Protein methylation in pea chloroplasts

The methylation of chloroplast proteins has been investigated by incubating intact pea (Pisum sativum) chloroplasts with [³H-methyl]-S-adenosylmethionine. Incubation in the light increases the amount of methylation in both the thylakoid and stromal fractions. Numerous thylakoid proteins serve as substrates for the methyltransfer reactions. Three of these thylakoid proteins are methylated to a significantly greater extent in the light than in the dark. One is a polypeptide with a molecular mass of 64 kD, a second has an M_r of 48 kD, and the third has a molecular mass of less than 10 kD. The primary stromal polypeptide methylated is the large subunit of ribulose bisphosphate carboxylase/oxygenase. One other stromal polypeptide, having a molecular mass of 24 kD, is also methylated much more in the light than in the dark. Two distinct types of protein methylation occur. One methyl-linkage is stable to basic conditions whereas a second type is base labile. The base-stable linkage is indicative of N-methylation of amino acid residues while base-lability is suggestive of carboxymethylation of amino acid residues. Labeling in the light increases the percentage of methylation that is base labile in the thylakoid fraction while no difference is observed in the amount of base-labile methylations in light-labeled and dark-labeled stromal proteins. Also suggestive of carboxymethylation is the detection of volatile [³H]methyl radioactivity which increases during the labeling period and is greater in chloroplasts labeled in the light as opposed to being labeled in the dark; this implies in vivo turnover of the [³H]methyl group.     

High-mobility group chromosomal proteins of wheat

Four proteins have been extracted from purified chromatin of wheat embryos with 0.35 M NaCl. These proteins are soluble in 2% (w/v) trichloroacetic acid and thus meet the original operational requirements to be classified as "high-mobility group" (HMG) chromosomal proteins. The proteins have been characterized by one- and two-dimensional electrophoresis, amino acid analysis, and peptide mapping. Three of the proteins (HMGb, c, and d) share the mammalian HMG characteristic of being rich in both acidic and basic amino acid residues. Unlike their putative mammalian counterparts, these plant HMG proteins contain less than 7 mol % proline. The fourth wheat protein (HMGa) is rich in both proline and in basic amino acid residues. This wheat protein, however, contains only about half the proportion of acidic residues found in mammalian HMG proteins--a characteristic also found in the trout testis HMG protein, H6. Comparative peptide maps show that none of the wheat HMG proteins are degradation products of other HMG proteins or the H1 histones. The peptide maps have not, however, been useful in establishing homologies with mammalian HMG proteins. Wheat HMG proteins are released from DNase I-treated nuclei and co-isolate with micrococcal nuclease-sensitive chromatin fractions. Similar observations concerning the HMG proteins of vertebrate animals have been considered consistent with a role for these proteins as structural components of actively transcribed chromatin.     

GLUTAMYL AND ASPARTYL MOIETIES OF CEREBRAL PROTEINS: ENRICHMENT IN MEMBRANE-CONTAINING MICROSOMAL SUBFRACTIONS

Abstract— The total mixed proteins (excluding proteolipids) were isolated from various tissues of the cat and from subcellular fractions of cat cerebral cortex and were compared for contents of total glutamyl and total aspartyl residues and total amide N. The proteins from renal cortex, testis and diaphragm were more acidic (proportionally more glutamyl and aspartyl residues with no increase of amide) than those from cerebral cortex, subcortical white matter and liver. Also the proteins isolated from cerebral cortical microsomes were more acidic than those from highly pure nuclear, mitochondrial and soluble subcellular fractions of cerebral cortex. Hepatic microsomal proteins gave very similar analyses.
Subfractionation of the microsomal preparations from cerebral cortex and liver into deoxycholate-insoluble (ribosomal) and deoxycholate-soluble ('membrane') fractions localized the acidic proteins to the latter, 'membrane' subtraction. The ribosomal proteins isolated from cerebral cortex (and liver) had an amino acid composition very similar to that reported previously for ribosomal proteins in a number of tissues from various species. Upon column chromatography on DEAE-celluIose, the deoxycholate-soluble ('membrane') subtraction of hepatic microsomes yielded a peak (eluted with 0-2 M-phosphate, pH 5-6, plus 0-5 M-NaCl) that contained an exceptionally high proportion (30 per cent) of glutamyl and aspartyl residues. We suggest that such acidic proteins may be characteristic of membranes and may subserve important metabolic functions therein.     

A New Form of Myelin Basic Protein Found in Human Brain

Human myelin basic protein was subjected to ion-exchange chromatography at high pH to separate the differently charged components. Polyacrylamide gel electrophoretic patterns of the fractions showed that the less basic fractions 3, 4, and 5 contained significant amounts of a protein somewhat smaller than the more common 18.5-kDa form. Fraction 3 consisted of approximately equal amounts of this smaller polypeptide and component 3, the 18.5-kDa form found in other mammalian myelin basic protein preparations. The two proteins in fraction 3 were separated by fast protein liquid chromatography. Both have blocked N termini and identical C termini (-Met-Ala-Arg-Arg). When the tryptic digests of the two proteins were fractionated by HPLC, the elution profiles were similar, except that four peaks found in the chromatogram of the larger protein were missing from the chromatogram of the smaller one. In addition, an extra peak was found in the elution pattern of the latter chromatogram. Amino acid analysis of the individual tryptic peptides indicated that the smaller protein lacked residues 106-116 (-Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg-Phe-Ser-Trp-). The deleted portion corresponds exactly to the amino acid sequence encoded by exon 5 of the mouse basic protein gene. This new form of myelin basic protein has a molecular weight of 17,200, calculated from its amino acid composition.     

Halophilic enzymes: proteins with a grain of salt

Halophilic enzymes, while performing identical enzymatic functions as their non-halophilic counterparts, have been shown to exhibit substantially different properties, among them the requirement for high salt concentrations, in the 1-4 M range, for activity and stability, and a high excess of acidic over basic amino residues. The following communication reviews the functional and structural properties of two proteins isolated from the extremely halophilic archaeon Haloarcula marismortui: the enzyme malate-dehydrogenase (hMDH) and the 2Fe-2S protein ferredoxin. It is argued that the high negative surface charge of halophilic proteins makes them more soluble and renders them more flexible at high salt concentrations, conditions under which non-halophilic proteins tend to aggregate and become rigid. This high surface charge is neutralized mainly by tightly bound water dipoles. The requirement of high salt concentration for the stabilization of halophilic enzymes, on the other hand, is due to a low affinity binding of the salt to specific sites on the surface of the folded polypeptide, thus stabilizing the active conformation of the protein.     

Sequence of the precursor to rat ornithine aminotransferase deduced from a cDNA clone

The nucleotide sequence of ornithine aminotransferase mRNA from rat liver, including the entire coding and 3' untranslated regions, was determined from two overlapping cDNA clones. The mRNA encodes a precursor polypeptide of 439 amino acid residues with a molecular weight of 48,332. The deduced amino acid composition of the proposed mature enzyme sequence (residues 35 through 439) was in good agreement with that reported for the purified protein. The amino-terminal segment of the precursor corresponding to residues 1 through 34 has an overall positive charge, containing 6 basic residues and only a single acidic residue, and is postulated to be the mitochondrial leader sequence. The first 22 amino acid residues of the proposed leader sequences share 54% homology with the leader peptide of rat ornithine transcarbamylase precursor and more limited homology to the leader peptides of other nuclear-encoded mitochondrial matrix proteins. Homology was also observed between residues 286 through 362 ornithine aminotransferase precursor and a region containing the pyridoxyl phosphate binding domain of mitochondrial aspartate aminotransferase.     

Molecular organization in bacterial cell membranes: IV. Isolation by preparative electrophoresis in sodium dodecylsulphate and properties of the two major polypeptide groups of a “soluble” fraction from Streptomyces albus membranes

Two polypeptide fractions have been purified from a “soluble” fraction of n-butanol-extracted Streptomyces albus membranes by preparative electrophoresis in sodium dodecylsulphate. They accounted for approx. 80% of the protein of the fraction (i.e. 20% of the total membrane protein). The ultraviolet spectrum of Group 1 (relative mobility 1.0) revealed the presence of nucleotide material, while that of Group 3 (relative mobility 0.65±0.05) showed the presence of a possibly aggregated protein-like material. About 100 and 30% of the protein contents (Lowry method) of Groups 3 and 1, respectively, were recovered as amino acid residues. These results confirm the protein nature of both fractions and suggest an overestimation of the protein value in Group 1. Both polypeptide groups can be classified as “extrinsic” membrane proteins on the basis of their similar amino acid composition (Vanderkooi, G. and Capaldi, R. A. (1972) Ann. N.Y. Acad. Sci. 195, 135–138). Three N-terminal amino acids were found in each fraction: one common (alanine), methionine, leucine or isoleucine (Group 3) and glutamic acid, lysine (Group 1). The sedimentation coefficients calculated were 2.46 S for Group 3 and 1.54 S for Group 1. Analysis of the isolated groups by gel electrophoresis under non-dissociating conditions or with Triton X-100, gave aggregate-like patterns.Sodium dodecylsulphate electrophoresis revealed an anomalous staining behaviour of Group 3 depending upon the dissociating conditions. The whole “soluble” fraction bound 0.40 mg dodecylsulphate /mg protein (0.55 mg detergent/mg protein corrected for overestimation). After dialysis, the fraction retained 10% of the bound dodecylsulphate. Circular dichroism of the isolated groups after exhaustive dialysis showed similar spectra, although of lower dichroism, to those obtained by other authors on soluble enzymes treated with sodium dodecylsulphate. Strong acid conditions were required to change the CD spectra of the polypeptides.     

NUCLEAR PROTEINS OF THE GUINEA PIG BRAIN

Abstract—

• 1 Chromatin protein fractions were separated from the nuclei from brain, liver and kidney of the guinea pig. The fractions were studied by electrophoretic methods and amino acid analysis.
• 2 Brain nuclear fractions were washed with 0.15m -NaCl and nuclear acidic proteins then removed by 0.35 m -NaCl. These 0.35 m -NaCl-extracted proteins were considered to be similar to the nuclear soluble acidic proteins.
• 3 Nonhistone-1, histone and nonhistone-2 fractions were obtained from 2.0 m -NaCl-soluble chromatin fractions by lowering the salt concentration and successive extraction with acid and alkali. The nonhistone-3 fraction was also extracted from the nuclear residue by alkaline solution.
• 4 The contents and characteristics of the nonhistone fractions of the brain, especially the nonhistone-1 fraction, differed among the three tissues. The histone fractions showed no obvious difference among the three tissues. The nonhistone-1 fraction of the brain, which comprised a low percentage of total nuclear protein, contained relatively high amounts of acidic proteins.

     

Halophilic β-lactamase as a new solubility- and folding-enhancing tag protein: production of native human interleukin 1α and human neutrophil α-defensin

The amino acid composition of halophilic enzymes is characterized by an abundant content of acidic amino acid, which confers to the halophilic enzymes extensive negative charges at neutral pH and high aqueous solubility. This negative charge prevents protein aggregation when denatured and thereby leads to highly efficient protein refolding. β-Lactamase from periplasmic space of moderate halophile (BLA), a typical halophilic enzyme, can be readily expressed as a native, active form in Escherichia coli cytoplasm. Similar to other halophilic enzymes, BLA is soluble upon denaturation by heat or urea treatments and, hence, can be efficiently refolded. Such high solubility and refolding efficiency make BLA a potential fusion partner for expression of aggregation-prone heterologous proteins to be expressed in E. coli. Here, we succeeded in the soluble expression of several “difficult-to-express” proteins as a BLA fusion protein and verified biological activities of human interleukin 1α and human neutrophil α-defensin, HNP-1.     

Regulation of concentrations of glycolytic enzymes and creatine-phosphate kinase in “fast-twitch” and “slow-twitch” skeletal muscles of the chicken

The distinctive contractile and metabolic characteristics of different skeletal muscle fiber types are associated with different protein populations in these cells. In the present work, we investigate the regulation of concentrations of three glycolytic enzymes (aldolase, enolase, glyceraldehyde-3-phosphate dehydrogenase) and creatine-phosphate kinase in “fast-twitch” (breast) and “slow-twitch” (lateral adductor) muscles of the chicken. Results of short-term amino acid incorporation experiments conducted both in vivo and with muscle explants in vitro showed that these enzymes turnover at different rates and that aldolase turns over 2 to 3 times faster than the other three enzymes. However, these differences in turnover rates were difficult to detect in long-term double-isotope incorporation experiments, presumably because extensive reutilization of labeled amino acids occurred during these long-term experiments. Mature muscle fibers synthesize these four cytosolic enzymes at very high rates. For example, 11 to 14% of the total labeled leucine incorporated into protein by breast muscle fibers was found in the enzyme aldolase. Results of short-term amino acid incorporation experiments also showed that the relative rates of synthesis of the three glycolytic enzymes were about fourfold higher in mature “fast-twitch” muscle fibers than in mature “slow-twitch” ones while the relative rates of synthesis of creatine-phosphate kinase were similar in the two fiber types. The relative rates of synthesis of these four enzymes and cytosolic proteins in general were found to be very similar in immature muscles of both types. More profound changes in the relative rates of synthesis of major cytosolic proteins, including the glycolytic enzymes, occurred during postembryonic maturation of fast-twitch fibers than occurred during maturation of slow-twitch fibers. Our work demonstrates that (1) the synthesis of creatine-phosphate is independently regulated with respect to the synthesis of the glycolytic enzymes in muscle fibers; and (2) the approximate fourfold higher steady-state concentrations of glycolytic enzymes in fast-twitch muscle fibers as compared with slow-twitch fibers are determined predominantly by regulatory mechanisms operating at the level of protein synthesis rather than protein degradation. Our demonstration that more profound changes in the relative rates of synthesis of major cytosolic proteins occur during maturation of fast-twitch fibers as compared with slow-twitch fibers is discussed in terms of the mode(s) of fiber-type differentiation proposed by others.     

HMG-like protein in barley and corn nuclei

Chromosomal proteins have been isolated from barley (Hordeum vulgare) and corn (Zea mays) nuclei by extraction with 5% perchloric acid. In each plant, one protein was shown to belong to the HMG proteins. Their molecular weights are very close to that of HMG 14 from chicken erythrocytes, as shown by electrophoretic mobility in SDS polyacrylamide gels. In acetic acid-urea-Triton polyacrylamide gels they migrate between HMG 1,2 and HMG 14, from chicken erythrocytes. Their amino acid compositions are typical of HMG proteins, with equivalent high values of acidic and basic residues. Extraction of HMG's from purified barley chromatin fractions with 0.35 M NaCl considerably reduces histone H2 contamination and increases the yield of HMG up to 0.7% of the total histones. In this technique a second protein was extracted which is soluble in 2% Trichloroacetic acid and shows electrophoretic mobility analogous to those of HMG 14 and 17 from chicken erythrocytes. Whether or not these proteins are counterparts of the animal HMG's 1–2 or HMG's 14–17 is discussed.     

The nuclear basic proteins of human testes and ejaculated spermatozoa

Human testicular nuclei were fractionated into two fractions according to their sedimentation in a sucrose density gradient. The nuclear basic proteins isolated from these two fractions were similar and also resembled electrophoretic mobilities and amino acid composition of the liver histones. Only quantitative differences among histone electrophoretic bands were observed. The nuclear basic proteins of ejaculated spermatozoa differed totally from those of the testes. The proteins could be divided into two categories on the basis of their electrophoretic mobilities, molecular weights and amino acid compositions. One group (SpH) was similar to testicular histones; another (HP) group was smaller, with nearly twice the electrophoretic mobility and a much higher arginine content. These proteins (HP) represent a new type of nuclear basic protein found in human tissues.     

A mechanism for the evolution of phosphorylation sites

Protein phosphorylation provides a mechanism for the rapid, reversible control of protein function. Phosphorylation adds negative charge to amino acid side chains, and negatively charged amino acids (Asp/Glu) can sometimes mimic the phosphorylated state of a protein. Using a comparative genomics approach, we show that nature also employs this trick in reverse by evolving serine, threonine, and tyrosine phosphorylation sites from Asp/Glu residues. Structures of three proteins where phosphosites evolved from acidic residues (DNA topoisomerase II, enolase, and C-Raf) show that the relevant acidic residues are present in salt bridges with conserved basic residues, and that phosphorylation has the potential to conditionally restore the salt bridges. The evolution of phosphorylation sites from glutamate and aspartate provides a rationale for why phosphorylation sometimes activates proteins, and helps explain the origins of this important and complex process.     

Physicochemical modifications of lysosomal hydrolases during intracellular transport

1. The following fractions were prepared from rat kidney and characterized ultrastructurally, biochemically and enzymically: (a) an ordinary rough microsomal (RM(1)) fraction; (b) a special rough microsomal (RM(2)) fraction enriched seven- to nine-fold in acid hydrolases over the homogenate; (c) a smooth microsomal (SM) fraction; (d) a Golgi (GM) fraction enriched 2.5-fold in acid hydrolases and 10-, 15- and 20-fold in sialyltransferase, N-acetyl-lactosamine synthetase and galactosyltransferase respectively; (e) a lysosomal (L) fraction enriched 15- to 23-fold in acid hydrolases. The frequency of Golgi sacs and tubules seen in the electron microscope and the specific activity of the three glycosyltransferases in these fractions increased in the order: RM(2)相似文献   

Purification and characterization of the major storage proteins of Phaseolus vulgaris seeds,and their intracellular and cotyledonary distribution

Several extraction and fractionation procedures have been employed to isolate the major storage proteins of mature seeds of Phaseolus vulgaris cv. “Seafarer”; three proteins which were soluble at pH 4,7, and one that was insoluble at that pH were identified. The characteristic subunits of the three pH 4.7 soluble proteins had MW's 50000 and 47000, 32000, and 23000 respectively; those of the pH 4.7 insoluble fractions had MW 60000 and 20000. Amino acid compositions, N-terminal amino acid residues and the presence of carbohydrate in these proteins have been determined. All these proteins occurred in the protein body fraction and their relative amounts were different in the outer and central parts of the cotyledons.     

A novel, highly phosphorylated protein, of the high-mobility group type, present in a variety of proliferating and non-proliferating mammalian cells

The present work describes a perchloric-acid-soluble high-mobility-group (HMG)-like protein present in HeLa and Ehrlich ascites cells, rat and calf liver. The protein is designated P1 and has, depending on the source, a molecular mass 48-53 kDa and an amino acid composition which, like the HMG proteins, is characterized by a high content of acidic and basic residues and of proline. The protein contains about 10 mol serine/100 mol amino acid residues, is highly phosphorylated and has, in contrast to the known HMG proteins, an acidic isoelectric point of 5.0. An estimate suggests that protein P1 in HeLa interphase cells contains 25-30 residues of phosphate. Like HMG 1 and 2 it is distributed between the nucleus and the cytoplasm. In HeLa metaphase cells P1 is further modified, resulting in an increase in apparent molecular mass from 53 kDa to 56 kDa.     

Studies on the relationship between the degradative rates of proteins in vivo and their isoelectric points

Acidic proteins tend to be degraded more rapidly than neutral or basic proteins in rat liver, skeletal muscle, kidney and brain and in mouse liver and skeletal muscle. We now report a similar relationship among soluble proteins from rat lung, heart and testes, and from human fibroblasts and mouse-embryo cells grown in culture. These findings indicate that the correlation between protein net charge and degradative rate is a general characteristic of intracellular protein degradation in mammals. This relationship between isoelectric point and half-life appears to be distinct from the previously reported correlation between subunit molecular weight and protein half-lives. The more rapid degradation of acidic proteins does not result from their being of larger molecular weight than neutral or basic proteins. Furthermore, proteins within specific isoelectric point ranges still exhibit a relationship between subunit size and half-life. Finally, a group of membrane or organelle-associated proteins that are insoluble in phosphate-buffered saline and water but soluble in 1% Triton X-100 exhibit a correlation between size and half-life, but not between net charge and half-life. The biochemical reasons for the relationship between protein isoelectric point and half-life are unclear, although several possible explanations are presented. It is not due to a greater sensitivity of acidic proteins to proteolytic attack since experiments with a variety of endoproteinases, including trypsin, chymotrypsin, Pronase, papain, chymopapain, Staphylococcus aureus V8 proteinase, pepsin and lysosomal cathepsins from rat liver, have failed to demonstrate more rapid digestion of acidic proteins.     

The occurrence of extended acidic sequences in nonhistone chromosomal proteins

Nonhistone chromosomal proteins and soluble cytoplasmic proteins from rat liver were treated with a combination of proteases and chemical reagents which split a variety of peptide bonds but do not attack sequences consisting predominantly or exclusively of acidic amino acid residues. Analysis of the resulting digests by gel filtration chromatography and column electrophoresis demonstrated that, relative to cytoplasmic proteins, nonhistone chromosomal proteins are rich in highly charged, acidic peptides up to 12 residues in length, but rarely contain very long peptides consisting exclusively of acidic residues such as are found in the nonhistone chromosomal proteins HMG1 and HMG2.     

Comparison of aragonitic molluscan shell proteins

Acidic macromolecules, as a nucleation factor for mollusc shell formation, are a major focus of research. It remains unclear, however, whether acidic macromolecules are present only in calcified shell organic matrices, and which acidic macromolecules are crucial for the nucleation process by binding to chitin as structural components. To clarify these questions, we applied 2D gel electrophoresis and amino acid analysis to soluble shell organic matrices from nacre shell, non-nacre aragonitic shell and non-calcified squid shells. The 2D gel electrophoresis results showed that the acidity of soluble proteins differs even between nacre shells, and some nacre (Haliotis gigantea) showed a basic protein migration pattern. Non-calcified shells also contained some moderately acidic proteins. The results did not support the correlation between the acidity of soluble shell proteins and shell structure.     

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A subunit I has been determined by automated Edman degradation of the cyanogen bromide fractions derived from the precursor protein. The activation peptide contains 94 amino acid residues in a unique sequence which precedes directly the amino-terminal alanine residue of carboxypeptidase A alpha. A notable feature of the activation peptide is the presence of acidic amino acid residues immediately preceding the site of activation. The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A shows extensive similarity to those of the corresponding porcine and rat enzymes.