Tunicamycin inhibits glycosylation of precursor polyprotein encoded by env gene of Rauscher murine leukemia virus.

The molecular weight of the precursor polyprotein to the envelope proteins of Rauscher murine leukemia virus is reduced from 85,000 to 68,000 daltons when synthesized in the presence of tunicamycin, a specific inhibitor of the synthesis of oligosaccharides that attach to glycoproteins via asparagine residues. The unglycosylated precursor protein (Pr68^$\text{env}$) is synthesized at a rate comparable to that of the normal carbohydrate-containing envelope precursor (gPr85^$\text{env}$). Pr68^$\text{env}$ is not proteolytically processed and remains undegraded in the cell. Thus, most if not all of the carbohydrate content of gPr85^$\text{env}$ is N-linked, and glycosylation appears to be necessary for normal processing of precursor proteins into viral particles.     

Promotion of DNA renaturation by a non-DNA factor in crown call tumor DNA preparation.

A 5400-fold excess of tobacco crown gall tumor DNA increased the renaturation rate of $\text{Agrobacterium tumefaciens}$ DNA, whereas, the same excess of healthy plant DNA had no effect on the rate or kinetics of renaturation. Since deoxyribonuclease treatment of the tumor DNA did not remove its ability to accelerate renaturation, the tumor tissue contains a non-DNA factor that increases the rate of renaturation of $\text{A. tumefaciens}$ DNA.     

Inhibition of β-lactamase-I by 6-β-sulfonamidopenicillanic acid sulfones: Evidence for conformational change accompanying the inhibition process

A number of 6-β-sulfonamidopenicillanic acid sulfones were examined for their ability to inhibit Bacillus cereus$\text{569}\text{H}$ β-lactamase I. Among these, 6-β-trifluoromethane sulfonamidopenicillanic acid sulfone was found to be the most potent inhibitor, effecting rapid and irreversible inactivation of the enzyme. Optical rotatory dispersion and differential scanning calorimetry were employed to probe the possible conformational changes accompanying the inactivation of B. cereus$\text{569}\text{H}$ β-lactamase 1 by 6-β-trifluoromethane sulfonamidopenicillanic acid sulfone. Optical rotatory dispersion measurements indicated the presence of approximately 29 and 17% helical structure in the native and inactivated enzyme, respectively. Differential scanning calorimetry determinations revealed that the inactivated enzyme was less thermostable than the native β-lactamase. The temperatures of maximum heat absorption were 48.4(±0.5) and 57.4(±0.1)°C for the inactivated and the native enzyme, respectively. Extensive conformational changes accompanying the interaction of the enzyme with the inhibitor may be responsible for the irreversible loss in the catalytic activity.     

Determination of zinc by flameless atomic absorption spectrophotometry

The flameless atomic absorption method described here is a simple, rapid, accurate microtechnique for determining zinc in aqueous solutions, serum, or urine. It requires no sample pretreatment, only 1.0 μl of sample per determination, no correction for viscosity differences between sample and standard solutions, and is not subject to ionic or organic interference. The average recovery of added zinc in serum is 97.5% and in urine is 97.6%. The values obtained for serum (mean ± SD: 94.6 ± 11.0 μAg/100 ml; N = 25) and urine (range: $\text{sol}\text{600–1000 μg}\text{24}\text{hr}$; N = 4) are comparable to the values reported in the literature. The coefficient of variation was less than 5.0% in all cases. The qualitative concentration limit was $\text{0.009}\text{μg}\text{100}\text{ml}$. The techniques and instrumentation described are also applicable to a number of trace minerals of common interest.     

The stability of deoxycytidine photohydrates in the mononucleotide, oligodeoxynucleotides and DNA

The stability of deoxycytidine photohydrates was determined for deoxycytidylic acid and deoxycytidine residues in oligodeoxynucleotides by optical measurements and in native and denatured DNA by a chemical assay. The half lives at 20° in $\text{10}{}^{\mathrm{?2}}\text{M}$ tris buffer, pH 7.7, were 102 min. for the mononucleotide, 128 min. for dpApGpG, 152 min. for MeOdpTpCpA, 51 min. for denatured $\text{E}\text{.}\text{coli}\text{DNA and 58 min. for native}\text{E}\text{.}\text{coli}$ DNA (at pH 8.1). It is concluded that the stability of deoxycytidine photohydrates is sufficient that they cannot at present be dismissed as lesions of possible biological importance in ultraviolet irradiated cells.     

Evidence of the presence of a latent active site in the large subunit of rat kidney gamma-glutamyl transpeptidase.

γ-Glutamyl transpeptidase (EC 2.3.2.2) of rat kidney is composed of two nonidentical polypeptide chains, the small and large subunits. The active site of this enzyme has previously been shown to be located in the small subunit [Inoue, M., Horiuchi, S. &; Morino, Y. (1977) Eur, J. Biochem. $\text{73}$, 335–342; Tate, S. S. &; Meister, A. (1977) Proc. Natl. Acad. Sci. U.S.A. $\text{74}$, 931–935] The denaturation of this oligomeric enzyme in 6 M urea, followed by chromatography on a Sephadex G-150, resulted in the separation of the large and small subunits. The removal of urea gave rise to an enzymatically active preparation from the denatured large subunit. Under several renaturation conditions, the small subunit polypeptide chain did not exhibit the enzymatic activity. Upon incubation with 6-diazo-5-oxo-L-[1,2,3,4,5-¹⁴C]norleucine, an affinity label for γ-glutamyl transpeptidase, the renatured preparation of the large subunit was covalently labeled with the affinity label with concomitant loss of the enzymatic activity. When the native enzyme was inactivated by the ¹⁴C-affinity label, radioactivity was selectively incorporated into the small subunit. These findings indicate that the isolated large subunit possesses an active site which is masked in the native state of the enzyme.     

Flash-induced absorption changes of the primary donor of photosystem II at 820 nm in chloroplasts inhibited by low pH or tris-treatment

A comparative study is made, at 15 °C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O₂ evolution was inhibited by low pH or by Tris-treatment.At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 μs.When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 180 μs) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 120 μs). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible.In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 μs.     

Mediated transport of anions in band 3-phospholipid vesicles

Band 3 protein, extracted from human erythrocyte membranes by Triton X-100, was recombined with egg lecithin/cholesterol mixtures to form small unilamellar vesicles at a yield of 15–20%. These systems exhibited sulfate fluxes which were inhibitable by stilbene disulfonates and other inhibitors. Maximal inhibition could only be obtained when inhibitors were present at both membrane surfaces. Inhibitor constants $\text{I}{}_{50}$ were higher than in the native membrane. Quantitatively, transport function was retained at least 60%, as related to the amount of protein involved. Sulfate transport in the recombinates resembled transport in the native membrane with respect to temperature dependence ($\text{E}{}_{\mathrm{<mtext>a</mtext>}}\text{= 29?32}\text{kcal}\text{/}\text{mol}$), pH dependence between pH 6.5 and 7.8, and the relationship between net and exchange fluxes. In contrast to the native cell, concentration dependence was linear up to 80 mM sulfate, which may be indicative of a lowered affinity for the substrate. Lactate transport in these systems, although substantial, was insensitive to stilbene disulfonates as well as to mercurials, indicating that band 3 is not involved in the specific monocarboxylate transfer in the erythrocyte. Anion transport in band 3-lipid recombinates was insensitive to cholesterol between 0 and 27 mol%. Treatment with proteases, while not affecting transport per se, abolished sensitivity to stilbene disulfonate inhibitors. These observations indicate a number of disturbances of band 3 after recombination, in spite of a preservation of the major transport properties.     

The interaction of chloride ions with human hemoglobin

The immobilization of glucose oxidase, a glycoenzyme from $\text{Aspergillus}$$\text{niger}$ consisting of 16% carbohydrate, has been achieved by oxidizing its carbohydrate residues with periodic acid followed by coupling the activated enzyme to water-insoluble $\text{p}$-aminostyrene. At pH 5.6 and 25°, approximately 60% of the carbohydrate residues are oxidized, but the enzyme retains full activity. No oxidation of any amino acid residue is evident. The enzyme-polymer conjugate derived from this activated enzyme retains full activity and even shows a slightly enhanced thermal stability at 60° compared with the soluble native and oxidized glucose oxidases.     

Autoxidation of native oxymyoglobin from bovine heart muscle

A method is described for the preparation of native oxymyoglobin from bovine heart muscle. The aqueous extract is gel filtered on Sephadex G-50 to isolate myoglobin from hemoglobin. Native oxymyoglobin is then separated from metmyoglobin by DEAE-cellulose chromatography.There is a marked effect of temperature on the autoxidation of native oxymyoglobin to metmyoglobin, with Q₁₀ values approximating 5.3 over the pH range of 5–10. The activation energies over this pH range are shown to be almost constant, i.e., 26.5 kcal·mole^?1.In contrast to the suggestions in earlier reports, the autoxidation rate of native oxymyoglobin estimated at physiological pH and temperature is quite high with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≤ 1.5}$ days under air saturation. This suggests the existence of an in vivo system(s) immediately reducing metmyoglobin formed to the ferrous state.     

"Super-reduction" of chromatium high-potential iron-sulphur protein in the presence of dimethyl sulphoxide

In solutions containing more than 70% dimethyl sulphoxide (DMSO) by volume, the normal reduced state of $\text{Chromatium}$ HIPIP can be further reduced by dithionite to give a form with a decreased absorption around 400 nm and an EPR signal similar to those of the reduced ferredoxins. These changes are reversible and the native HIPIP is recovered on removal of DMSO. These observations favour the hypothesis that the four-iron cluster in ferredoxins and HIPIP can exist in three oxidation states.     

Comparison of sequence homology of poly(A) and non-poly(A) containing 34S RNA of AKR murine leukemia virus.

AKR MuLV 70S RNA was separated on Poly(U)-Sepharose into poly(A) and non-poly(A) containing 34S subunits. The ratio of the two fractions was 2:1, respectively. Both fractions were hybridized to AKR MuLV [³H]cDNA, and the hybrids were assayed by nuclease S₁ and cesium sulfate centrifugation. The poly(A) and non-poly(A) subunits hybridized to [³H]cDNA to the same extent (80%), with identical $\text{CO}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values; and the hybrids of both fractions had identical T_m values (81°C in 0.15 M NaCl). These results demonstrate that the poly(A) and non-poly(A) containing subunits of the AKR genome have identical or very similar base sequences in the heteropolymeric regions.     

A novel subunit separation procedure for cryptomonad phycoerythrin 545.

A method to separate phycoerythrin 545, isolated from the cryptomonad alga, $\text{Rhodomonas}\text{lens}$, into two subunits has been developed. The method uses no denaturants (urea, guanidine, detergent) but relies on dissociation of the dimeric protein and subsequent aggregation of the β subunit at pH 3.0. The absorption spectra and amino acid composition of the subunits are presented. The spectra of the α subunit was red-shifted relative to β in both pH 3.0 buffer and in acidic 8.0 M urea.     

The mechanism of anion inhibition of pork heart aspartate aminotransferase

The mechanism of anion inhibition of the reaction of the pork heart extramitochondrial aspartate aminotransferase (EC 2.6.1.1) with erythro-β-hydroxy-l-aspartate was investigated. This reaction produces a mixture of complexes, one of which is characterized by an absorption maximum at 492 nm. Spectrophotometric analysis of equilibrium mixtures of aspartate aminotransferase and erythro-β-hydroxy-l-aspartate, in different buffers, indicated that acetate, chloride, and cacodylate were competitive inhibitors of hydroxyaspartate binding. Pyrophosphate, however, was not a competitive inhibitor. Between pH 4.5 and 9.0 the affinity of the enzyme for the monovalent anions decreased as the pH increased. The data indicated that the anion binding group had a pK_a in the range from pH 6 to 7, depending upon the anion studied. From pH 4.5 to 9.0, the substrate dissociation constant and the distribution of enzyme-substrate complexes were both unaffected by pH. By stopped-flow spectrophotometry, an initial rapid relaxation ($\text{t}\text{1}\text{2}\text{= 2–8}\text{ms}$) was associated with an increase in absorbance at 492 nm, and this rate depended upon both substrate and buffer concentrations. A slower relaxation ($\text{t}\text{1}\text{2}\text{= 180}\text{ms}$) was associated with a decrease in the absorbance at 492 nm to approximately 70% of the value attained in the first rapid reaction. The rate of this slower reaction was largely independent of substrate and buffer concentrations. Kinetic analysis of the rates of the first relaxation in several different concentrations of Tris-acetate buffer of pH 8 showed that the rate of association decreased with increasing acetate concentration whereas the reaction rate for dissociation was unaffected. Thus, acetate appears to exert its inhibitory effect by preventing the formation of the enzyme-substrate complex rather than by displacing the substrate from the enzyme.     

Selectivity of oxidase and reductase activity of horse heart cytochrome c

The ascorbate-TMPD-cytochrome $\text{c}$ oxidase and succinate cytochrome $\text{c}$ reductase activities and the redox potentials of native and chemically modified cytochromes $\text{c}\text{—NBS-cytochrome}\text{c}$ with modification of Trp-59 and Met-65, nitro-cytochrome $\text{c}$ with modification of Tyr-67, and a new preparation, Chloramine-T-cytochrome m$\text{c}$ with modification of Met-80 and -65 to methionine sulfoxide—have been compared at pH 7.8 in 25 mM cacodylate-Tris buffer. These modifications exhibit (i) a slight lowering of redox potential, from 260 mV to 180, 215 and 170 mV, respectively, (ii) destabilization of the cytochrome $\text{c}$-reductase complex, 6 to 12 fold, but without alteration of the cytochrome $\text{c}$-oxidase complex, and (iii) a slight lowering of the maximum velocity for both the oxidase and reductase reactions. The selective destabilization of the cytochrome $\text{c}$-reductase complex is interpreted as an indication of a two-path, two-function model for the oxido-reduction function of cytochrome $\text{c}$.     

Effect of pH, carbamylation and other hemoglobins on deoxyhemoglobin S aggregation inside intact erythrocytes as detected by proton relaxation rate measurements.

Measurement of the transverse water proton relaxation rate has been used to study the effect of pH, carbamylation, and other hemoglobins on the aggregation of deoxyhemoglobin S inside intact erythrocytes. Upon complete deoxygenation, cyanate-treated ($\text{S}\text{S}$) erythrocytes and erythrocytes heterozygous with respect to hemoglobin S ($\text{A}\text{S}$, $\text{C}\text{S}$, and $\text{S}\text{D}$) have high transverse water proton relaxation rates very similar to the values obtained with homozygous ($\text{S}\text{S}$) erythrocytes. These results suggest extensive intermolecular interactions between deoxyhemoglobin S molecules and a resultant increase in the correlation time for the small fraction of “irrotationally bound” water. When the transverse relaxation rate in deoxygenated ($\text{S}\text{S}$) erythrocytes was measured as a function of pH, the maximum rate was observed between pH 7.0 and 7.5. Upon increasing the pH beyond this range the observed relaxation rate decreases as does the number of sickled cells. Upon decreasing the pH, the observed transverse relaxation rate also decreases but the ratio of values from $\text{deoxy}\text{oxy}$ ($\text{S}\text{S}$) erythrocytes remains in the normal range of 4–6 and the number of sickled cells does not change. Therefore, the deoxyhemoglobin S aggregate inside sickled erythrocytes, as observed by water proton relaxation rates, is not altered by carbamylation or by the presence of nongelling hemoglobins. In addition, the enhancement of the relaxation rates as a function of pH is consistent with the number of sickled forms observed.     

Effects of temperature,lipid modification and pH on the mobility of the major proteins of the receptor-rich membranes from Torpedo marmorata

The factors influencing the overall mobility of the major proteins of the acetylcholine receptor-rich membranes from Torpedo marmorata have been investigated by saturation transfer ESR spectroscopy and the lateral distribution of these proteins has been studied by electron microscopy. A spin-labelled derivative of maleimide, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (MSL), was used under various conditions of incubation, enabling us to attach it mainly to either an extrinsic protein of 43 kdaltons, or an intrinsic protein (40 kdaltons) bearing the α-toxin-binding site. (1) The direct reaction of MSL with the membrane fragments resulted in almost exclusive labelling of the 43 kdalton protein, an extrinsic protein located on the inner face of the receptor-rich membranes. (2) After the free SH groups were blocked with $\text{N-}\text{ethylmaleimide}$ and the disulfide bridges opened with the reducing agent dithiothreitol, MSL reacted with both the 40 and 43 kdalton proteins ($\text{6.0±0.6}\text{MSL}$ molecules per α-toxin-binding site). (3) After the latter labelling procedure membranes were exposed to pH 11, resulting in extraction of the 43 kdalton protein and leaving $\text{2.2 ± 0.4}\text{MSL}$ molecules per α-toxin-binding site; sodium dodecyl sulfate polyacrylamide gel electrophoresis performed with $\text{N-[}{}^{14}\text{C}\text{]}\text{ethylmaleimide}$ suggested that MSL was bound mainly to the 40 kdalton polypeptide chain of the acetylcholine receptor. The following conclusions were made with the native and alkaline-treated membranes: In the native membranes, saturation transfer ESR does not reveal any significant protein rotational diffusion (itrotational correlation time $\text{τ}{}_{\mathrm{c}}\text{> 1}\text{ms}$). Temperature variations and/or lipid modifications obtained by fusion of exogenous lipids and/or cholesterol exchange have little influence on the saturation transfer ESR spectra. Electron microscopy reveals that upon lipid addition, proteins remain in the form of clusters while areas depleted of proteins appear. On the other hand, alkaline treatment strikingly enhances the motion of the MSL-labelled proteins in the membrane ($\text{100 ? τ}{}_{\mathrm{c}}\text{? 120 μ}\text{s}$). Furthermore, the rotational diffusion of the MSL-labelled proteins (mainly the 40 kdalton protein) becomes sensitive to temperature, lipid composition and the lipid-to-protein ratio. Electron microscopy shows that alkaline extraction does not cause large reorganization of the acetylcholine receptor in the plane of the membrane. However, when phospholipids are added to pH 11 treated membranes, a dispersion of the receptor rosettes is observed. In contrast, cholesterol enrichment of the latter membranes induces clustering of the receptor and immobilization as judged by saturation transfer ESR. Upon reassociation of the pH 11 soluble proteins with the alkaline-treated membranes, the restriction of the acetylcholine receptor rotational mobility is also restored ($\text{τ}{}_{\mathrm{c}}\text{? 1}\text{ms}$).     

Alkali-labile oligosaccharides from bovine milk fat globule membrane clycoprotein

Phenol extraction of bovine milk fat globule membrane gave a glycoprotein fraction which, in sodium dodecyl sulphate electrophoresis, showed three major bands, all staining for both protein and carbohydrate. Alkaline borohydride treatment and desialylation of the glycoprotein fraction released the reduced disaccharide β-d-galactosyl(1 → 3)-$\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ (T-antigen), which was identified by gas chromatography using a standard. All of the disaccharide units in the native glycoprotein were shown to be substituted by sialic acid, and a tetrasaccharide containing the disaccharide plus two molecules of sialic acid was isolated following alkaline borohydride treatment of the glycoprotein and gel filtration. Periodate oxidation of native and desialylated glycoprotein, together with paper chromatography of alkali degraded oligosaccharide fragments, indicated that the major alkali-labile oligosaccharide of the glycoprotein fraction is a tetrasaccharide containing $\text{β-}\text{d}\text{-galactosyl(1 → 3)}\text{-N-}\text{acetyl-}\text{d}\text{-galactosamine}$ substituted by sialic acid at position C3 of the galactosyl and position C6 of the $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ residue. Evidence was also obtained for the presence of small amounts of unsubstituted alkali-labile $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ linked directly to protein in the native glycoprotein.Serological evidence using agglutinins from Vicia graminea, Arachis hypogoea and human anti-T serum confirmed the presence in the native glycoprotein of a sialic acid substituted T-antigen. Similar evidence using agglutinins from Helix pomatia and Cepaea hortensis also confirmed the presence of terminal alkali-labile $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ in the native glycoprotein.     

Preparation of pyridoxal 5'-phosphate-bound sepharose and its use for immobilization of tryptophanase

Pyridoxal 5′-phosphate-bound Sepharose (SP) was prepared by coupling pyridoxal 5′-phosphate (PLP) to diazotized p-aminobenzamidohexyl-Sepharose. A derivative of pyridoxine having an absorption maximum at $\text{ca.}$ 316 nm (possibly, 6-amino-pyridoxine 5′-phosphate) was liberated from SP by treatment with 0.1 M sodium dithionite at pH 9.0. SP catalyzed the cleavage of tryptophan in the presence of Cu²⁺, a typical non-enzymatic model of tryptophanase reaction. From the spectrophotometric data and catalytic activity, it was estimated that SP contained about 1.5 μmoles of bound PLP per gram of Sepharose. Tetrameric apotryptophanase was immobilized by incubation with SP, followed by reduction with NaBH₄. The resulting immobilized tryptophanase retained $\text{ca.}$ 60 % of the catalytic activity of free tryptophanase used. This method was much superior to other methods used commonly for preparation of immobilized enzymes.