractions, designated iron reductase A and iron reductase B, with molecular weights of 41,000 and 32,000, respectively. Both of these soluble cytoplasmic enzymes required the presence of flavin mononucleotide for activity and utilized NADH to reduce iron supplied as ferric citrate. Iron reductase B was responsible for the majority of activity in crude extracts and was purified 556-fold by conventional protein purification techniques. The apparent Km values of iron reductase B for NADH, Fe3+, and flavin mononucleotide were determined to be 18.2, 8.3, and 3.2 microM, respectively.

Pyruvate carboxylase activity in subacute necrotizing encephalopathy (Leigh's disease).

Leigh's disease is a heterogeneous group of disorders, in which clinical and biochemical features suggest abnormal pyruvate metabolism. In two patients with Leigh's disease, diagnosed according to rigorous clinical, radiographic, and histologic criteria, we tested the hypothesis that pyruvate carboxylase deficiency might be the primary etiology. Pyruvate carboxylase specific activities in extracts of cultured skin fibroblasts from both patients were in the normal range. These results, together with other evidence, suggest that isolated pyruvate carboxylase deficiency does not cause the Leigh's disease phenotype.

Short-term regulation of tyrosine hydroxylase in tonically-active and in tonically-inactive dopamine neurons: Effects of haloperidol and protein phosphorylation

Dopamine (DA)-containing neurons of retina were employed as an experimental model for studying the short-term regulation of tyrosine hydroxylase (TH) in tonically-active and tonically-inactive neurons. These DA-containing neurons are trans-synaptically activated by light. Two mechanisms have been observed in this system for regulation of TH activity. A short-term activation of TH that is characterized by a decreased apparent K m for pteridine cofactors occurs in response to rapid increases of neuronal activity. A second mechanism occurs in response to prolonged, tonic changes of neuronal activity and is characterized by changes of V max. Both the K m changes and V max changes represent changes of specific activity of TH rather than enzyme induction. To determine the effects of short-term increases of neuronal activity on TH in tonically-active and tonically-inactive neurons, the effects of acute administration of haloperidol were examined in rats that were continuously light-exposed or light-deprived for 4 days. Haloperidol increased TH activity in both light-exposed and light-deprived retinas. The drug elicited the same percent stimulation in both experimental conditions. However, because the basal activity of TH was higher in the light-exposed than the light-deprived retinas, the absolute increase of TH specific activity was greater in the light-exposed samples. The effect of protein phosphorylation on TH activity in extracts of chronically light-exposed or light-deprived retinas was also examined to determine if the differences in the response to haloperidol might be due to a difference in the amount of TH available for short-term activation. Phosphorylation by endogenous cyclic AMP-dependent protein kinase (APK) or by purified catalytic subunit of APK resulted in larger increases of TH specific activity in extracts of light-exposed retinas than in those of light-deprived retinas. As was observed for haloperidol-induced activation, the percent stimulation elicited by phosphorylation was similar in extracts of light-exposed and light-deprived retinas. These observations suggest that more enzyme is available for short-term activation in tonically-active neurons than in those that are tonically inactive. A hypothetical model is proposed in which TH exists in active and inactive forms, the ratio of which depends on the tonic level of neuronal activity.

ATP Synthesis in Cell-Free Extracts of Peanut (Arachis hypogaeaL.) Seeds

Cell-free extracts of peanut (Arachis hypogaea L., cv. Shulamit) seeds, incubated with various substrates, synthesized ATP. Significant synthesis occurred in the presence of AMP + PEP, NADH2 + PEP and NAD + PEP. When the activities were examined in extracts prepared with 0-3 M mannitol, the rates were 0-6, 0-1 and 0-04 nmol min-1 mg-1 protein, respectively. The activities under such conditions were linear with time up to 90 min incubation at 30 °C. In the presence of PEP + NADH2 there was a higher specific activity in extracts from non-dormant seeds than from dormant seeds. No such difference was found when PEP + AMP or NAD + PEP was used as the substrate. The temperature dependence of the activity showed a relatively high energy of activation (Ea) for AMP + PEP and a low one if NADH2 + PEP or NAD + PEP was used as substrate. In buffer extracts of seeds ATP was synthesized in the presence of the above-mentioned substrate combinations but the rate of activity exhibited a lag phase at the early time of incubation, after which higher rates of activities (as compared with mannitol extracts) were obtained. The activities were Co+-dependent, with a Km of about 0-7 mM. In the buffer extracts relatively high activities of adenylate kinase (EC 2.7.4.3 (AK) and pyruvate kinase (EC 2.7.1.50) (PK) were found. AK was stimulated by ethephon (ethylene). This effect is temperature-dependent and occurs in both directions: in the presence of ADP (->ATP + AMP) as well as if ATP + AMP is used as substrate to synthesize ADP. PK is Co+-dependent, and unaffected by ethephon. Both activities were stimulated by malonate.

Genetic and biomedical studies demonstrating a second gene coding for asparagine synthetase in Escherichia coli.

Genetic experiments have indicated that asparagine auxotrophs of Escherichias coli K-12 can be made asparagine prototrophs at either of two sites on the chromosome and that wild-type strains require both sites to be mutated to produce asparagine auxotrophy. The former asn locus is now called asnA, and the new gene is designated asnB. The asnB gene is located near gal.AsnA+ asnB and asnA asnB+ strains were constructed, and the asparagine synthetic reaction was characterized in extracts. These studies revealed that the asnA gene codes for the enzyme previously described (H. Cedar and J.H. Schwartz, J. Biol. Chem. 244: 4112-4121, 1969), whereas the asnB gene is involved in the production of an enzyme which differs from the one previously described in its specific activity in extracts, its stability at low and high temperatures, and its apparent ability to use either glutamine or ammonia as amide nitrogen donor. Physiological studies showed that either enzyme alone is sufficient to allow a maximal growth rate under conditions of asparagine limitation.

UMP pyrophosphorylase of Tetrahymena pyriformis: Partial purification and properties

UMP pyrophosphorylase (EC 2.4.2.9, UMP:pyrophosphate phosphoribosyltransferase) was purified approximately 85-fold from exponentially growing cells of Tetrahymena pyriformis GL-7. It was found to have a molecular weight of 36,000, and was active over a broad pH range, with an optimum at 7.5. The enzyme exhibited a temperature optimum at 40 °C, above which irreversible inactivation began to occur. The apparent K m values for uracil and phosphoribosyl pyrophosphate (PRPP) were 0.4 and 6.9 m, respectively. The pyrophosphorylase exhibited a pyrimidine base specificity for uracil, although 5-fluorouracil was utilized by the enzyme. Neither cytosine, orotic acid, nor 6-azauracil competed with uracil for the enzyme or inhibited the production of UMP from uracil and PRPP. Although most triphosphates had little effect on pyrophosphorylase activity, UTP and dUTP, each at a concentration of 1 m m, depressed UMP formation by 86 and 59%, respectively. Thus, UMP pyrophosphorylase may be sensitive to feedback inhibition by the product of the pathway it initiates. UMP pyrophosphorylase specific activity in extracts of Tetrahymena grown in a medium containing uracil as the sole pyrimidine source was threefold higher than that in extracts of cells grown on uridine or UMP.

Adenosine metabolism in phytohemagglutinin-stimulated human lymphocytes.

The association of a human genetic deficiency of adenosine deaminase activity with combined immunodeficiency prompted a study of the effects of adenosine and of inhibition of adenosine deaminase activity on human lymphocyte transformation and a detailed study of adenosine metabolism throughout phytohemagglutinin-induced blastogenesis. The adenosine deaminase inhibitor, coformycin, at a concentration that inhibited adenosine deaminase activity more than 95%, or 50 muM adenosine, did not prevent blastogenesis by criteria of morphology or thymidine incorporation into acid-precipitable material. The combination of coformycin and adenosine, however, substantially reduced both the viable cell count and the incorporation of thymidine into DNA in phytohemagglutinin-stimulated lymphocytes. Incubation of lymphocytes with phytohemagglutinin for 72 h produced a 12-fold increase in the rate of deamination and a 6-fold increase in phosphorylation of adenosine by intact lymphocytes. There was no change in the apparent affinity for adenosine with either deamination or phosphorylation. The increased rates of metabolism, apparent as early as 3 h after addition of mitogen, may be due to increased entry of the nucleoside into stimulated lymphocytes. Increased adenosine metabolism was not due to changes in total enzyme activity; after 72 h in culture, the ratios of specific activities in extracts of stimulated to unstimulated lymphocytes were essentially unchanged for adenosine kinase, 0.92, and decreased for adenosine deaminase, 0.44. As much as 38% of the initial lymphocyte adenosine deaminase activity accumulated extracellularly after a 72-h culture with phytohemagglutinin. In phytohemagglutinin-stimulated lymphocytes, the principal route of adenosine metabolism was phosphorylation at less than 5 muM adenosine, and deamination at concentrations greater than 5 muM. In unstimulated lymphocytes, deamination was the principal route of adenosine metabolism over the range of adenosine concentrations studied (0.5-250 muM). These studies demonstrate the dependence of both the unstimulated and stimulated lymphocyte on adenosine and may account for the observed sensitivity of mitogen-stimulated lymphocytes to the toxic effects of exogenously supplied adenosine in the presence of the adenosine deaminase inhibitor coformycin. A single case of immunodeficiency disease has been reported in association with purine nucleoside phosphorylase deficiency. The catabolism of guanosine was also found to be enhanced in stimulated normal lymphocytes; phosphorolysis of guanosine to guanine by intact lymphocytes increased six fold after 72-h culture with phytohemagglutinin. The specific activity of purine nucleoside phosphorylase in extracts, with guanosine as substrate, was essentially the same in stimulated and unstimulated lymphocytes after 72 h of culture.

The autotrophic growth of Micrococcus denitrificans on Methanol.

Ribulose bisphosphate carboxylase is present at a high specific activity in extracts of methanol-grown Microccus denitrificans. Enzymic and physiological evidence indicates that, during growth on methanol, the ribulose bisphosphate cycle is the route of carbon assimilation.

Inorganic pyrophosphatase of Streptococcus faecium F24.

The acid Co2+-activated and the alkaline Mg2+-activated inorganic pyrophosphatases of Streptococcus faecium F24 were studied by a variety of techniques to determine whether they are activities of two distinct proteins or of a single protein. Both enzyme activities were found to be cryptic and soluble. The specific activities of both enzymes increased coordinately 1.5- to 3-fold during exponential growth at 37C, and then decreased as the cells approached stationary phase. Similar shifts in specific activity did not occur upon growth at 30C. The specific activities in extracts of cells in stationary phase varied coordinately over a 2-fold range depending on nutritional conditions. Diethylaminoethyl cellulose (DEAE-cellulose) and Sephadex G-100 column chromatography and starch–gel electrophoresis did not resolve the two activities. Slight differences in the thermal inactivation kinetics of the two activities were observed. It was concluded that the two pyrophosphatase activities are most probably those of a single protein under different assay conditions.

Nitrogenase activity in cell-free extracts of the blue-green alga, Anabaena cylindrica.

Cell-free extracts with high nitrogenase activity were prepared by sonic oscillation and French press treatment from the blue-gree alga Anabaena cylindrica. Extracts were prepared from cells grown on a 95% N(2)-5% CO(2) gas mixture followed by a period of nitrogen starvation under an atmosphere of 95% argon-5% CO(2). No increase in the specific activity of extracts was achieved by breaking heterocysts. Activity (assayed by acetylene reduction) was found to be dependent on adenosine triphosphate (ATP), an ATP-generating system, and a low-potential reductant. Na(2)S(2)O(2) employed as reductant supports higher rates of nitrogenase activity than reduced ferredoxin. The activity is associated with a small-particle fraction that can be sedimented by ultracentrifugation. In contrast to the particulate nitrogenase of Azotobacter, which is stable in air, the A. cylindrica nitrogenase is an oxygen sensitive as nitrogenase prepared from anaerobic bacteria.

Biochemical and cytochemical evidence for the polar concentration of periplasmic enzymes in a "minicell" strain of Escherichia coli.

A number of "surface" enzymes of Escherichia coli (i.e., among those selectively released by osmotic shock) all displayed higher specific activities in extracts of minicells than in extracts of typical rod forms; these enzymes included alkaline phosphatase, cyclic phosphodiesterase, acid hexose monophosphatase, 5'-nucleotidase, and ribonuclease I. In addition, alkaline phosphatase, cyclic phosphodiesterase, and acid hexose monophosphatase were cytochemically localized to regions of minicell periplasm that resembled reactive polar enlargements of the periplasm in rod forms. In contrast, a number of "internal" cytoplasmic enzymes (inorganic pyrophosphatase, beta-galactosidase, glutamine synthetase, polynucleotide phosphorylase, and ribonuclease II) showed elevated or similar specific activities in extracts of rod forms versus extracts of minicells. A specific heat-labile inhibitor for 5'-nucleotidase, known to occur in the cytoplasm, also showed no enrichment in minicells. These findings indicate that the "surface" enzymes are segregated in vivo into the terminal minicell buds, possibly because these enzymes are concentrated in the polar enlargements of the periplasm in typical rod forms.