Unique human glycoprotein, alpha1-microglycoprotein: isolation from the urine of a cancer patient and its characterization.

A human glycoprotein was isolated from the urine of a patient with plasma cell leukemia. It appears pure and homogeneous when examined by immunoelectrophoresis, sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis, gel filtration in 6 M guanidine hydrochloride (Gdn.HCl), and NH2-terminal amino acid sequence analysis. It has a brown color due to a tightly (most likely covalently) bound chromophore group(s) and migrates to the alpha1 region in immunoelectrophoresis. A molecular weight (mol wt) of 27 000 was obtained for the glycoprotein by gel filtration in 6 M Gdn.HCl. Its approximate mol wt determined by Na-DodSO4-polyacrylamide gel electrophoresis is 29 000 on 5% and 7.5% and 10% gels. Amino acid and hexosamine analyses showed that it is a glycoprotein and indicated that it contains four half-cystine residues per molecule. Based on the above observations we designated it "alpha1-microglycoprotein" (alpha1-MGP). Isoelectric focusing of alpha1-MGP showed a significant charge heterogeneity, although only a single NH2-terminal amino acid sequence was obtained for alpha1-MGP, i.e., Gly-Pro-Val-Pro-( )-Pro-Pro-Asx-Asx-Ile-Glx-Val-Glx-Glx-Asx-Phe-Phe-Ile-(Ser or Ala)-Arg. The alpha1-MGP was found in significant concentrations in the urine of many patients with neoplastic diseases.     

Recombinant rat guanidinoacetate methyltransferase: structure and function of the NH2-terminal region as deduced by limited proteolysis

Recombinant rat liver guanidinoacetate methyltransferase, a monomeric protein with Mr 26,000, is inactivated upon incubation with low concentrations of trypsin. Examination of the reaction products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography followed by amino acid analysis and sequencing of isolated peptides reveals that the inactivation is due to the cleavage of the NH2-terminal segment after Arg20. The cleaved peptide is not tightly associated with the rest of the protein. The rate of inactivation is not affected by the presence of either S-adenosylmethionine (AdoMet) or guanidinoacetate, but a substantial retardation of inactivation is observed when both substrates are present. The cleavage at Arg20 is also slowed by cross-linking Cys15 and Cys90 by a disulfide bond. An equilibrium binding study shows that guanidinoacetate methyltransferase in the free form binds AdoMet but not guanidinoacetate. The trypsin-modified enzyme, despite having no catalytic activity, can weakly bind AdoMet and guanidinoacetate in the presence of AdoMet. Chymotrypsin rapidly hydrolyzes the peptide bond after Trp19, and elastase cleaves the bond after Ala24, leading in both cases to loss of activity. The results obtained in this study suggest that the portion of the methyltransferase around residues 19-24 is highly exposed to the solvent and flexible. The results also indicate that the NH2-terminal region is not directly involved in substrate binding but plays a role in catalysis.     

Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin.

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.     

The primary structure of monkey pituitary growth hormone

Monkey growth hormone (MGH) has been purified by reverse-phase high-performance liquid chromatography (HPLC). Tryptic digests of MGH were separated by HPLC and paper electrophoresis. From amino acid composition, from NH2-terminal residue and sequence analyses of these tryptic peptides, and from their alignment with those of human growth hormone, the primary structure of MGH was proposed. There are only four residues which are different in these two growth hormone molecules.     

Purification and characterization of an N alpha-acetyltransferase from Saccharomyces cerevisiae

N alpha-Acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of proteins and peptides, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 4,600-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, hydroxylapatite, DE52 cellulose, and Affi-Gel blue. The Mr of the native enzyme was estimated to be 180,000 +/- 10,000 by gel filtration chromatography, and the Mr of each subunit was estimated to be 95,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 9.0, and its pI is 4.3 as determined by chromatofocusing on Mono-P. The enzyme catalyzed the transfer of an acetyl group to various synthetic peptides, including human adrenocorticotropic hormone (ACTH) (1-24) and its [Phe2] analogue, yeast alcohol dehydrogenase I (1-24), yeast alcohol dehydrogenase II (1-24), and human superoxide dismutase (1-24). These peptides contain either Ser or Ala as NH2-terminal residues which together with Met are the most commonly acetylated NH2-terminal residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). Yeast enolase, containing a free NH2-terminal Ala residue, is known not to be N alpha-acetylated in vivo (Chin, C. C. Q., Brewer, J. M., and Wold, F. (1981) J. Biol. Chem. 256, 1377-1384), and enolase (1-24), a synthetic peptide mimicking the protein's NH2 terminus, was not acetylated in vitro by yeast acetyltransferase. The enzyme did not catalyze the N alpha-acetylation of other synthetic peptides including ACTH(11-24), ACTH(7-38), ACTH(18-39), human beta-endorphin, yeast superoxide dismutase (1-24). Each of these peptides has an NH2-terminal residue which is rarely acetylated in proteins (Lys, Phe, Arg, Tyr, Val, respectively). Among a series of divalent cations, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate and N-bromosuccinimide.     

Purification and properties of UDP-glucuronyltransferase from kidney microsomes of beta-naphthoflavone-treated rat

Rat kidney microsomal UDP-glucuronyltransferase activities toward phenoic xenobiotics were enhanced about 4-5-fold by treatment of the animal with beta-naphthoflavone. The transferase activity toward serotonin, an endogenous substrate, was also enhanced about 7.5-fold. A form of UDP-glucuronyltransferase was purified from kidney microsomes of beta-naphthoflavone-treated rat by solubilization with sodium cholate and two steps of column chromatography, the first with DEAE-Toyopearl (fast flow rate liquid chromatography:FFLC) and the second with UDP-hexanolamine Sepharose 4B (affinity chromatography). These procedures gave about 39-fold purification and 11.5% yield of the transferase activity toward 1-naphthol. The preparation, tentatively termed "GT-2," was highly purified as judged from the single protein band (Mr 54,000) on sodium dodecylsulfate (SDS)-polyacrylamide slab gel electrophoresis. It catalyzed the glucuronidation of not only phenolic xenobiotics such as 1-naphthol, 4-nitrophenol, and 4-methylumbelliferone but also serotonin. From the result that apparent molecular weight of GT-2 was reduced to 50,000 by endo-beta-N-acetylglucosaminidase H (Endo H)-treatment, GT-2 was found to be a 50,000 Da polypeptide carrying "high mannose" type oligosaccharide chain(s). The NH2-terminal sequence of 20 residues of GT-2 was determined to be Asp-Lys-Leu-Leu-Val-Val-Pro-Gln-Asp-Gly-Ser-His-Trp-Leu-Ser-Met-Lys-Glu- Ile-Val . It was observed that there are two amino acids substitutions in the seven NH2-terminal residues in comparison with GT-1, which was purified from liver microsomes of 3-methylcholanthrene-treated rat. The NH2-terminal sequence of GT-2 was found to be homologous with the NH2-terminal sequence from the 26th to 46th amino acid residue of various UDP-glucuronyltransferase cloned by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of the alpha-chain cross-link acceptor sites of human fibrin

The potential cross-link acceptor sites of fibrin were specifically labeled with the fluorescent, substitute cross-link donor monodansyl cadaverine (MDC). Several fluorescent alpha-chain peptides generated from enzymatic and cyanogen bromide (CNBr) cleavage of the labeled fibrin were identified by sodium dodecyl sulfate disc gel electrophoresis; they were isolated and then characterized by amino acid analysis, NH2-terminal sequence analysis, and chromatographic and electrophoretic analyses of their digestion products. Ancrod cleavage of MDC-labeled fibrin produced a series of six alpha-chain peptides of molecular weights 34,000 to 12,000, each of which contained an MDC-labeled acceptor site, and an NH2-terminal alpha-chain derivative of molecular weight 37,500. The latter remains disulfide bound in the residual fibrin and has two MDC-labeled sit-s which are separable by CNBr cleavage. Mild plasmin digestion of MDC-labeled fibrin generated fluorescent alpha-chain peptides of molecular weights 45,000, 42,000, 35,000, 23,000, 21,000, and 2,500 in the supernatant and a nonfluorescent NH2-terminal alpha-chain derivative of molecular weight 25,000 which remained in the insoluble residual fibrin. The alignment of these plasmic supernatant peptides was determined from NH2-terminal sequence analyses which indicated that an MDC acceptor site was located at approximately residue 255 of the Aalpha-chain. Cleavage of the MDC-labeled alpha-chain by CNBr, however, localized most of its fluorescence (approximately 80%) to a fragment of molecular weight 29,000 which had the same NH2-terminal sequence as the labeled plasmic peptide of molecular weight 21,000. Both peptides were cleaved by ancrod into two acceptor site-containing peptides of approximately equal fluorescence. The preliminary NH2-terminal sequence analyses of these peptides, when combined with the above findings, indicated that these two other cross-link acceptor sites are in a peptide segment which comprises the middle 17% of the Aalpha-chain.     

Studies of the structure of fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase

Some physicochemical properties of a homogeneous preparation of a bifunctional enzyme, fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase, were determined. The molecular weight of the enzyme is 101 000 as determined by high-speed sedimentation equilibrium. The molecular weight of dissociated enzyme is 55 000 in 6 M guanidinium chloride by sedimentation equilibrium and in sodium dodecyl sulfate by polyacrylamide gel electrophoresis. A value of 4.7 was observed for the isoelectric point. Tryptic peptide maps and high-performance liquid chromatography of the trypsin-digested enzyme revealed approximately 60 peptides. Amino acid analysis of the enzyme shows that it contains 27 lysine and 36 arginine residues per 55 000 daltons. No free N-terminal amino acid residue was detectable, suggesting that it is blocked. Hydrolysis of the enzyme by carboxypeptidases A and B releases tyrosine followed by histidine and arginine, indicating that the amino acid sequence at the carboxyl terminus is probably -Arg-His-Tyr. Tryptic digestion of [32P]phosphofructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase yields a 32P-labeled peptide detected by tryptic peptide mapping and high-performance liquid chromatography. Thermolysin digestion of CNBr-cleaved 32P-enzyme also yields a single 32P-peptide. These results indicate that fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase is a dimer of 55 000 daltons and the subunits are very similar, if not identical.     

Evidence for preferential proteolytic cleavage of one of the two fibronectin subunits and for immunological localization of a site distinguishing them

Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin-binding and heparin-binding fractions were analyzed in NaDodSO4-polyacrylamide gel electrophoresis followed by immunoblotting with a defined monoclonal anti-fibronectin antibody. In early cathepsin G digests, several gelatin-binding fragments were detected: a few large (Mr greater than or equal to 150 000) polypeptides and fragments of Mr = 85 000, 72 000, 64 000 and 40 000. The 85 000-Mr and 64 000-Mr fragments appeared as closely spaced doublets and reacted with the antibody while the 72 000-Mr and 40 000-Mr fragments did not. Therefore the 64 000-Mr fragments are likely to be derived from the 85 000-Mr fragments. Three large fragments that bound to heparin, but not to gelatin were detected: Mr = 145 000, 135 000 and 120 000. Of these only the 135 000-Mr peptide reacted with the antibody. When fibronectin was digested with thrombin, polypeptides of Mr = 180 000-200 000 and a 30 000-Mr NH2-terminal fragment were produced. Cathepsin G added to this mixture further cleaved the fragments to a digestion pattern resembling that obtained from intact fibronectin except that the 85 000-Mr and 64 000-Mr fragments appeared as single bands and the amount of the 72 000-Mr fragment was reduced. The results suggest that thrombin cleaves the 30 000-Mr fragment preferentially from the NH2-terminal end of one of the two subunits of fibronectin and that the 85 000-Mr, 72 000-Mr and 64 000-Mr fragments obtained by the additional cathepsin G digestion were derived from the other chain. The results are consistent with the model that the antigenic determinant resides 72 000-85 000 Da from the NH2-terminus and is cleaved by cathepsin G alternatively at one of its sides. Thus, the components of the 85 000-Mr and 64 000-Mr doublets are derived from different subunits and the region located by the antibody may be responsible for the difference in their migration in the polyacrylamide gel.     

Comparative studies on the primary structure of human cystatin as from epidermis, liver, spleen, and leukocytes

We have studied the primary structure of human cystatin As from epidermis, liver, spleen, and leukocytes. These molecules were indistinguishable on PAGE in the presence and absence of SDS, by fast protein liquid chromatography (FPLC) chromatofocusing on a Mono P column, and in amino acid composition. The NH2- and COOH-terminal amino acid sequences of human cystatin As from epidermis, liver, and spleen were identical with those of human leukocyte cystatin A previously reported except for the lack of the NH2-terminal methionine residue in human epidermal cystatin A. The peptides obtained upon digestion of four human cystatin As with Achromobacter protease I (AP) showed identical peptide maps on HPLC except for different retention times of the NH2-terminal peptides. Furthermore, the amino acid compositions of corresponding separated peptide quartets were identical. We also determined the complete amino acid sequence of human epidermal cystatin A by sequencing peptides obtained from AP digestion and cyanogen bromide (CNBr) cleavage. It consisted of 97 amino acid residues, and was identical with those of human cystatin As from liver, spleen, and leukocytes except for the lack of the NH2-terminal methionine residue.     

Identification of kex2-related proteases in chromaffin granules by partial amino acid sequence analysis

We have characterized glycoprotein H (GpH) from bovine adrenal medullary chromaffin granules. Two-dimensional gel electrophoresis was used to purify GpH from an insoluble fraction obtained following extraction of chromaffin granule membranes with lithium diiodosalicylate. The GpH material was recovered from two-dimensional gel spots by concentration and recovery on a one-dimensional gel followed by electro-blotting to a poly(vinylidene difluoride) membrane. This material was subjected to in situ tryptic digestion. The released peptides were purified by microbore high performance liquid chromatography and sequenced. The peptide sequences revealed extensive similarity to the mammalian kex2/subtilisin-related proteases (PC2 and PC3) which have been characterized recently by molecular cloning and sequence analysis (Smeekens, S. P., and Steiner, D. F. (1990) J. Biol. Chem. 265, 2997-3000; Smeekens, S. P., Avruch, A. S., LaMendola, J., Chan, S. J., and Steiner, D. F. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 340-344). The sequence similarity included regions that contain residues equivalent to the aspartic acid and histidine residues which are involved in the active site of the subtilisin family of serine proteases. The sequence data revealed the presence of tryptic peptides derived from both PC2 and PC3. NH2-terminal sequence analysis of GpH gave two sequences which were aligned with residues 110-121 of PC2 and PC3. It is likely that these sequences represent the mature form of PC2 and PC3 in chromaffin granules. These forms would be generated by cleavage at a site which is conserved in mammalian kex2-related enzymes and which would result in the release of approximately 80-residue propeptides. It was concluded that the spot identified as GpH by two-dimensional gel electrophoresis contains the bovine counterparts of both PC2 and PC3. The direct identification of these components in chromaffin granules supports their role in the processing of protein precursors.     

Human tumor necrosis factor. Production, purification, and characterization

Human tumor necrosis factor (TNF) was purified to homogeneity from serum-free tissue culture supernatants of the HL-60 promyelocytic leukemia cell line induced by 4 beta-phorbol 12-myristate 13-acetate. The purification scheme consisted of controlled-pore glass and DEAE-cellulose chromatography, Mono Q-fast-protein liquid chromatography, and reverse-phase high performance liquid chromatography. The purified protein was homogeneous by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and NH2-terminal sequence analysis. The specific activity of purified tumor necrosis factor is approximately 10(8) units/mg. The protein has a molecular weight of approximately 17,000, an isoelectric point of 5.3, and contains two cysteines involved in a disulfide bridge. Approximately 50% homology between TNF and another cytolytic lymphokine, lymphotoxin, exists when the NH2-terminal 34 residues of TNF and internal sequence generated by tryptic, Staphylococcus aureus V8 protease, and chymotryptic digests of TNF are aligned with the complete amino acid sequence of lymphotoxin.     

Purification and characterization of a Ca2+/calmodulin-dependent protein kinase from rat brain

A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.     

Characterization of pepsin-resistant peptides present in a glycoprotein isolated from lung lavage of normal rabbit

A glycoprotein of Mr 36 000 has been isolated from lung lavage of normal rabbit and purified to homogeneity by gel chromatography. Three peptides containing hydroxyproline and nearly 30% glycine have been isolated and purified from pepsin-digested native glycoprotein. Partial NH2-terminal amino acid sequence analysis on one of the peptides indicated the presence of -Gly-Pro-Hyp-Gly- sequence in the peptide chain, suggesting that collagen-like region(s) may be present in this glycoprotein.     

Structural studies on a glycoprotein isolated from alveoli of patients with alveolar proteinosis.

A major glycoprotein 36 000 molecular weight) has been isolated from lung lavage of patients with alveolar proteinosis and found to contain five residues of hydroxyproline, fifty residues of glycine, three residues of methionine, 3 mol of sialic acid, 4.4 mol of mannose, 4.0 mol of galactose, 6.0 mol of glucosamine, and 1 mol of fucose. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted, as expected, in four peptides of apparent molecular weights of 18 000, 12 000, 5000 and 1000, respectively. The chemical compositions of the CNBr peptides indicate the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contain carbohydrate. Gel filtration, acrylamide gel electrophoresis and end-group analyses of the native glycoprotein and its CNBr peptides indicate that the peptides are homogeneous. End-group analyses of the CNBr cleavage products assign the 18 000 molecular weight peptide to the NH2-terminal portion and the 1000 molecular weight peptide to the COOH-terminal portion of the native glycoprotein molecule. Pronase digestion of the 36 000 molecular weight glycoprotein, followed by gel filtration and cation exchange chromatography, resulted in two fractions. One fraction was acidic and contained all the carbohydrate, a high content of aspartic acid and no hydroxyproline. The other fraction was basic and contained 8.4% hydroxyproline, 14% proline, 28% glycine and no carbohydrate, suggesting the presence of collagen-like sequence in the peptide chain. Paper electrophoresis of the basic fraction demonstrated two components, the amino acid compositions of which are identical to those of collagen. Partial amino-terminal sequence analysis of one of the CNBr peptides (18 000 molecular weight) indicated the presence of -Fly-Pro-HyP-Gly-sequence in the peptide chain, which confirms our suggestion that collagen-like regions are present in the native glycoprotein molecule. Limited acid hydrolysis of the acidic fraction and subsequent fractionation of the acid hydrolysate using Dowex column yielded a fraction which produced brown colour with ninhydrin reagent. Paper chromatography of this fraction demonstrated a large component which also stained brown with ninhydrin reagent. After acid hydrolysis, this component was found to consist of equal amounts of asparitic acid and glucosamine, indicating that the N-acetylglucosamine of the oligosaccharides is linked to the asparagine residue of the peptide. No serine or threonine linkages are present.     

Biosynthesis of human preapolipoprotein A-IV

The primary translation product of human intestinal apolipoprotein A-IV mRNA was purified from ascites and wheat germ cell-free systems. Comparison of its NH2-terminal sequence with mature, chylomicron-associated apo-A-IV revealed that apo-A-IV was initially synthesized with a 20-amino acid long NH2-terminal extension: Met-X-Leu-X-Ala-Val-Val-Leu-X-Leu-Ala-Leu-Val-Ala-Val-Ala-Leu-X-X-Ala. Co-translational cleavage of the cell-free product as well as Edman degradation of the stable intracellular form of the protein recovered from Hep G2 cells indicated that this entire 20-amino acid sequence behaved as a signal peptide. There is at least 55% sequence homology between the rat and human apo-A-IV signal peptides and 33% homology between the human A-I and A-IV presegments. Agarose gel chromatography of Hep G2 culture media indicated that neither apo-A-IV nor -A-I is associated with particles that have physical properties resembling any of the plasma lipoprotein density classes. Incubation of plasma with Hep G2 media resulted in transfer of A-I but not A-IV to lipoproteins. Since the NH2 termini of co-translationally cleaved and chylomicron-associated apo-A-IV are identical, it is apparent that 1) this polypeptide does not undergo NH2-terminal post-translational proteolysis like proapo-A-II or proapo-A-I, and 2) regulation of A-IV-lipoprotein interaction is not dependent on any NH2-terminal proteolytic processing event.     

Amino acid sequence of histone H1 at the ADP-ribose-accepting site and ADP-ribose X histone-H1 adduct as an inhibitor of cyclic-AMP-dependent phosphorylation

The ADP-ribosylation site of histone H1 from calf thymus by purified hen liver nuclear ADP-ribosyltransferase was determined and effects of the ADP-ribose X histone-H1 adduct on cAMP-dependent phosphorylation of the histone H1 were investigated. ADP-ribosylated histone H1 was prepared by incubation of histone H1, 1 mM [adenylate-32P]NAD and the purified ADP-ribosyltransferase. N-Bromosuccinimide-directed bisection of ADP-ribosylated histone H1 showed that the NH2-terminal fragment (Mr = 6000) was modified and contained serine residue 38, the site of phosphorylation by cAMP-dependent protein kinase. Digestion of the NH2-terminal fragment with cathepsin D and trypsin, and purification of this fragment, using high-performance liquid chromatography, yielded a radiolabelled single peptide corresponding to residues 29-34 of histone H1, containing the arginine residue as the ADP-ribosylation site. These results indicate that ADP-ribosylation of histone H1 occurs at the arginine residue 34, sequenced at the NH2-terminal side of the phosphate-accepting serine residue 38. Phosphorylation of histone H1 from calf thymus by cAMP-dependent protein kinase was markedly reduced when histone H1 was ADP-ribosylated. Kinetic studies of phosphorylation revealed that ADP-ribosylated histone H1 was a linear competitive inhibitor of histone H1 and a linear non-competitive inhibitor of ATP.     

Naturally occurring peptidoglycan variants of Streptococcus pneumoniae.

Analysis by high-performance liquid chromatography of the stem peptide composition of cell walls purified from a large number of pneumococcal strains indicates that these bacteria produce a highly conserved species-specific peptidoglycan independent of serotype, isolation date, and geographic origin. Characteristic features of this highly reproducible peptide pattern are the dominance of linear stem peptides with a monomeric tripeptide, a tri-tetra linear dimer, and two indirectly cross-linked tri-tetra dimers being the most abundant components. Screening of strains with the high-performance liquid chromatography technique has identified two naturally occurring peptidoglycan variants in which the species-specific stem peptide composition was replaced by two drastically different and distinct stem peptide patterns, each unique to the particular clone of pneumococci producing it. Both isolates were multidrug resistant, including resistance to penicillin. In one of these clones--defined by multilocus enzyme analysis and pulsed-field gel electrophoresis of the chromosomal DNAs--the linear stem peptides were replaced by branched peptides that most frequently carried an alanyl-alanine substituent on the epsilon amino group of the diamino acid residue. In the second clone, the predominant stem peptide species replacing the linear stem peptides carried a seryl-alanine substituent. The abnormal peptidoglycans may be related to the altered substrate preference of transpeptidases (penicillin-binding proteins) in the pneumococcal variants.     

Isolation of a 5,300-dalton peptide containing a pyridoxal phosphate binding site from the 38,000-dalton domain of band 3 of human erythrocyte membranes

A hydrophobic 5,300-dalton peptide was isolated from the 38,000-dalton domain of Band 3 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography. The peptide was affinity labeled with pyridoxal phosphate and sodium [3H]borohydride when erythrocytes were incubated in vitro. The peptide was not labeled with these agents when cells were incubated in the presence of a specific inhibitor of anion transport, suggesting that the peptide contains at least a part of the active center for the anion transport system in the cell membrane. The peptide was eluted from a reversed-phase high-performance liquid chromatography column with a high concentration of acetonitrile (more than 65%), although the elution pattern of the hydrophobic peptide was not as sharp as that of the soluble peptides. However, a satisfactory separation was achieved when this procedure was employed in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Isolation and characterization of a 60-kilodalton glycoprotein esterase from liver microsomal membranes

A glycoprotein having a subunit weight of approximately 60,000 was isolated from rabbit liver microsomes. It is a predominant component of the hepatic microsomal membrane and reacts rapidly with diisopropylphosphorofluoridate (DFP), resulting in the loss of enzymatic activity toward artificial substrates such as acyl esters of o-nitrophenols. Automated Edman degradation of this protein together with sequence analysis of peptides provided the NH2-terminal sequence of some 70 residues as follows: His-Pro-Ser- Ala-Pro-Pro-Val-Val-Asp-Thr-Val-Lys-Gly-Lys-Val- Leu-Gly-Lys-Phe-Val-Ser-Leu-Glu-Gly-Phe-Ala-Gln- Pro-Val-Ala-Val-Phe-Leu-Gly-Val-Pro-Phe-Ala-Lys- Pro-Pro-Leu-Gly-Ser-Leu-Arg-Phe-Ala-Pro-Pro-Gln- Pro-Ala-Glu-Ser-Trp-Ser-His-Val-Lys-Asn (CHO)- Thr-Thr-Ser-Tyr-Pro-Pro-Met-Cys-Ser-Ser. A carbohydrate attachment was identified at asparaginyl residue 61. The COOH-terminal peptide of the protein was isolated from two independent enzymatic digests, and its sequence was established as Arg-Glu-Thr-Glu-His-Ile-Glu-Leu. In order to isolate the DFP binding peptide, liver microsomes were labeled with [3H]DFP and the 60-kDa protein containing covalently bound DFP isolated in pure form. Following reduction and carboxymethylation, the DFP-labeled protein was fragmented with trypsin and the digest subjected to gel filtration. Digestion of the labeled peptide preparations with chymotrypsin followed by chromatography of the digest yielded two diisopropylphosphoryl (DIP) peptides. Automated Edman degradation of these peptides provided the following amino acid sequences: Gly-Glu-DIPSer- Ala-Gly-Gly-Gln-Ser-Val-Ser-Ile-Leu-Leu-Leu-Ser- Pro and Thr-Val-Ile-Gly-Asp-DIPHis-Gly-Asp-Glu-Ile-Phe. The active site serine peptide of the 60-kDa protein shows some 70% similarity to the active center region of choline esterases. While the postulated active histidyl residue in choline esterases has not been identified, it is proposed that the DFP binding histidine of the 60-kDa protein corresponds to His-438/440 of choline esterases.