Selectivity in Protein Degradation during Sporulation of Bacillus subtilis

The breakdown of cellular protein was investigated in Bacillus subtilis labeled with glycine-2-³H or L-phenylalanine-U-¹⁴C at different stages of vegetative growth and sporulation. In cells labeled with l-phenylalanine-U-¹⁴C, multiple protein turnover was observed. However, in cells labeled with glycine-2-³H, the patterns of protein turnover were quite different in the stages of growth and sporulation; proteins which were labeled at the early stationary phase were degraded rapidly, but those labeled at the late sporulation stage were hardly degraded. It was found that glycine incorporated into cells at the late sporulation stage was mainly utilized for biosynthesis of the spore coat protein. These data suggest that the spore coat protein which contains relatively large amounts of glycine is little subject to further degradation.     

A study of the dynamic state of histidine residues in tryptophan synthetase subunit by 13 C nuclear magnetic resonance

Tryptophan synthetase α subunit in which the histidine C₂ (ring) positions are enriched in ¹³C and labeled with deuterium was prepared by incorporation of labeled histidine into protein of Escherichia coli. ¹³C nuclear magnetic resonance studies of the specifically labeled enzyme demonstrate that all four histidine residues of α subunit are highly immobilized within the protein matrix.     

Interaction of a radiolabeled cytokinin photoaffinity probe with a receptor protein

A photoreactive analog of the cytokinin 6-benzylaminopurine was prepared by the method of Theiler $\text{et. al.}$ (1) modified so as to include a radioactive atom in the final product, [methylene-¹⁴C] 2-azido-6-benzylaminopurine. The affinity of this doubly labeled cytokinin probe for a previously described cytokinin receptor protein (2,3) is very nearly the same as for the parent cytokinin. The cytokinin probe was covalently incorporated into the receptor protein by irradiation with ultraviolet light, and its presence there was quantitatively established by assaying for non-dialyzable ¹⁴C. The labeled protein was subjected to SDS polyacrylamide gel electrophoresis and the subunits assayed for radioactivity by fluorography. Each of the four subunits of the receptor protein was labeled with ¹⁴C to some extent. The data suggest that all four subunits of the protein either actively participate in the formation of the cytokinin binding site or exist in close proximity to it.     

In vivo labeling of myelin lipids and proteolipid protein with [3H]myristate, [14C]linoleate,and [14C]linolenate

To investigate the incorporation of essential fatty acids into myelin components, 24-day-old rabbits were injected intracerebrally with [¹⁴C]linoleate, [¹⁴C]linolenate, or [³H]Myristate for comparison. Animals were killed 22 hr later and myelin was isolated. [³H]myristate labeled all myelin lipids including monogalactosyl diglyceride, with the exception of sulfatides. With¹⁴C-essential fatty acids, only glycerophospholipids were efficiently labeled and their specific activities were in the following decreasing orders: PC>PI>PE>PS with [¹⁴C]linoleate, and PE>PC>PI=PS with [¹⁴C]linolenate. Among myelin proteins, PLP and DM-20 were labeled with all 3 precursors. PLP was purified from myelin labeled with¹⁴C-essential fatty acids. The label was then cleaved from the protein by alkaline methanolysis and was identified as a dienoic ([¹⁴C]linoleate) or a tetraenoic ([¹⁴C]linolenate) fatty acid. MBP was not labeled with [³H]myristate, but was slightly labeled with both¹⁴C-essential fatty acids. The signification of the latter result is discussed.Abbreviations FA fatty acid(s) - HPTLC high-performance thin-layer chromatography - MBP myelin basic protein - PLP proteolipid protein - PC phosphatidylcholine - PE phosphatidylethanolamine and ethanolamine plasmalogens - PI phosphatidylinositol - PS phosphatidylserine - SDS sodium dodecylsulfate     

Comparative rates of protein synthesis of rat kidney medulla and cortex in vitro

The $\text{in}\text{vitro}$ rate of ¹⁴C-leucine incorporation into protein has been examined in rat kidney tissue. The presence of a marked gradient was observed. Thus, the white medulla was the most active in this respect followed by, in descending order, red medulla and cortex. ¹⁴C-Leucine incorporation into protein was completely abolished in the presence of cycloheximide. The distribution of labeled protein between the medium and slice suggests a high degree of cellular integrity and little secretion of labeled protein from slice to medium. The pattern of ¹⁴C-leucine incorporation amongst the different zones of kidney of hypophysectomized rats was similar to that noted in normal rats.     

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies¹. Since poliovirus is sensitive to conformational conversion² when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch^3,4 is the micro protein A-immunoprecipitation test⁵. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies^6,7. However, as another opportunity, these interesting and stable single-domain antibodies⁸ can be easily engineered with different tags. The widely used (His)₆-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with ³⁵S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.     

Cell-specific fatty acylation of proteins in cultured cells of neuronal and glial origin

Distinct sets of cellular proteins were labeled with [³H]myristic and [³H]palmitic acids in primary (rat neurons and astroglia) and continuous (murine N1E-115 neuroblastoma and rat C6 glioma) cell cultures derived from the nervous system. Both soluble and membrane proteins were modified by myristate in a hydroxylamine-stable (amide) linkage, while palmitoylated proteins were esterlinked and almost exclusively membrane bound. Chain elongation of both labeled fatty acids prior to acylation was observed, but no protein amide-liked [³H]myristate originating from [³H]palmitate was detected. Fatty acylation profiles differed considerably among most of the cell lines, except for rat astroglial and glioma cells in which myristoylated proteins appeared to be almost identical based on SDS gel electrophoresis. An unidentified 47 kDa myristoylated protein was labeled to a significantly greater extent in astroglial than in glioma cells; the expression of this protein could be related to transformation or development in cells of glial origin.     

CRP,immunologically cross-reacting protein of prolyl hydroxylase. Its role in assembly of active prolyl hydroxylase and cellular localization in L-929 fibroblasts

There are two forms of prolyl hydroxylase in L-929 flbroblasts. One is the enzymatically active tetramer having two α- and two β-subunits. The other is monomeric cross-reacting protein which is enzymatically inactive but is structurally related to β-subunit of the enzyme. Cultured L-929 fibroblasts at mid-log phase were labeled by ³H-labeled amino acid mixture and the radioactivity was chased for 24 h while cells were harvested and plated at higher cell densities in cultures. The results indicated that both α-subunit of the tetrameric prolyl hydroxylase and cross-reacting protein were labeled, but the β-subunit of the tetrameric active prolyl hydroxylase was not labeled until the cells were crowded for 24 h. Using immunofluorescent techniques with antibodies directed against pure tetrameric prolyl hydroxylase, capping or patching was observed when the cells were incubated at 37 °C. Also, it was found that phagosomes prepared from L-929 flbroblasts contained about 30% of total enzyme protein as determined immunologically but contained no significant prolyl hydroxylase activities. Labeling cells with ¹²⁵I by lactoperoxidase, cross-reacting protein was labeled but both α- and β-subunits of tetrameric active prolyl hydroxylase were not labeled. The results indicate that cross-reacting protein can be utilized as the precursor of β-subunit by the cells to form tetrameric active prolyl hydroxylase and that cross-reacting protein is found associated with cytoplasmic membranes.     

The Phosphorylation of Ribosomal Protein in Lemna minor

Sterile cultures of Lemna minor have been labeled with ³²P₁, and the ribosomal proteins have been examined for radioactivity. In relatively short term labeling a radioactive protein was found which ran as a single component in both urea/acetic acid and sodium lauryl sulfate gel electrophoresis. Acid hydrolysis of the labeled protein permitted the isolation of serine phosphate. After labeling to equilibrium with ³²P₁, calculation indicated only 0.6 to 0.75 atom of this protein phosphorus per ribosome.     

Reversal of axonal transport at a nerve crush.

Abstract— —We have compared retrograde axonal transport of ³H-labeled protein in normal rat motor and sensory axons, and axons which were injured by a distal ligation of the sciatic nerve. After injection of L-[³H]leucine into the vicinity of the neuron cell bodies, labeled protein was transported into the axons. A premature return of protein towards the cell bodies occurred in the injured axons, which we interpret as a reversal of axonal transport occurring at the site of injury. We estimate that reversal of transport occurred within 1.9–2.4 h of the arrival of labeled protein at the injury, and that the minimum velocity of the subsequent retrograde transport was 112–133 mm day^?1. The ability of the injured axons to reverse transport developed about 0.8 h after making the injury. A large fraction of the orthograde transported protein was returned towards the cell body: it is estimated that by 28 h after labeled protein in sensory axons reached the injury, 46% of the³H-labeled protein originally transported to the injury site had been returned. In intact sensory nerves at this time only 15% of the transported protein had returned. It is suggested that axonal injury produces a sudden increase in the return of newly synthesized protein to the cell body, and that this might serve as a signal for chromatolysis.     

Biosynthetic site-specific <Superscript>13</Superscript> C labeling of the light-harvesting 2 protein complex: A model for solid state NMR structure determination of transmembrane proteins

Partly biosynthetic site-directed isotopically ¹³C enriched photosynthetic light-harvesting 2(LH2) complexes have been prepared from Rhodopseudomonas acidophila strain 10050 by using chemically labeled [1,2,3,4–¹³C], [1,4–¹³C] and [2,3–¹³C] succinic acid as a precursor in the growth medium. Two-dimensional proton driven spin diffusion (PDSD) solid state NMR correlation spectroscopy has been used to trace each individual ¹³C isotope from the labeled succinic acid precursor to its destination into the protein and into the embedded major light-absorbing bacteriochlorophyll cofactors. For both the residues of the protein and for the cofactors distinct labeling patterns have been deduced, for protein complexes prepared from [1,4–¹³C]-succinic acid or [2,3–¹³C]-succinic labeled media. All residues, except isoleucine and leucine, have been labeled almost homogeneously by the succinic acid precursor. Carbonyl carbons in the protein backbone were labeled by [1,4–¹³C]-succinic acid, while the C and C carbons of the residues were labeled by [2,3 ¹³C]-succinic acid. Leucine and isoleucine residues were labeled using a uniformly labeled amino acid mixture in the medium. The pattern labeling yields an increase of the resolution and less spectral crowding. The partial labeling technique in combination with conventional solid state NMR methods at ultra high magnetic fields provides an attractive route to resolve chemical shifts for -helical transmembrane protein structures.     

Sequential protein expression and selective labeling for in-cell NMR in human cells

BackgroundIn-cell NMR is a powerful technique to investigate proteins in living human cells at atomic resolution. Ideally, when studying functional processes involving protein–protein interactions by NMR, only one partner should be isotopically labeled. Here we show that constitutive and transient protein expression can be combined with protein silencing to obtain selective protein labeling in human cells.MethodsWe established a human cell line stably overexpressing the copper binding protein HAH1. A second protein (human superoxide dismutase 1, SOD1) was overexpressed by transient transfection and isotopically labeled. A silencing vector containing shRNA sequences against the HAH1 gene was used to decrease the rate of HAH1 synthesis during the expression of SOD1. The levels of HAH1 mRNA and protein were measured as a function of time following transfection by RT-PCR and Western Blot, and the final cell samples were analyzed by in-cell NMR.ResultsSOD1 was ectopically expressed and labeled in a time window during which HAH1 biosynthesis was strongly decreased by shRNA, thus preventing its labeling. In-cell NMR spectra confirmed that, while both proteins were present, only SOD1 was selectively labeled and could be detected by ¹H–¹⁵N heteronuclear NMR.Conclusions and general significanceWe showed that controlling protein expression by specifically silencing a stably expressed protein is a useful strategy to obtain selective isotope labeling of only one protein. This approach relies on established techniques thus permitting the investigation of protein–protein interactions by NMR in human cells.     

Optimization of amino acid type-specific <Superscript>13</Superscript>C and <Superscript>15</Superscript>N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm

We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273–6279 (1982)), types of amino acids are labeled with ¹³C or/and ¹⁵N such that cross peaks between ¹³CO(i – 1) and ¹⁵NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with ¹³C and the second with ¹⁵N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B₂R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin.     

A rapid sensitive collagenase assay.

A rapid, sensitive collagenase assay has been developed using¹⁴C-acetylated collagen as a substrate. Acid-soluble calfskin collagen was labeled with [1-¹⁴C]acetic anhydride at pH 8. The acetylated collagen had a specific activity of 6.25 × 10⁵ dpm/mg protein. Collagen was not denatured as evidenced by its resistance to nonspecific proteolysis and sensitivity to bacterial collagenase. Polyacrylamide gel electrophoresis of the acetylated protein showed that the radioactivity was present in the three bands corresponding to the α, β, and γ components of collagen. The rate of release of ¹⁴C from labeled collagen by Clostridium histolyticum collagenase was proportional to enzyme and substrate concentration.     

Dark CO(2) Fixation and its Role in the Growth of Plant Tissue

Experiments were designed to determine the significance of dark CO₂ fixation in excised maize roots, carrot slices and excised tomato roots grown in tissue culture. Bicarbonate-¹⁴C was used to determine the pathway and amounts of CO₂ fixation, while leucine-¹⁴C was used to estimate protein synthesis in tissues aerated with various levels of CO₂.

Organic acids were labeled from bicarbonate-¹⁴C, with malate being the major labeled acid. Only glutamate and aspartate were labeled in the amino acid fraction and these 2 amino acids comprised over 90% of the ¹⁴C label in the ethanol-water insoluble residue.

Studies with leucine-¹⁴C as an indicator of protein synthesis in carrot slices and tomato roots showed that those tissues aerated with air incorporated 33% more leucine-¹⁴C into protein than those aerated with CO₂-free air. Growth of excised tomato roots aerated with air was 50% more than growth of tissue aerated with CO₂-free air. These studies are consistent with the suggestion that dark fixation of CO₂ is involved in the growth of plant tissues.

     

Content of nonhistone protein in nuclei after hyperthermic treatment

When nuclei were isolated from Chinese hamster ovary cells after being heated, there was a large increase in the amount of ³H-tryptophan labeled nonhistone protein in the nucleus relative to the whole cell. After 15 min or 30 min of heating at 45.5°C, the nuclear nonhistone protein content increased by 1.6 or 1.8, respectively. In contrast, when the nuclear nonhistone protein content was determined in the intact cell by using autoradiography to quantify ³H-tryptophan labeled protein in the nucleus and cytoplasm in sections of fixed cells, the nuclear nonhistone protein content increased by only 1.14 or 1.28 for 15 or 30 min at 45.5°C, respectively. Therefore, heat does not induce a massive movement of cytoplasmic protein into the nucleus. © 1993 Wiley-Liss, Inc.     

Transfection: enhancement by assembly protein of bacteriophage R17.

Assembly protein was isolated by DEAE cellulose chromatography from disrupted R17 bacteriophage and reconstituted with purified R17 phage RNA. Following reconstitution, ¹²⁵I labeled assembly protein co-sediments with 27S R17 phage RNA in a sucrose gradient. SDS-polyacrylamide gel analysis of the 27S ¹²⁵I labeled protein-RNA complex confirmed that assembly protein was the only phage protein associated with the RNA. The specific infectivity (PFU/μg RNA) of the R17 phage RNA-assembly protein complex was 35-fold greater than that of R17 phage RNA when assayed on $\text{Escherichia coli}$ spheroplasts. Infectivity of both preparations was destroyed by treatment with pancreatic ribonuclease A. Furthermore, the assembly protein-RNA complex was infectious for intact cells whereas phage RNA was not infectious. Infectivity of this 27S complex for intact cells was totally eliminated by pretreatment with ribonuclease.     

Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes

Incubation of turkey erythrocyte membranes with cholera toxin and [³²P]NAD caused toxin-dependent incorporation of ³²P into a 42,000 M_r peptide which could be distinguished from toxin-independent ³²P incorporation into other membrane proteins. The radiolabeled 42,000 M_r peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 M_r labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 M_r peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes.     

Arginine Metabolism in Developing Soybean Cotyledons : II. Biosynthesis

Tracer kinetic experiments were performed using [ureido-¹⁴C] citrulline, [1-¹⁴C]ornithine, and isotope trapping techniques to determine if arginine is synthesized via the urea cycle in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with the ¹⁴C-solution and incubated in sealed vials containing a CO₂ trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. In the ¹⁴C-citrulline feeding experiment argininosuccinate was the most highly labeled compound after 5 minutes and it was the first compound to lose ¹⁴C later in the time course. Carbon-14 was also recovered in free arginine, protein arginine, and CO₂ up to 4 hours after introduction of label. All of the ¹⁴C in free and protein arginine could be accounted for in the C-6 position. Metabolism of ¹⁴C-ornithine resulted in ¹⁴C-incorporation into citrulline and free and protein arginine and the evolution of ¹⁴CO₂. Citrulline was the most highly labeled compound after 15 minutes and was the first compound to reach a steady state level of ¹⁴C. With the addition of 800 nanomoles unlabeled citrulline to the ¹⁴C-ornithine feeding solution citrulline was the only compound labeled after 5 minutes and the steady state level of ¹⁴C-citrulline increased 12-fold. The appearance of ¹⁴C in free arginine and protein arginine was also delayed. In both ¹⁴C-ornithine feedings all of the ¹⁴C in free and protein arginine could be accounted for in the C-1 position. Together, the data support the reaction sequence: ornithine → citrulline → argininosuccinate → arginine → protein arginine.     

Studies on the biogenesis of an enzymatically active complex III of the respiratory chain from yeast mitochondria

Complex III isolated from yeast mitochondria catalyzed an antimycin A and Diuron-sensitive coenzyme QH₂-cytochrome c reductase activity with a turnover number of 15.7 sec^?1 and contained 10 nmoles of cytochrome b and 4.6 nmoles of cytochrome c₁ per mg of protein. Electrophoresis in sodium dodecyl sulfate acrylamide gels resolved Complex III into 10 bands with apparent molecular weights of 50,000, 40,000, 30,000, 29,000, 24,000, 17,000, 16,000, 12,000, 8,400, and 5,800. Yeast cells were labeled under nongrowing conditions with (³⁵S)-methionine in the absence or presence of inhibitors of cytoplasmi? or mitochondrial protein synthesis. Labeled Complex III was isolated by immunoprecipitation from detergent-solubilized mitochondria using antiserum raised against the purified complex. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis revealed that a 30,000-dalton protein, cytochrome b, as well as 16,000-dalton protein were labeled in the presence of cycloheximide, indicating that they are products of mitochondrial protein synthesis. Immunoprecipitates from mitochondria obtained from cells labeled in the presence of chloramphenicol contained a new radioactive peak with a molecular weight of 100,000. In addition, significant decreases in the labeling of the proteins with molecular weights of 50,000, 40,000, 30,000, and 16,000 were observed. When Complex III was isolated by immunoprecipitation from intact spheroplasts after a 5-minute pulse with (³⁵S)-methionine, the 100,000-dalton protein was labeled in the immunoprecipitate whether or not chloramphenicol was present; however, after a 1-hour chase with unlabeled methionine, decreased labeling of the 100,000-dalton protein was observed concomitant with an increased labeling of the 50,000- and 40,000-dalton proteins. These results suggest that a protein with a molecular weight of 100,000 may either be a precursor or a partially assembled form of other proteins of Complex III, most probably the two largest polypeptides.