Arginyl-tRNA-protein transferase in eucaryotic protists

Soluble extracts of $\text{Saccharomyces cerevisiae}$ and $\text{Blastocladiella emersonii}$ were found to catalyze the specific transfer of arginine from a mixture of [¹⁴C] aminoacyl-tRNAs into protein. Arginine transfer was stimulated by bovine serum albumin. Glu-Ala, Asp-Ala and cystinyl-$\text{bis}$-Ala inhibited incorporation into protein, whereas dipeptides with other NH₂-terminal residues linked to alanine did not. These results indicate the presence of an enzyme in eucaryotic protists with the same donor and acceptor specificity as mammalian arginyl-tRNA-protein transferase.     

Interferon induction of polyamine-dependent protein kinase activity in Ehrlich ascites tumor cells

Treatment of Ehrlich ascites tumor cell cultures $\text{in}\text{vitro}$ with interferon induces a protein kinase activity that is activated by the polyamines, spermidine and spermine. Putrescine antagonizes the activation. The protein kinase yields a phosphorylated endogenous polypeptide of M_r 68,000–70,000. The polyamine-dependent protein kinase activity cofractionates with a double-stranded RNA-dependent protein kinase activity during affinity chromatography on poly (I) ·poly (C) - agarose or by chromatography on phosphocellulose. The double-stranded RNA-dependent protein kinase also phosphorylates an endogenous polypeptide of M_r 68,000–70,000. Unsuccessful attempts to discriminate between these two protein kinase activities on the bases of their respective capacities to be activated by either double-stranded RNA or spermidine/spermine, suggest that a single protein kinase enzyme may be activated by these strikingly dissimilar modifiers.     

Phospholipase A2 inactivation of microsomal 17β-hydroxysteroid oxidoreductase: Rates of phospholipid hydrolysis and enzyme inactivation,effects of hydrolysis products and properties of the phospholipase A2-treated enzyme

Exposure of guinea pig liver microsomes to phospholipase A₂ resulted in the nearly complete loss of 17β-hydroxy-steroid oxidoreductase (17β-HSD) activity, the time course of which correlated with phospholipid hydrolysis and lysolecithin formation. Lysolecithin and unsaturated fatty acids added to microsomes also inactivated 17β-HSD indicating that they may contribute to the inactivation by phospholipase A₂.If exposure to lysolecithin and fatty acids was minimized by including serum albumin in the reaction mixture, phospholipids were rapidly hydrolyzed; but in this case the extent of 17β-HSD inactivation was less and the rate of loss was significantly slower. The data suggest that phospholipid hydrolysis $\text{per se}$ results in a destabilization of 17β-HSD resulting in the subsequent activity loss.The inactivation of 17β-HSD by lysolecithin and fatty acids has not been reported previously and is suggestive of a possible control mechanism $\text{in vivo}$.     

Bacillus subtilis aminopeptidase: Specificity toward amino acid amides,dipeptides, and oligopeptides

Bacillus subtilis aminopeptidase hydrolyzed amino acid amides with a specificity similar to that determined using amino acyl-β-naphthylamides, but at much greater catalytic rates. Neutral and basic amino acid amides were the best substrates. A series of Leu and Lys NH₂-terminal dipeptides hydrolyzed by Co²⁺-activated aminopeptidase showed that the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ ratios for the Lys substrates were fourfold greater than the corresponding Leu substrates and that catalytic differences reflected the identity of COOH terminal residues. Greatest catalytic rates were obtained when aromatic residues were in the COOH terminal position of the substrate (Trp, Tyr, Phe); but, significant hydrolysis was achieved when aliphatic residues were COOH-terminal in the dipeptide. The Co²⁺-activated enzyme would not hydrolyze peptide bonds composed of the imide nitrogen of Pro, thus, bradykinin was not a substrate. However, the Co²⁺-activated enzyme removed sequentially the first four residues from eledoisin-related peptide and the A chain of bovine insulin.     

Phosphorylation of tyrosine aminotransferase in vitro by cyclic nucleotide-dependent protein kinases

An intraperitoneal injection of either leucine (1.57 mg/g body wt) or valine (2 mg/g body wt) into newborn mice led to a rapid accumulation of inactive monoribosomes in their brains. $\text{In}\text{vitro}$ measurements of protein synthesis by the remaining active ribosomes in leucine-treated mice revealed that polypeptide chain elongation was also inhibited. When a mixture of the seven amino acids from the leucine transport system was injected (0.15 mg each amino acid/g body wt) following the valine or leucine treatment, brain monoribosomes did not accumulate and elongation rates in the leucine-treated mice were only slightly altered.     

Solubility of the intramitochondrially synthesized protein and other membrane proteins of rat liver mitochondria in acidic or neutral chloroform-methanol

Almost all protein species of submitochondrial particles from rat liver identified by SDS-polyacrylamide gel electrophoresis were extracted into acidic /2 mM/HCl/ chloroform:methanol /2:1, $\text{v}\text{v}$/, whereas a single protein /or lipoprotein/ with molecular weight of 9.000 was extracted into neutral chloroform-methanol mixture. Evidence for intramitochondrial synthesis of this hydrophobic protein in rat liver $\text{in vivo}$ is presented.     

Transport of [14C]Gly-Pro in a proline peptidase mutant of Salmonella typhimurium

The transport of [¹⁴C]Gly-Pro was examined using a mutant of Salmonella typhimurium (strain TN87) deficient in an X-Pro dipeptidase and an X-Pro-Y iminopeptidase. The dipeptide was taken up by one saturable transport system having a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of $\text{5.3 · 10}{}^{\mathrm{?7}}\text{M}$ and a $\text{V}$ of 1.4 nmol/mg dry wt cell per min. The uptake of Gly-Pro was not inhibited by amino acids or tripeptides and the transport system exhibited a rather broad side chain specificity for dipeptides. Dipeptides containing hydrophobic residues were the most potent inhibitors of this dipeptide transport system exhibiting $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values between 10^?8 and 10^?7 M. In contrast, dipeptides containing glycine residues were particularly weak inhibitors. Finally, Gly-Pro was found to be in the intact form inside the cell and was concentrated more than 1000-fold.     

Hydrolysis of Protein and Model Dipeptide Substrates by Attached and Nonattached Marine Pseudomonas sp. Strain NCIMB 2021

Rates of substrate hydrolysis by nonattached bacteria and by bacteria attached to particles derived from marine diatom frustules were estimated by using two substrates, a dipeptide analog and a protein. Adsorption of the two substrates onto the particles was also evaluated. Methyl-coumarinyl-amide-leucine (MCA-leucine) was used to estimate hydrolysis of dipeptides by measuring an increase in fluorescence as MCA-leucine was hydrolyzed to leucine and the fluorochrome methylcoumarin. To examine hydrolysis of a larger molecule, we prepared a radiolabeled protein by ¹⁴C-methylation of bovine serum albumin. The rate of protein hydrolysis in samples of particle-attached or nonattached bacteria was estimated by precipitating all nonhydrolyzed protein with cold trichloroacetic acid and then determining the trichloroacetic acid-soluble radiolabeled material, which represented methyl-¹⁴C-peptides and -amino acids. About 25% of the MCA-leucine adsorbed to the particles. MCA-leucine was hydrolyzed faster by nonattached than attached bacteria, which was probably related to its tendency to remain dissolved in the liquid phase. In contrast, almost 100% of the labeled protein adsorbed to the particles. Accordingly, protein was much less available to nonattached bacteria but was rapidly hydrolyzed by attached bacteria.     

Stimulation of ornithine decarboxylase synthesis in the rat thyroid

High titer antiserum to hepatic ornithine decarboxylase was prepared by employing enzyme·monospecific antibody complex as the immunizing antigen. This new antiserum preparation was successfully labeled with ¹²⁵I and was found to retain its specific immune properties. Iodinated antiserum was used to precipitate thyroid ornithine decarboxylase induced by a mixture of thyroid stimulating hormone and methyl xanthine in rat thyroids $\text{in vitro}$. ¹²⁵I-labeled antibody incorporation into the enzyme antibody complex after induction $\text{in vitro}$ showed an increase which paralleled the increase in enzymatic activity and thus suggested $\text{de novo}$ synthesis of thyroid enzyme protein.     

Aminopeptidases in the Acidic Fraction of the Yeast Autolysate

Two aminopeptidases, I and II, were found in the acidic fraction of the yeast autolysate, adsorbed on DEAE-cellose and DEAE-Sephadex A&50. Aminopeptidase I was purified as a single protein with a molecular weight of 200,000. The enzyme required Zn for its activity and hydrolyzed dipeptides, and a polypeptide (glucagon). It also hydrolyzed amides, naphthylamides and the p-nitroanilide of amino acids. The enzyme was strongly inhibited by sulfhydryl reagents. Aminopeptidase II seemed also to be a metal enzyme with a molecular weight of 34,000. The enzyme hydrolyzed the dipeptide and tetrapeptide but not leucine-p-nitroanilide.     

Estimation of DNA, RNA and 125 I- and 3 H-labelled DNA in the same sample

Estimation of DNA, RNA, and the specific activity of DNA after labelling with [³H]thymidine and/or [¹²⁵I]iodeoxyuridine has been accomplished using a recently developed procedure for the estimation of DNA with p-nitrophenylhydrazine (pNPH). Samples of the pNPH reaction mixture are used for RNA estimation by th orcinol procedure and for ¹²⁵I and tritium measurement. Correction for ¹²⁵I contribution to the tritium measurement in double labelling experiments is accomplished either by use of a simple calibration curve (for high ${}^3\text{H}{}^{125}\text{I}$ ratios) or by removal of ¹²⁵I by hydrolysis and precipitation as AgI; in the latter procedure the efficiency of removal of ¹²⁵I was greater than 99%.     

The substrate specificity of juvenile hormone esterase from Manduca sexta haemolymph

Haemolymph of $\text{Manduca sexta}$ fifth instar larvae contains a high esterase activity capable of hydrolyzing $\text{Hyalophora cecropia}$ C₁₈ juvenile hormone (JH). In an attempt to characterize the substrate specificity of the enzyme (s) involved, dilute haemolymph was incubated $\text{in vitro}$ with a series of JH-analogs. Ethyl esters with or without the 10,11-epoxide group were hydrolyzed readily but isopropyl esters and the (2Z)-isomer of JH were not affected. Indirect evidence was obtained for the hydrolysis of aziridine analogs of JH. Correlations of these results to biological activities are discussed.     

Esterase activity of rabbit pulmonary angiotensin converting enzyme

A series of depsipeptides have been synthesized and used to demonstrate the esterase activity of rabbit pulmonary angiotensin converting enzyme. Among the esters studied, Bz-Phe-OPhe-Ala was found to have the highest $\text{k}{}_{\mathrm{cat}}\text{K}{}_{\mathrm{m}}$ which is about $\text{1}\text{5}$ that of its exact peptide analog, Bz-Phe-Phe-Ala. Esters such as Bz-Gly-OGly-Phe, Bz-Gly-OPhe-Phe and Bz-Gly-OLeu-Ala were also hydrolyzed but at much lower rates. Normal Michaelis-Menten behavior is observed and the kinetic parameters obtained indicate that the esters and their peptide analogs bind to the enzyme equally well, but that peptides are hydrolyzed at much higher rates. Studies on the pH-rate profiles, chloride ion effect, inhibition and chemical modifications detect no mechanistic differences between ester and peptide hydrolysis.     

Mitochondrial heterogeneity in foetal and suckling rat liver: differential leucine incorporation into the proteins of two mitochondrial populations.

Iodination of intact mitochondria with ¹²⁵I results in the labeling of essentially one polypeptide with an approximate MW of 30 000. This polypeptide seems to be a component of the inner boundary membrane as it can not be removed from the mitochondria by procedures which destroy the outer membrane (e.g. incubation with digitonin). The amount of the radio-active label which can be bound to this polypeptide is determined by ADP, atractylate, and bongkrekate, components which act on the functional state and the position of the $\text{ADP}\text{ATP}$ carrier in the membrane. [1,2]     

Involvement of 50S ribosomal proteins L6 and L10 in the ribosome dependent GTPase activity of elongation factor G

CsCI-prepared 50S cores in the presence of groups of individual split proteins were tested for their capacity to support EF-G dependent GTP hydrolysis. The activity of cores prepared at 40 mM Mg²⁺ could be restored by adding $\text{L7}\text{L12}$ and L10 together, each in an amount of two copies per 50S particle, which abolishes the difference in activity between L7 and L12. In the range of 20-2 mM Mg²⁺, 50S cores lose the protein L6, which is also required for GTP hydrolysis. L10 cannot replace L6, or $\text{vice versa}$.     

Major intrinsic polypeptide of lens membrane: Biochemical and immunological characterization of the major cyanogen bromide fragment

A protein of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 has been shown to be the major component of eye-lens junctions, which are similar but not identical to the gap junctions of liver and other tissues. Cyanogen bromide cleavage of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide from bovine lenses yields a major fragment of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 15 000 (fragment 1). However, if the junctions are first treated with trypsin or carboxypeptidase Y, cyanogen bromide treatment yields a fragment of reduced molecular weight. Since protease treatment has been shown to cleave residues almost exclusively from the carboxy-terminal end of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide, it follows that fragment 1 represents the carboxy-terminal half of this molecule, part of which is exposed to proteolytic attack outside the membrane. This latter result is corroborated by the fact that antisera which recognize both the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide and fragment 1 fail to do so after preadsorption with intact membranes. In addition, comparative amino acid and partial sequence analyses of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide and fragment 1 indicate that fragment 1 is more hydrophilic in character, suggesting that much of the amino-terminal half of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide is buried within the lipid bilayer.     

Metabolic control of lactose entry in Escherichia coli

A general method has been developed for determining the rate of entry of lactose into cells of Escherichia coli that contain β-galactosidase. Lactose entry is measured by either the glucose or galactose released after lactose hydrolysis. Since lactose is hydrolyzed by β-galactosidase as soon as it enters the cell, this assay measures the activity of the lactose transport system with respect to the translocation step. Using assays of glucose release, lactose entry was studied in strain GN2, which does not phosphorylate glucose. Lactose entry was stimulated 3-fold when cells were also presented with readily metabolizable substrates. Entry of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ (ONPG) was only slightly elevated (1.5-fold) under the same conditions. The effects of arsenate treatment and anaerobiosis suggest that lactose entry may be limited by the need for reextrusion of protons which enter during H⁺/sugar cotransport. Entry of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ is less dependent on the need for proton reextrusion, probably because the stoichiometry of H⁺/substrate cotransport is greater for lactose than for ONPG.     

Detection of proteins which recognize and bind oriC sequences

Extracts from $\text{E.}$$\text{coli}$ capable of supporting $\text{in}$$\text{vitro}$$\text{oriC}$-dependent DNA replication have been examined with a protein blotting protocol to identify DNA-binding proteins. Four polypeptide chains with apparent affinity for $\text{oriC}$ DNA were detected.     

Resolution of the light-harvesting chlorophyll ab-protein of Vicia faba chloroplasts into two different chlorophyll-protein complexes

Thylakoids of Vicia faba chloroplasts disaggregated by sodium dodecyl sulfate were separated by means of different electrophoretic systems. Under the conditions of a high resolving gel system the chlorophyll containing zone previously termed chlorophyll-protein complex II or light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ was found to be inhomogeneous. It represents a mixture of two distinct chlorophyll-proteins characterized by different spectral properties and different apoproteins. One chlorophyll-protein exhibits a chlorophyll $\text{a}\text{b}$ ratio of 0.9 and is associated with polypeptides of 24 000 and 23 000 daltons. The 24 000 dalton band is proved to bind chlorophyll and has a light-harvesting function. The function of the 23 000 dalton band is unknown. The second chlorophyll-protein has a chlorophyll $\text{a}\text{b}$ ratio of 2.1 and an additional absorption maximum in the position of 637 nm. It is associated with only one polypeptide which has an apparent molecular weight of 23 000. The two 23 000 dalton polypeptides occurring in both complexes are not identical.