Thermal analysis of bacteriophage T4.

The process of bacteriophage T4 morphogenesis was studied using a heat leakage scanning calorimeter. Thermograms of defective mutant 49 (am NG727) in permissive and non-permissive cells of $\text{Escherichia coli}$ showed a difference in thermal properties between packaged and non-packaged DNA molecules. $\text{In vivo}$, non-packaged DNA carried out their thermal transition at 85°C, the same temperature as that of T4 DNA melting measured in the standard saline citrate buffer, while the packaged DNA gave a sharper peak at 87°C due to some interaction with the head shell structure. Empty head shells showed a sharp heat absorption peak at 89°C both $\text{in vivo}$ and $\text{in vitro}$, indicating the high degree of cooperativity in their conformational changes.     

Two forms of glutamine synthetase in free-living root-nodule bacteria.

Cell-free extracts of $\text{Rhizobium japonicum}$ 61A76 contain two forms of glutamine synthetase (EC 6.3.1.2) which can be easily separated by isoelectric focusing. The more acid form (pI = 5.4), like the enzyme from $\text{E. coli}$, is stable at 50° and catalyses an ADP-dependent transferase reaction, whose inhibition by excess Mg⁺⁺ can be relieved by snake venom phosphodiesterase. The more alkaline form (pI = 6.1) is labile at 50°, and catalyses and ADP-dependent transferase reaction which is strongly inhibited by Mg⁺⁺ regardless of phosphodiesterase treatment. We have also observed the two forms of the enzyme in a nitrogenaseless mutant of 61A76, and in other strains of rhizobia, but only the acid form in $\text{E. coli}$ W, $\text{A. vinelandii}$ OP, and $\text{K. pneumoniae}$ M 51A.     

Protease of adenovirus type 2. In vitro processing of core protein

Protease activity associated with temperature sensitive mutant ts3 of adenovirus type 2 was studied. This activity was induced only when ts3 was propagated at 33°C. By $\text{in vivo}$ and $\text{in vitro}$ experiments the enzyme was found to cleave main core polypeptide PVII to VII. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protease activity of ts3 was partially characterized. Phenylmethylsulfonyl fluoride (1mM) and SDS (0.5%) inhibited its activity completely. EDTA (10 mM) did not seem to inhibit its activity. Protease activity was completely abolished after 10 min. of incubation at 60°C. Autocatalytic cleavage of PVII to VII was not observed.     

Studies on the action of porphyrinogenic trace metals on the activity of hepatic uroporphyrinogen decarboxylase

Trace metals which produce experimental uroporphyrinuria in animals during prolonged exposure inhibit uroporphyrinogen (uro) decarboxylase in rat liver extracts $\text{in}\text{,}\text{vitro}$. Inhibitory effects are prevented by sulfhydryl reagents, suggesting metal binding to sulfhydryl groups of the enzyme as the likely mode of action. Mercury, the most potent of the metals tested with respect to sulfur binding kinetics, produces the greatest inhibition of enzyme activity. In contrast, iron, which is considered to play a role in the etiology of porphyria cutanea tarda (PCT) via inhibition of uro decarboxylase, did not inhibit the enzyme in the present test system, suggesting an indirect mode of action $\text{in}\text{,}\text{vivo}$. These results suggest that direct inhibition of uro decarboxylase underlies uroporphyrinuria produced by prolonged trace metal exposure. Experimental inhibition of uro decarboxylase by trace metals may serve as a model for studying metal-induced uroporphyrinuria and PCT in humans.     

Evidence for the regulation of pyridoxal 5′-phosphate formation in liver by pyridoxamine (pyridoxine) 5′-phosphate oxidase

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The K_i, 3 μM, is considerably lower than the K_m for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The K_i determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition $\text{in}\text{vivo}$ is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than K_i, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the $\text{in}\text{vivo}$ rate of pyridoxal 5′-phosphate formation.     

Regulation of guanylate cyclase activity during cytodifferentiation of Blastocladiella emersonii.

Levels of guanylate cyclase activity in extracts of the unicellular eukaryote $\text{Blastocladiella}$$\text{emersonii}$ differed by at least 100-fold at different stages of the cell cycle, paralleling changes in the cyclic GMP content of this organism (Proc. Natl. Acad. Sci. U.S.A. $\text{72}$, 442 (1975)). Extracts of vegetative cells lacked appreciable guanylate cyclase activity, whereas the specific activity of the enzyme in zoospore extracts was 2 nmol cyclic GMP synthesized/min/mg protein at 35°. Guanylate cyclase activity increased at least 50-fold during the period of zoospore formation when cyclic GMP begins to accumulate $\text{in}$$\text{vivo}$. Since actinomycin D or cycloheximide added at the beginning of this period blocked any increase in enzyme activity, it appears that $\text{de}$$\text{novo}$ synthesis of guanylate cyclase during sporulation is responsible for the accumulation of cyclic GMP that occurs at that time.     

Mechanism of suppression in Drosophila: II. Enzymatic discrimination of wild-type and suppressor tyrosine transfer RNA

Previous studies had shown that two principle forms of tyrosine transfer RNA of Drosophila melanogaster were present in wild-type adult flies but that the second form was virtually absent in a suppressor mutant, su(s)². Current results are at variance with the previous ones, in that the suppressor mutant has significant amounts of the second form of tRNA^Tyr. A second chromatography system for separating these forms of tRNA^Tyr is described, RPC-5, and is compared to the system used previously, RPC-2. Both systems indicate that wild-type flies contain the two forms of tRNA^Tyr in a ratio of $\text{40}\text{60}$, the suppressor mutant in a ratio of $\text{60}\text{40}$. The difference between current and previous results can be attributed to the procedures used in the preparation of the enzyme that is used as a source of tyrosyl-tRNA ligase. The enzyme activity can be separated into two fractions on DEAE-cellulose chromatography. With suppressor tRNA as substrate, one enzyme fraction charges both forms of tRNA^Tyr but the second enzyme fraction charges the first form preferentially or nearly exclusively in some cases, as was seen in the previous experiments. With wild-type tRNA as substrate both enzyme fractions charge both forms of tRNA^Tyr. Storage results in the loss of the enzyme's ability to discriminate against the second form of tRNA^Tyr from the suppressor mutant, while the enzymatic activity is retained. We postulate that the su(s)⁺ locus produces an enzyme that modifies the second isoacceptor of tRNA^Tyr and that, when such modification fails to occur (as in the su(s)² mutant), the tRNA is unable to accept tyrosine from one form of tyrosyl-tRNA ligase. How the discrimination against the second isoacceptor by the ligase may be important metabolically is not apparent.     

Crystallography of Bacillus subtilis levansucrase

Levansucrase, an exocellular enzyme, has been isolated from a high producer mutant, the BS5C4 constitutive strain, of Bacillus subtilis. Three crystalline forms have been obtained, all three belonging to the orthorhombic space group P2₁2₁2₁. The most suitable form for a three-dimensional structure investigation has cell dimensions, $\text{a = 68}\text{A}\text{?}$, $\text{b = 125}\text{A}\text{?}$, $\text{c = 54}\text{A}\text{?}$. There is one molecule in the asymmetric unit.     

Coenzyme A: fatty acid synthetase apoenzyme 4'-phosphopantetheine transferase in yeast

A mixture of two pantetheine-free mutant fatty acid synthetases was dissociated and recombined $\text{in}$$\text{vitro}$ to form a hybrid apoenzyme complex. $\text{In}$$\text{vivo}$ the corresponding $\text{Saccharomyces}$$\text{cerevisiae}$$\text{fas}$-mutants exhibit interallelic complementation when crossed with each other and the enzyme synthesized in the resulting diploid contains pantetheine and exhibits overall fatty acid synthetase activity. Accordingly, the hybrid apoenzyme formed $\text{in}$$\text{vitro}$ could be activated to holo-fatty acid synthetase when incubated with coenzyme A and a partially purified yeast cell extract. The enzyme coenzyme A: fatty acid synthetase apoenzyme 4′-phosphopantetheine transferase has thus been identified in yeast. Further studies on the mechanism of fatty acid synthetase holoenzyme formation will now be possible.     

Increased acridine orange uptake and lysosomal enzyme levels in rat liver after steroid administration

A steroid which stabilizes lysosomes $\text{in vitro}$ and a pyrogenic steroid which labilizes lysosomes $\text{in vitro}$ were compared with respect to their ability to modify lysosomal uptake and lysosomal enzyme levels $\text{in vivo}$. Cortisone acetate increased the uptake of acridine orange by rat liver lysosomes when the dye was administered by intrathoracic injection. The steroid increased and accelerated the uptake of acridine orange so that, in liver lysosomes from treated rats, the maximum uptake was double that of controls and was reached at 2h, whereas in controls the maximum uptake was at 4h after the injection of the dye. This large elevation of uptake is specific to the lysosomal fraction and is not seen in other subcellular fractions of rat liver. The specific activities of a lysosomal enzyme β-N-acetylglucosaminidase were increased in lysosomal fractions from cortisone acetate-treated rats. Etiocholanolone, a steroid which labilizes lysosome $\text{in vitro}$, similarly accelerated and increased acridine orange uptake by lysosomes but had little effect on lysosomal β-N-acetylglucosaminidase levels. Thus the ability of steroids to stabilize or labilize lysosomes $\text{in vitro}$ does not correlate with their effect on lysosomal uptake of injected substances $\text{in vivo}$, or with their ability to induce increased specific activities of lysosomal enzymes.     

Catalytic function of thymidylate synthase is confined to S phase due to its association with replitase

Catalytic activity of thymidylate synthase, as measured $\text{in}\text{,}\text{vivo}$, is tightly linked to S phase of the cell cycle in Chinese hamster embryo fibroblast cells. This activity, as measured $\text{in}\text{,}\text{vitro}$, is found in all parts of the cell cycle. Thymidylate synthase activity in nuclear (karyoplast) extracts increased as the cells progressed from $\text{G}{}_0\text{G}{}_1$ to S phase. This enzymatic activity in the nuclei of S phase cells is associated with the multienzyme complex (replitase) that also contained DNA polymerase and other enzymes of DNA replication and precursor synthesis. The degree of association of thymidylate synthase with replitase, which increased co-ordinately as the cells progressed from $\text{G}{}_0\text{G}{}_1$ phase to S phase, coincided strongly with the level of $\text{in}\text{,}\text{vivo}$ activity of the enzyme.     

Excision of gamma-ray damaged thymine by E. coli extracts is due to the 5'leads to 3' exonuclease associated with DNA polymerase I

Extracts of $\text{E. coli pol}$Aexl which contains a temperature sensitive 5′→3′ exonuclease function of polymerase I accomplish the selective excision of products of the 5,6-dihydroxy-dihydrothymine type from γ-irradiated DNA and OsO₄-oxidized polyd(A-T) at the permissive temperature (30°) but not at the nonpermissive temperature (42°). The 5′→3′ exonuclease activity of polymerase I, therefore, acts as a repair exonuclease in γ-ray excision repair.     

Multiple forms of starch branching enzyme of maize: Evidence for independent genetic control

Purification of starch branching enzymes from kernels of two nonlinked mutants of maize, $\text{sugary}$ and $\text{amylose-extender}$, showed the basis of the two mutations to be associated with branching enzymes I and IIb, respectively. Branching enzyme I from $\text{sugary}$ kernels purified as nonmutant branching enzyme I, but had an altered pattern of activity when amylose was used as a substrate. In addition to the typical fall in absorbance at high wavelengths (550–700 nm) of the amylose-iodine complex, branching of amylose by $\text{sugary}$ branching enzyme I caused an increase in absorbance at low wavelengths (400–550 nm). Branching enzyme IIb was undetected in extracts of $\text{amylose-extender}$ kernels, while branching enzymes I and IIa appeared unaltered. Low umprimed starch synthase activity was also observed in DEAE-cellulose fractions of $\text{amylose-extender}$ maize, but this activity was regenerated by the addition of any branching enzyme.     

Metabolism of progesterone in mouse vaginal tissue

The $\text{in vitro}$ and $\text{in vivo}$ metabolism of 1,2- ³H-progesterone was studied in estrogen-stimulated and control vaginae of ovariectomized mice. Employing two-dimensional thin-layer chromatography, gas-liquid chromatography and metabolite “trapping” techniques, the major and minor pathways for progesterone metabolism were determined $\text{in vitro}$ and shown to involve saturation of the Δ⁴-double bond to yield 5α-pregnane compounds and reduction of the C20 and C3 ketone groups to form 20α- and 3α- and 3β-hydroxy derivatives, respectively. The quantities of 20β-hydroxy metabolites and 5β-epimers that were detected were considered not to be significant. The major metabolites formed by untreated tissues following $\text{in vitro}$ incubation in the presence of both high (10^?6M) and low (10^?8M) progesterone concentrations were 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione. Although these two derivatives were also found in sizable quantities in estrogen-treated tissues, a marked increase (5-fold) in the rate of C20 ketone reduction at high progesterone concentrations (10^?6M) to yield 20α-hydroxy-4-pregnen-3-one was demonstrated. Following intravaginal administration of ³H-progesterone $\text{in vivo}$, only progesterone and 3α-hydroxy-5α-pregnan-20-one were retained in appreciable quantities through 2 hr, suggesting rapid loss of 20α-hydroxy-4-pregnen-3-one and the 5α-pregnanediols from this tissue under $\text{in vivo}$ conditions.     

Biosynthesis of 5-methylaminomethyl-2-thiouridylate. I. Isolation of a new tRNA-methylase specific for 5-methylaminomethyl-2-thiouridylate

A new enzyme, which catalyzes the transfer of a methyl group to tRNA to form 5-methylaminomethyl-2-thiouridylate, was isolated from $\text{E.}$$\text{coli}$ by a procedure including affinity chromatography. The purified enzyme was nearly homogeneous upon disc electrophoresis. Using methyl-deficient tRNA^Glu of $\text{E.}$$\text{coli}$ as substrate, the 5-methylaminomethyl-2-thiouridylate residue synthesized was mostly found in the anticodon loop, showing a coincidence of the modification site $\text{in}$$\text{vitro}$ with that $\text{in}$$\text{vivo}$.     

Esterification of arylhydroxylamines: Evidence for a specific gene product in mutagenesis

Characterization of a $\text{Salmonella}\text{typhmurium}$ mutant strain (TA98/1,8-DNP₆) resistant to the mutagenicity of nitrated polycyclic aromatic hydrocarbons (nitroarenes) revealed that it was also non-responsive to the mutagenic action of nitroso- and N-hydroxylaminoarenes. The mutant strain was fully sensitive to the mutagenic action of the corresponding hydroxamic acid ester. These results suggest that TA98/1,8-DNP₆ is deficient in a specific esterifying enzyme and that esterification of the penultimate mutagenic metabolites of nitro- and aminoarenes ($\text{e.g.}$, arylhydroxylamines) to form potent electrophiles is controlled by a specific gene.     

Catalytic inhibition of gamma-aminobutyric acid - alpha-ketoglutarate transaminase of bacterial origin by 4-aminohex-5-ynoic acid, a substrate analog.

γ-Aminobutyric acid-α-ketoglutarate transaminase from $\text{Pseudomonas fluorescens}$ is irreversibly inhibited by 4-aminohex-5-yhoic acid, a new structural analog of GABA. The fact that this inhibition requires the pyridoxal form of the holoenzyme, and the formation of a Michaelis complex is in support of a catalytic mechanism. The compound is also active $\text{in vitro}$ and $\text{in vivo}$ on the same enzyme from mammalian brain.     

Selective killing of Leishmania infected mouse macrophages by 6-methylpurine 2′-deoxyriboside

6-methylpurine 2′-deoxyriboside killed mouse macrophages infected with amastigotes of $\text{Leishmania donovani}$ and $\text{Leishmania mexicana}$, but did not affect the growth of non-parasitized cells. $\text{Leishmania}$ extracts cleaved the non-toxic 6-methylpurine 2′-deoxyriboside to 6-methylpurine, a potent adenine antimetabolite for mammalian cells. By eliminating macrophages latently infected with $\text{Leishmania donovani}$ amastigotes, 6-methylpurine 2′-deoxyriboside could augment the effects of leishmanicidal agents $\text{in vivo}$.     

Pyruvate and nitrogenase activity in cell-free extracts of the blue-green alga Anabaena cylindrica

When extracts of $\text{Anabaena cylindrica}$ are prepared in the absence of dithionite, they catalyze pyruvate-dependent acetylene reduction, a reaction not observable in assays containing dithionite. Ferredoxin and coenzyme-A, but not NADP and ferredoxin-NADP reductase, are required for maximal pyruvate-dependent activity. These acetylene-reducing extracts do not exhibit NADP-pyruvate dehydrogenase activity. However, pyruvate:ferredoxin oxidoreductase is present at levels of activity sufficient to support the $\text{in vitro}$ rate of pyruvate-supported acetylene reduction. These $\text{in vitro}$ data support earlier $\text{in vivo}$ evidence that pyruvate:ferredoxin oxidoreductase transfers electrons from pyruvate to nitrogenase in $\text{A. cylindrica}$.     

Characteristics of Methylobacillus flagellatum KT mutants, deficient for phosphoglucoisomerase, an enzyme of the ribulose monophosphate cycle of obligate methylotrophic bacteria

The activities of the key enzymes of ribulose monophosphate cycle for formaldehyde oxidation and assimilation were tested in crude extracts from temperature sensitive mutants of obligatemethylotroph M. flagellatum KT. Two mutants deficient in phosphoglucoisomerase activity were identified during this screening. Phosphoglucoisomerase of T525 pgi-1 mutant was active both at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. Complete inactivation of the enzyme at 42 degrees C occurred in 2 h in vitro, while in vivo incubation at nonpermissive temperature for more than 10 h was required for the enzyme inactivation. Phosphoglucoisomerase activity of T566 pgi-2 was 5-fold lower as compared with the one from the parent strain incubated at 30 degrees C. The enzyme was inactivated in 2 min. in crude extract at nonpermissive temperature.