Terminal deoxynucleotidyltransferase. Alignment of alpha- and beta-subunits of the core enzyme along the primary translation product.

Terminal deoxynucleotidyltransferase exists in multiple Mr forms, all apparently generated from a single polypeptide of 62kDa. On isolation and purification, the smallest catalytically active protein of this enzyme consists of two subunits, alpha (12kDa) and beta (30kDa). Recently a complementary-DNA nucleotide sequence has been reported for a portion of the enzyme from human lymphoblast. We have pinpointed the locations of the alpha- and beta-subunits within the elucidated nucleotide sequence. From these data, the portions of the nucleotide sequence coding for the catalytically important area of the transferase can be estimated. Here the amino acid sequence of a number of tryptic peptides from calf alpha- and beta-subunits is presented. Because of the striking homology between the amino acid sequence of the calf enzyme and that predicted for human lymphoblast enzyme, it is possible for us to conclude that the alpha-subunit was generated from the C-terminus of the precursor protein and the beta-subunit was non-overlapping and proximal.     

Isolation and partial characterization of an Mr 60,000 subunit of a type 2A phosphatase from rabbit reticulocytes

A Mr 60,000 peptide that modulates the activity of the Mr 35,000 catalytic subunit of a type 2A phosphatase has been isolated from rabbit reticulocytes and partially characterized. The peptide appears to be a subunit of the intact phosphatase that has been isolated under nondenaturing conditions. The Mr 60,000 peptide itself is catalytically inactive. However, it binds to the Mr 35,000 catalytic subunit causing a decrease in its activity for dephosphorylation of phosphorylated 40 S ribosomal subunits, but an increase in dephosphorylation of peptide initiation factor 2 phosphorylated in its alpha subunit. Reassociation of the Mr 60,000 and the Mr 35,000 peptides yields a two-subunit phosphatase with a Stokes radius of 42 A; sedimentation coefficient, S20,w of 5.1 S; molecular weight of 89,000. These parameters are compared to those of the native three-subunit enzyme and those of the isolated Mr 35,000 and 60,000 peptides.     

Structure of proteoheparan sulfates from fibroblasts. Confluent and proliferating fibroblasts produce at least three types of proteoheparan sulfates with functionally different core proteins

[3H]Leucine- and [35S]sulfate-labeled proteoheparan sulfates were isolated from postconfluent or proliferating cultures of human skin fibroblasts. Cell layers were solubilized by Triton X-100, and transferrin-binding macromolecules were isolated by affinity chromatography. Proteoglycans with no affinity for transferrin were purified by using ion-exchange and gel permeation chromatography. Postconfluent cells synthesize a proteoheparan sulfate of Mr 350,000 (as determined by gel permeation chromatography) which has affinity for transferrin as well as for octyl-Sepharose. Its core protein (Mr 180,000) consists of two disulfide-bonded polypeptides of Mr 90,000. This species was not detected in cultures of proliferating cells. Proliferating and confluent cells also synthesize other forms of proteoheparan sulfates (Mr 200,000-400,000) which have no affinity for transferrin. However, most of them have affinity for octyl-Sepharose. The core protein of proteoheparan sulfates made by proliferating cells has Mr 50,000. A smaller form (Mr 250,000) of this proteoglycan was solubilized by Triton X-100, whereas a larger form (Mr 400,000) remained associated with the pericellular matrix. A third type of proteoheparan sulfate (Mr 200,000) without affinity for transferrin nor octyl-Sepharose was associated with postconfluent cell layers but not with proliferating ones. Its core protein has Mr 35,000. Heparan sulfate oligosaccharides (Mr 6,000 or higher) were found in proliferating cells but not in postconfluent ones.     

Relative molecular mass determination of a major, highest relative molecular mass extracellular amelogenin of developing bovine enamel

Proteins of developing bovine enamel were fractionated by molecular sieving and ion-exchange chromatography. The major fraction corresponding to the highest Mr amelogenin of Mr approximately 26 000-30 000 was isolated and its Mr determined by SDS-PAGE, molecular sieving on G-100 resin and high performance liquid chromatography and by sedimentation-equilibrium ultracentrifugation, the latter three procedures in guanidine hydrochloride. SDS-PAGE and HPLC molecular sieving, employing commonly used Mr standards, gave Mr values of approximately 22 000-26 000. SDS-PAGE and HPLC molecular sieving, using proline-rich CNBr peptides of collagen as standards, and sedimentation-equilibrium ultracentrifugation, gave Mr values of approximately 15 000-18 000 and approximately 17 385, respectively. These latter values correspond well with those reported earlier and with the Mr of the major amelogenin computed from recent amino acid sequence data (approximately 19 000). It is concluded that the recently described, highest Mr amelogenin of Mr = 26 000-30 000 is not a new component but is identical to the proline-rich components having relative molecular masses ranging from 15 000 to 18 000 described much earlier by several groups of workers.     

Identification and characterization of cathepsin D in a highly purified sialidase from starfish A. pectinifera

A sialidase [EC 3.2.1.18] from the ovary of starfish Asterina pectinifera was isolated and highly purified by preparative PAGE. The SDS-PAGE separation of the purified enzyme revealed two natures of protein bands, upper (50 kDa) and a lower (47 kDa). To identify the protein, N-terminal amino acid sequence of the upper band was done. The sequence matched with the N-terminal amino acid sequence of human lysosomal mature cathepsin D and cathepsin D activity was also found in all the preparation steps. Protease inhibitor pepstatin A inhibited the proteolysis activity of cathepsin D against a synthetic substrate. The two enzymes sialidase and cathepsin D were separated from each other by using high-performance gel-filtration chromatography. The Western blot analysis and isoelectric focusing showed the co-purified cathepsin D is a 50 kDa protein with a PI value of 4.2.     

Primary structure and functional expression of h-caldesmon complementary DNA

Recently, the two Mr forms of caldesmon (Mr's in the range of 120-150kDa and 70-80kDa as judged by SDS-PAGE) have been identified. h-Caldesman (high Mr 120-150kDa caldesmon) is predominantly expressed in smooth muscles, and l-caldesmon (low Mr 70-80kDa caldesmon) in non-muscle cells. In this paper, we report the nucleotide sequence of chick embryo gizzard h-caldesmon cDNA and its translation into amino acid sequence. This sequence predicts a protein of 771 amino acids with a Mr of 88,743. The central portion of this sequence is composed of a 10-fold repeat of conserved amino acid sequence containing 13-15 amino acids. Further, a recombinant protein produced in Escherichia coli containing the full-length h-caldesmon cDNA has been characterized. Although the Mr of h-caldesmon predicted from amino acid sequence is 88,743, native and recombinant proteins show the same mol. wt. with 150kDa as measured by SDS-PAGE. This discrepancy may be due to the acidic amino acid-rich sequences at the N-terminal and central portions. A recombinant protein produced in E. coli possesses calmodulin-, F-actin- and tropomyosin-binding abilities in common with the native h-caldesmon.     

Complete amino acid sequence of human liver cytosolic alanine aminotransferase (GPT) determined by a combination of conventional and mass spectral methods

The complete amino acid sequence of human liver cytosolic alanine aminotransferase (GPT) (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. Isolated peptides were analyzed with a protein sequencer or with a plasma desorption time of flight mass spectrometer and placed in the sequence on the basis of their molecular mass and homology to the sequence of rat GPT. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The Mr of the subunit was calculated to be 54,479, which was in good agreement with a Mr of 55,000 estimated by SDS-PAGE, and also indicated that the active enzyme with a Mr of 114,000 was a homodimer composed of two identical subunits. The amino acid sequence is highly homologous to that of rat GPT (87.9% identity) recently determined [Ishiguro, M., Suzuki, M., Takio, K., Matsuzawa, T., & Titani, K. (1991) Biochemistry 30, 6048-6053]. All of the crucial amino acid residues are conserved in human GPT, which seem to be hydrogen bonding to pyridoxal 5'-phosphate in rat GPT by the sequence homology to other alpha-aminotransferases with known tertiary structures.     

Heavy riboflavin synthase of Bacillus subtilis. Primary structure of the beta subunit

Heavy riboflavin synthase is a 1,000,000-Da protein catalyzing the last two reactions of riboflavin biosynthesis. The enzyme complex consists of 60 beta subunits (Mr = 16,200) and approximately three alpha subunits (Mr = 23,000). beta subunits were isolated and cleaved with cyanogen bromide. Fragments were isolated and further digested with trypsin and staphylococcal protease. Peptides were isolated by high performance liquid chromatography. Sequences were determined by automated liquid-phase Edman degradation. The complete sequence of the beta subunit (154 amino acids) was established by direct sequencing of the NH2 terminus, sequencing of overlapping peptides, and carboxypeptidase degradation of the COOH terminus. The sequence shows no detectable homologies to other proteins. A computer prediction of secondary structure elements indicates 34% alpha helix and 30% beta sheet.     

Cytovillin and other microvillar proteins of human choriocarcinoma cells

Microvilli were isolated from cultured human JEG-3 choriocarcinoma cells using a gentle shearing method. The protein components of the isolated microvilli were examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The major Mr 42,000 and Mr 100,000 polypeptide bands reacted with anti-actin and anti-alpha-actinin antisera, respectively. Extraction of the isolated JEG-3 microvilli with Triton X-100 left an insoluble cytoskeletal residue containing mainly actin, alpha-actin, and polypeptides of Mr 200,000, 55,000 and 35,000. The Mr 35,000 polypeptide remained insoluble only at high concentrations of free Ca2+. Immunoblotting analysis of the JEG-3 microvilli indicated that they were devoid of tropomyosin, although the total JEG-3 protein lysates gave a strong positive reaction with anti-tropomyosin antiserum. The different subcellular localization of cytovillin and tropomyosin was also shown by indirect immunofluorescence microscopy. Cytovillin, an Mr 75,000 microvillus-specific membrane protein of JEG-3 cells, existed in an oligomeric form (dimer or trimer) as shown by gel filtration of Triton X-100 solubilized microvillar proteins and by native polyacrylamide gel electrophoresis of purified cytovillin. Disulfide bridges were not involved in the aggregation, because the mobility of cytovillin was similar under reducing and nonreducing conditions in SDS-PAGE. Cytovillin was shown to be closely related to ezrin, a minor component of chicken intestinal brush border microvilli.     

The lower molecular weight acid phosphatase from the frog liver: isolation of homogeneous AcPase III and IV representing glycoforms with different bioactivity

1. AcPase III and AcPase IV, the major enzyme forms of the LMW AcPase of the frog (Rana esculenta) liver were resolved and purified to homogeneity. 2. AcPase III and IV showed a single protein band on SDS-PAGE corresponding to a mol. wt (Mr) of about 35,000. The Mr of the native enzyme forms were 33,200 (gel electrophoresis) and 38,200 +/- 5000 (gel filtration). This indicates that they are monomeric proteins sharing the same protein molecule. 3. AcPase III and IV differ essentially in thermostability and the activating effect of ConA binding. 4. AcPase III and IV are considerably activated with DTT but they differed markedly by the extent of this activation and the accompanying changes of their pH-activity curves. 5. It is suggested that the frog liver LMW AcPase represents a set of glycoforms whose different bioactivity is determined by the redox states of their essential cysteine residues.     

Plasma-desorption mass spectrometry as an aid in protein sequence determination. Application of the method on a cuticular protein from the migratory locust (Locusta migratoria).

The complete amino acid sequence of a structural protein, protein 8, isolated from the pharate cuticle of the locust Locusta migratoria was determined. Protein 8 contains 148 amino acid residues and has an Mr of 15,224. By the extensive use of information obtained by plasma-desorption mass spectrometry (p.d.m.s.) it was possible to reduce the need for conventional sequence determination and to improve the reliability of the results. On the basis of the determined Mr of the intact protein all the peptides that constitute the complete sequence could be isolated from a time-course enzymic digestion. The isolated peptides were sequenced by using a combination of Edman degradation and carboxypeptidase digestion monitored by p.d.m.s. The alignment of the peptides was established from the time-course digestion and further verified by a second enzymic digestion. The primary structure of the protein consists of two hydrophilic and two hydrophobic regions. The hydrophobic regions are enriched in alanine, valine and proline and dominated by a repetitive sequence Ala-Ala-Pro-(Ala/Val). The sequence strengthens the view that the cuticle proteins belong to a unique family of structural proteins.     

Purification and characterization of rat liver minoxidil sulphotransferase.

Minoxidil (Mx), a pyrimidine N-oxide, is used therapeutically as an antihypertensive agent and to induce hair growth in patients with male pattern baldness. Mx NO-sulphate has been implicated as the agent active in producing these effects. This paper describes the purification of a unique sulphotransferase (ST) from rat liver cytosol that is capable of catalysing the sulphation of Mx. By using DEAE-Sepharose CL-6B chromatography, hydroxyapatite chromatography and ATP-agarose affinity chromatography, Mx-ST activity was purified 240-fold compared with the activity in cytosol. The purified enzyme was also capable of sulphating p-nitrophenol (PNP) at low concentrations (less than 10 microM). Mx-ST was purified to homogeneity, as evaluated by SDS/PAGE and reverse-phase h.p.l.c. The active form of the enzyme had a molecular mass of 66,000-68,000 Da as estimated by gel exclusion chromatography and a subunit molecular mass of 35,000 Da. The apparent Km values for Mx, 3'-phosphoadenosine 5'-phosphosulphate and PNP were 625 microM, 5.0 microM and 0.5 microM respectively. However, PNP displayed potent substrate inhibition at concentrations above 1.2 microM. Antibodies raised in rabbits to the pure enzyme detected a single band in rat liver cytosol with a subunit molecular mass of 35,000 Da, as determined by immunoblotting. The anti-(rat Mx-ST) antibodies also reacted with the phenol-sulphating form of human liver phenol sulphotransferase, suggesting some structural similarity between these proteins.     

The protein encoded by gvpC is a minor component of gas vesicles isolated from the cyanobacteria Anabaena flos-aquae and Microcyctis sp.

The proteins present in gas vesicles of the cyanobacteria Anabaena flos-aquae and Microcystis sp. were separated by SDS-polyacrylamide gel electrophoresis. Each contained a protein of Mr 22K whose N-terminal amino acid sequences showed homology with that of the Calothrix sp. PCC 7601 gvpC gene product. The gvpC gene from A. flos-aquae was cloned and sequenced. The derived amino acid sequence for the gene product indicated a protein, GVPc, of 193 residues and Mr 21985 containing five highly conserved 33 amino acid repeats. The sequence was identical at the N-terminus to that of the Mr 22K protein present in gas vesicles and showed correspondence to seven tryptic peptides isolated from gas vesicles. This establishes that GVPc forms a second protein component of the gas vesicle, in addition to the main constituent, the 70 residue GVPa. Quantitative amino acid analysis of entire gas vesicles reveals that GVPc accounts for only 2.9% of the protein molecules and 8.2% of the mass present: this is insufficient to form the conical end caps of the gas vesicles. It is suggested that GVPc provides the hydrophilic outer surface of the gas vesicle wall; the 33 amino acid repeats may interact with the periodic structure provided by GVPa.     

Purification and some properties of the extracellular alpha-amylase-pullulanase produced by Clostridium thermohydrosulfuricum.

The novel alpha-amylase-pullulanase produced by Clostridium thermohydrosulfuricum E 101-69 was purified as two forms (I and II) from culture medium, by using gel filtration in 6 M-guanidine hydrochloride as the final step. Renatured alpha-amylase-pullulanase I and II had apparent Mr values of 370,000 +/- 85,000 and 330,000 +/- 85,000 respectively, as determined by native polyacrylamide-gradient-gel electrophoresis. Both forms appear to be dimers of two similar subunits, with Mr values of 190,000 +/- 30,000 for enzyme I and 180,000 +/- 30,000 for enzyme II according to SDS/polyacrylamide-gradient-gel electrophoresis. The two forms had similar amino acid compositions, the same N-terminal sequence (Glu-Ile-Asp-Thr-Ala-Pro-Ala-Ile) and the same pI of 4.25. Both forms contained sugars having mobilities identical with those of rhamnose, glucose, galactose and mannose. The amount of neutral hexoses relative to protein was 11-12% (w/w) for both forms.     

Isolation and characterization of a high molecular weight protein phosphatase from rabbit skeletal muscle

A high molecular weight protein phosphatase (phosphatase H-II) was isolated from rabbit skeletal muscle. The enzyme had a Mr = 260,000 as determined by gel filtration and possessed two types of subunit, of Mr = 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On ethanol treatment, the enzyme was dissociated to an active species of Mr = 35,000. The purified phosphatase dephosphorylated lysine-rich histone, phosphorylase a, glycogen synthase, and phosphorylase kinase. It dephosphorylated both the alpha- and beta-subunit phosphates of phosphorylase kinase, with a preference for the dephosphorylation of the alpha-subunit phosphate over the beta-subunit phosphate of phosphorylase kinase. The enzyme also dephosphorylated p-nitrophenyl phosphate at alkaline pH. Phosphatase H-II is distinct from the major phosphorylase phosphatase activities in the muscle extracts. Its enzymatic properties closely resemble that of a Mr = 33,500 protein phosphatase (protein phosphatase C-II) isolated from the same tissue. However, despite their similarity of enzymatic properties, the Mr = 35,000 subunit of phosphatase H-II is physically different from phosphatase C-II as revealed by their different sizes on sodium dodecyl sulfate-gel electrophoresis. On trypsin treatment of the enzyme, this subunit is converted to a form which is a similar size to phosphatase C-II.     

Cloning, expression, and catalytic mechanism of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.

The first representative of a group of mammalian, low molecular weight phosphotyrosyl protein phosphatases was cloned, sequenced and expressed in Escherichia coli. Using a 61-mer oligonucleotide probe based on the amino acid sequence of the purified enzyme, several overlapping cDNA clones were isolated from a bovine heart cDNA library. A full-length clone was obtained consisting of a 27-bp 5' noncoding region, an open reading frame encoding the expected 157 amino acid protein, and an extensive 3' nontranslated sequence. The identification of the clone as full length was consistent with results obtained in mRNA blotting experiments using poly(A)+ mRNA from bovine heart. The coding sequence was placed downstream of a bacteriophage T7 promoter, and protein was expressed in E. coli. The expressed enzyme was soluble, and catalytically active and was readily isolated and purified. The recombinant protein had the expected Mr of 18,000 (estimated by SDS-PAGE), and it showed cross-reactivity with antisera that had been raised against both the bovine heart and the human placenta enzymes. The amino acid sequence of the N-terminal region of the expressed protein showed that methionine had been removed, resulting in a sequence identical to that of the enzyme isolated from the bovine tissue, with the exception that the N-terminal alanine of the protein from tissue is acetylated. A kinetically competent phosphoenzyme intermediate was trapped from a phosphatase-catalyzed reaction. Using 31P NMR, the covalent intermediate was identified as a cysteinyl phosphate. By analogy with the nomenclature used for serine esterases, these enzymes may be called cysteine phosphatases.     

Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis: application to peptide mapping of human complement component C3

The combination of high-resolution Tricine-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (H. Sch?gger and G. von Jagow (1987) Anal. Biochem. 166, 368-379) and electroblotting onto polyvinylidene difluoride (PVDF) membranes represents a powerful technique for the isolation of small amounts of peptides and protein fragments (Mr 1000-20,000) in a suitable form for amino acid sequencing, directly on the blotting membrane. Conditions for electrophoresis and electroblotting were optimized with respect to high transfer yield and suitability for both amino acid analysis and sequence determination of stained PVDF-bound peptides. Transfer yields were 50-80%, amino acid compositions including Cys were correct, and picomole quantities were sequenced with initial and repetitive yields as high as those we normally obtain for peptides in solution. The method was used for peptide mapping of polymorphic forms of human complement component C3.     

Cloning and sequence analysis of the cDNA for human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase catalyzes the oxidation of many prostaglandins at C-15, resulting in a subsequent reduction in their biological activity. We report the isolation of the cDNA for this enzyme. A human placental lambda gt11 cDNA library was screened using polyclonal antibodies prepared against the human placental enzyme. A 2.5-kilobase cDNA containing the entire coding region for the enzyme was isolated. The cDNA encodes for a protein of 266 amino acids with a calculated Mr of 28,975. Identification of the cDNA as that coding for 15-hydroxyprostaglandin dehydrogenase was based on the comparison of the deduced amino acid sequence with the amino acid sequence of two peptides, one from the rabbit lung enzyme and the other from the human placental enzyme. This cDNA hybridizes with two species of poly(A+) RNA isolated from human placenta: one of 3.4 kilobases and the other of 2.0 kilobases. Isolation of the cDNA for 15-hydroxyprostaglandin dehydrogenase should facilitate studies on the structure, function, and regulation of this enzyme.     

Isolation of cDNA clones encoding an enzyme from bovine cells that repairs oxidative DNA damage in vitro: homology with bacterial repair enzymes.

Ionizing radiation and radiomimetic compounds, such as hydrogen peroxide and bleomycin, generate DNA strand breaks with fragmented deoxyribose 3' termini via the formation of oxygen-derived free radicals. These fragmented sugars require removal by enzymes with 3' phosphodiesterase activity before DNA synthesis can proceed. An enzyme that reactivates bleomycin-damaged DNA to a substrate for Klenow polymerase has been purified from calf thymus. The enzyme, which has a Mr of 38,000 on SDS-PAGE, also reactivates hydrogen peroxide-damaged DNA and has an associated apurinic/apyrimidinic (AP) endonuclease activity. The N-terminal amino acid sequence of the purified protein matches that reported previously for a calf thymus enzyme purified on the basis of AP endonuclease activity. Degenerate oligonucleotide primers based on this sequence were used in the polymerase chain reaction to generate from a bovine cDNA library a fragment specific for the 5' end of the coding sequence. Using this cDNA fragment as a probe, several clones containing 1.35 kb cDNA inserts were isolated and the complete nucleotide sequence of one of these determined. This revealed an 0.95 kb open reading frame which would encode a polypeptide of Mr 35,500 and with a N-terminal sequence matching that determined experimentally. The predicted amino acid sequence shows strong homology with the sequences of two bacterial enzymes that repair oxidative DNA damage, ExoA protein of S. pneumoniae and exonuclease III of E. coli.     

The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product

The sequence of a 1.56-kb DNA fragment containing the tyrosinase gene (mel) from Streptomyces antibioticus was determined and the Mr (30612) and amino acid (aa) sequence of the protein were deduced from the nucleotide (nt) sequence. Intracellular and extracellular tyrosinase from S. antibioticus, transformed with pIJ702 (containing mel), were purified to homogeneity; the Mr (29 500), as determined by Sephadex G-75 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was consistent with the value derived from the nt sequence. Edman degradation established that the N-terminal sequence of both the intracellular and extracellular forms of tyrosinase are identical and correspond to the aa sequence derived from the structural gene. In addition, this sequence exhibits striking homology to the N-terminal region of the intracellular and extracellular enzyme purified from Streptomyces glaucescens (Crameri et al., 1982). An additional open reading frame (ORF438) upstream of the mel gene, was also identified that appears to code for a protein (Mr = 14 754) with a putative signal sequence.