Partial Tryptic Digestion Research Articles

High-performance liquid chromatography of amino acids, peptides and proteins : XCIX. Comparative study of the equilibrium refolding of bovine, procine and human growth hormone by size-exclusion chromatography

The equilibrium refolding of bovine, porcine and human growth hormone and ovine prolactin in guanidine hydrochloride has been investigated using high-performance size-exclusion chromatography (HPSEC). It was found that bovine an porcine growth hormones exhibited very similar refolding behaviour. However, the renaturation of human growth hormone followed a different pathway. In particular, the folding transition of human growth hormone occured at 4.7 M guanidine hydrochloride compared to 3.8 and 3.5 M for the bovine and porcine molecules, respectively, and 3.5 M for ovine prolactin. The refolding mechanism of an internally clipped fragment derived from partial tryptic digestion, exhibited similar folding properties to the corresponding intact molecule. The internally clipped analogue existed as a relatively larger molecule under fully denaturing conditions. Reduction followed by carboxymethylation resulted in growth hormone molecules with significantly reduced stability and altered folding properties. The results have been correlated with differences in structure to further demonstrated the utility of HPSEC in the study of protein folding and stability.

Hereditary Pyropoikilocytosis and Elliptocytosis in a White French Family With the Spectrin αI/74 Variant Related to a CGT to CAT Codon Change (Arg to His) at Position 22 of the Spectrin al Domain

We describe a white French family in which 12 subjects presented with hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP). Eight of these subjects were shown to be heterozygous for a spectrin (Sp) alpha I/74 variant, as demonstrated by analysis of partial tryptic digestion fragments of spectrin. This abnormal peptide pattern was associated with a decreased ability of Sp dimers to self-associate. In this kindred, in which four generations were available for study, the clinical expression varied from mild HE to HPP with an intermediate status of hemolytic HE. The severity of the disease appeared to be correlated both with the estimated amount of variant Sp (42% to 65%) and the excess of Sp dimers found in the membrane (30% to 51%, with a normal value of 3.7% +/- 1.6%). Reassociation studies using isolated Sp alpha and beta chains from an affected patient and an unaffected control subject showed that the Sp alpha I/74 Kd abnormal tryptic peptide resulted from a defect in the Sp alpha chain. Partial amino acid sequencing showed that the Sp alpha I/74 Kd peptide resulted from cleavage at lysine residue 42 of the Sp alpha I/80 Kd domain. Knowledge of the exon/intron organization of the human alpha Sp gene allowed us to amplify by the polymerase chain reaction the second exon of the alpha Sp gene in total cellular DNA of the HPP proposita. The amplified fragment was subcloned and sequenced. We found a G to A base substitution in the 22nd codon (CAT for CGT), which changes the normal arginine to a histidine. Hybridization of amplified DNAs with allele-specific oligonucleotides corresponding to the normal and mutant sequences confirmed the presence of the mutation in six other HE and HPP members of the family. The identification of this mutation at the DNA level confirmed the transmission of the same molecular defect in Sp through four generations but with different patterns of clinical expression.

Hereditary pyropoikilocytosis and elliptocytosis in a white French family with the spectrin alpha I/74 variant related to a CGT to CAT codon change (Arg to His) at position 22 of the spectrin alpha I domain

We describe a white French family in which 12 subjects presented with hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP). Eight of these subjects were shown to be heterozygous for a spectrin (Sp) alpha I/74 variant, as demonstrated by analysis of partial tryptic digestion fragments of spectrin. This abnormal peptide pattern was associated with a decreased ability of Sp dimers to self-associate. In this kindred, in which four generations were available for study, the clinical expression varied from mild HE to HPP with an intermediate status of hemolytic HE. The severity of the disease appeared to be correlated both with the estimated amount of variant Sp (42% to 65%) and the excess of Sp dimers found in the membrane (30% to 51%, with a normal value of 3.7% +/- 1.6%). Reassociation studies using isolated Sp alpha and beta chains from an affected patient and an unaffected control subject showed that the Sp alpha I/74 Kd abnormal tryptic peptide resulted from a defect in the Sp alpha chain. Partial amino acid sequencing showed that the Sp alpha I/74 Kd peptide resulted from cleavage at lysine residue 42 of the Sp alpha I/80 Kd domain. Knowledge of the exon/intron organization of the human alpha Sp gene allowed us to amplify by the polymerase chain reaction the second exon of the alpha Sp gene in total cellular DNA of the HPP proposita. The amplified fragment was subcloned and sequenced. We found a G to A base substitution in the 22nd codon (CAT for CGT), which changes the normal arginine to a histidine. Hybridization of amplified DNAs with allele- specific oligonucleotides corresponding to the normal and mutant sequences confirmed the presence of the mutation in six other HE and HPP members of the family. The identification of this mutation at the DNA level confirmed the transmission of the same molecular defect in Sp through four generations but with different patterns of clinical expression.

Sequence and exon-intron organization of the DNA encoding the alpha I domain of human spectrin. Application to the study of mutations causing hereditary elliptocytosis.

We have determined the exon-intron organization and the nucleotide sequence of the exons and their flanking intronic DNA in cloned genomic DNA that encodes the first 526 amino acids of the alpha I domain of the human red cell spectrin polypeptide chain. From the gene sequence we designed oligonucleotide primers to use in the polymerase chain reaction technique to amplify the appropriate exons in DNA from individuals with three variants of hereditary elliptocytosis characterized by the presence of abnormal alpha I spectrin peptides, 46-50 and 65-68 kD in size, in partial tryptic digests of spectrin. The alpha I/68-kD abnormality resulted from a duplication of leucine codon 148 in exon 4: TTG-CTG to TTG-TTG-CTG. The alpha I/50a defect was associated in different individuals with two separate single base changes in exon 6: CTG to CCG (leucine to proline) encoding residue 254, and TCC to CCC (serine to proline) encoding residue 255. In another individual with the alpha I/50a polypeptide defect, the nucleotide sequence encoding amino acid residues 221 through 264 was normal. The alpha I/50b abnormality resulted from a single base change of CAG (glutamine) to CCG (proline) encoding residue 465 in exon 11 in two unrelated individuals. In a third individual with alpha I/50b-kD hereditary elliptocytosis, the entire exon encoding residues 445 through 490 was normal. The relationship of the alpha I domain polypeptide structure to these mutations and the organization of the gene is discussed.

Induction of ovalbumin-specific cytotoxic T cells by in vivo peptide immunization.

CTL recognize peptide forms of processed, foreign antigens in association with class I molecules encoded by the MHC and are usually directed against endogenously synthesized "cellular antigens," such as those expressed by virus-infected cells. In vitro studies have shown that small exogenous peptides can directly associate with class I molecules on the cell surface and mimic the target complex derived by intracellular processing and presentation. We have recently generated OVA-specific, H-2Kb-restricted CTL by immunizing C57BL/6 mice with a syngeneic tumor line transfected with the OVA cDNA. The CTL recognize the OVA transfectant E.G7-OVA and the synthetic peptide OVA258-276, but fail to recognize the native protein. We reasoned that given the potential for direct peptide/class I association observed in vitro, OVA258-276 may induce CTL after in vivo priming. However, we found that this is not the case. OVA258-276 and peptides of increasing lengths up to OVA242-276 and OVA242-285, which are all able to form the target complex in vitro, are inefficient at priming E.G7-OVA-specific CTL responses after intravenous injection. This is also true for both native and denatured OVA. In contrast to these results the synthetic peptide OVA229-276 corresponding to a peptide in a partial tryptic digestion of OVA can efficiently prime C57BL/6 mice in vivo after intravenous injection. This peptide elicits CTL that appear identical to those derived from animals immunized with syngeneic cells producing OVA endogenously. These results are discussed in terms of separate class I and class II antigen presentation pathways and the ability of only certain, exogenous antigens to enter the cytoplasmic, class I pathway.

High-yield purification of platelet-derived endothelial cell growth factor: structural characterization and establishment of a specific antiserum

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa protein that stimulates the growth of endothelial cells [Miyazono, K., et al. (1987) J. Biol. Chem. 262, 4098-4103]. Here, we describe a method to purify large quantities of PD-ECGF from human platelet lysate at a high yield (14% overall recovery). The purification method involves five steps, using high-performance liquid chromatography grade hydroxylapatite and hydrophobic chromatographies as the two final steps. The purified material contained two major components of apparent molecular weight values of 46,000 and 44,000. These components coeluted in a high-resolving reversed-phase chromatography and were found to give similar peptide maps after treatments with staphylococcal V8 protease, suggesting that the 44-kDa form is related to the 46-kDa molecule. Partial tryptic digestion of native PD-ECGF revealed that the molecule contains a trypsin-resistant domain of 37-39 kDa. A rabbit antiserum was produced against the purified material and was found to specifically recognize PD-ECGF in immunoblotting. When added to the cell culture medium, an immunoglobulin fraction of the antiserum neutralized the activity of purified PD-ECGF. Furthermore, it completely neutralized the endothelial cell mitogenic activity of platelet lysate, indicating that PD-ECGF is the only mitogen in platelet lysate for this cell type.

Nucleolin (C23), a physiological substrate for casein kinase II

Nucleolin (C23), a 110 kDa phosphoprotein, which is mainly found in the nucleolus has been shown to be a physiological substrate for casein kinase II (CKII). Nucleolin was identified and characterized by immunodetection using an anti-nucleolin antibody. Phosphopeptide patterns from nucleolin phosphorylated by purified casein kinase II and of phosphorylated nucleolin which had been isolated from tumor cells grown in the presence of [32P]-o-phosphate, were identical. The partial tryptic digest revealed nine phosphopeptides. Nucleolin isolated from Krebs II mouse ascites cells was phosphorylated by purified casein kinase II with about two moles phosphate per one mole of nucleolin.

Properties of a human insulin receptor with a COOH-terminal truncation. I. Insulin binding, autophosphorylation, and endocytosis.

In order to test the contribution of the insulin receptor COOH terminus to insulin action, a truncation of 43 COOH-terminal amino acids was engineered by cDNA-based deletion mutagenesis. This cDNA (HIR delta CT), as well as cDNA encoding the complete receptor (HIRc) was transfected into Rat 1 fibroblasts. Cells expressing 6.4 X 10(3) and 1.25 X 10(6) normal receptors and 2.5 X 10(5) HIR delta CT receptors, as well as control Rat 1 fibroblasts were selected for further analysis. All cell lines exhibited insulin binding of similar affinity. Partial tryptic digestion and immunoprecipitation by region-specific antibodies verified that the HIR delta CT receptors were truncated at the COOH terminus. Purified HIRc and HIR delta CT receptors underwent autophosphorylation with similar insulin and ATP sensitivity, although the HIR delta CT receptors were slightly more active in the absence of insulin. Transfected HIRc and HIR delta CT receptors undergo endocytosis in a normal fashion. Insulin internalization and degradation in both HIRc and HIR delta CT cells is increased in proportion to receptor number. Intracellular insulin processing, degradation, and release were qualitatively comparable among the transfected cell lines. Complete and truncated receptors internalize, recycle, and down-regulate normally. We conclude the following: 1) the COOH-terminal portion of the insulin receptor is not necessary for partial autophosphorylation or endocytosis; 2) following internalization the intracellular itinerary of the receptor and ligand appear normal with the truncated receptor; and 3) truncation of the COOH terminus does not impair recycling of the receptor or retroendocytosis of internalized ligand.

Drosophila melanogaster H1 histone is phosphorylated stably.

Phosphorylation of histone H1 is developmentally regulated in Drosophila spp. It cannot be detected in preblastoderm embryos or polytene salivary gland cells, but in cellular blastoderm, postblastoderm embryo, and amitotic adult head nuclei, it occurs with a frequency of roughly 4 x 10(5) molecules per nucleus. We used pulse-labeling to study the relationship between H1 synthesis and modification in cultured cells. These results reveal that the H1-associated phosphate is stable and suggest that Drosophila H1 is synthesized, translocated to the nucleus, associated with chromatin, and then phosphorylated. Partial tryptic digestion of Drosophila H1 revealed that the phosphorylation site is located within the globular, central domain of the protein. Thus, the developmentally regulated phosphorylation of Drosophila H1 presents two contrasts with previously studied H1 phosphorylation. It is not correlated with DNA replication, and it is located in the central domain of the protein.

Drosophila melanogaster H1 histone is phosphorylated stably.

Phosphorylation of histone H1 is developmentally regulated in Drosophila spp. It cannot be detected in preblastoderm embryos or polytene salivary gland cells, but in cellular blastoderm, postblastoderm embryo, and amitotic adult head nuclei, it occurs with a frequency of roughly 4 x 10(5) molecules per nucleus. We used pulse-labeling to study the relationship between H1 synthesis and modification in cultured cells. These results reveal that the H1-associated phosphate is stable and suggest that Drosophila H1 is synthesized, translocated to the nucleus, associated with chromatin, and then phosphorylated. Partial tryptic digestion of Drosophila H1 revealed that the phosphorylation site is located within the globular, central domain of the protein. Thus, the developmentally regulated phosphorylation of Drosophila H1 presents two contrasts with previously studied H1 phosphorylation. It is not correlated with DNA replication, and it is located in the central domain of the protein.

Mapping regions of the matrix protein of vesicular stomatitis virus which bind to ribonucleocapsids, liposomes, and monoclonal antibodies

The matrix (M) protein of vesicular stomatitis virus (VSV) appears to function as a bridge between the ribonucleocapsid (RNP) core and the envelope in assembly of the virion. Two such properties would necessitate at least one site for interaction with the nucleocapsid and one with the envelope. In this study M protein was found to mediate the in vitro binding to RNP cores of phospholipid vesicles, representing membrane structures. The M protein could bind initially to either the vesicles or the RNP cores to promote RNP-vesicle association. A trypsin-resistant fragment (MT) of M protein, missing the initial 43 amino acids from its amino terminus, reconstituted with acidic phospholipid vesicles with the same binding efficiency as did whole M protein, suggesting that the carboxy-terminal 81% retained those regions of the M protein which interact with a lipid bilayer. The MT protein, however, was considerably less efficient than intact M protein as an inhibitor of in vitro virus transcription; almost 2.5-fold more MT protein than intact M protein was required for 50% inhibition of VSV transcription, indicating that a site for interaction with the RNP core may have been lost. A monoclonal antibody which is able to reverse the in vitro inhibition of transcription by M protein did not react by immunoblotting with MT protein. Partial tryptic digests of the M protein probed with this monoclonal antibody indicated that epitope 1 lies between amino acid residues 18 and 43. This region appears to be a site that promotes interaction of the M protein with the RNP core of VSV. Monoclonal antibodies to epitopes 2 and 3, which exhibit some overlap in binding to M protein but do not reverse transcription inhibition, were mapped by cleavage with N-chlorosuccinimide at regions in a carboxy direction from epitope 1.

Bombesin-like peptides in human endocrine tumors: quantitation, biochemical characterization, and secretion.

Bombesin-like immunoreactivity (BLI) in 20 human endocrine tumors was studied using an antiserum directed toward the C-terminal region of bombesin. Additionally, plasma BLI was assayed in normal subjects and patients with known BLI-containing tumors. In 7 tumors (medullary carcinoma of the thyroid, n = 2; carcinoid of the lung, n = 3; hepatic carcinoid, n = 1; pheochromocytoma, n = 1), the BLI content ranged from 6-2000 pg/mg wet wt of tissue. Sephadex G-50 gel chromatography of tumor extracts under acid-dissociating conditions revealed 2 peaks of BLI: 1 coeluting with porcine gastrin-releasing peptide (GRP) and 1 with bombesin. Reverse phase ODS silica HPLC analysis of the G-50 peaks using a methanol-trifluoroacetic acid gradient showed that human tumor BLI more closely resembled porcine GRP and its C-terminal fragment GRP-(14-27) than bombesin itself. Partial tryptic digestion of the tumor GRP-like peptide generated a product which, on HPLC, was similar to GRP-(14-27). Elevated plasma BLI was detected in the peripheral circulation of three subjects and in the vessels draining the tumor metastases of one of these patients. BLI was undetectable in normal subjects. These results indicate 1) that BLI is present in and may be secreted by various human endocrine tumors, and 2) that human tumor BLI closely resembles porcine GRP and its C-terminal fragment GRP-(14-27).

Mammalian single-stranded DNA binding proteins and heterogeneous nuclear RNA proteins have common antigenic determinants.

Antibodies were raised in rabbit against a pure subset of calf thymus single-stranded DNA binding proteins (ssDBPs) and purified by affinity chromatography on antigen-Sepharose. In Western blot experiments these antibodies were shown to react to the same extent with the whole family of bovine ssDBPs, as well as with ssDBPs from HeLa cells. When used to stain total cell extracts from both calf thymus and HeLa cells the antibodies reacted only with bands corresponding to the ssDBPs and with a set of bands of higher molecular weight, whose electrophoretic pattern matched that of the 40S hnRNP core proteins. In effect we observed that purified 40S hnRNP core proteins from HeLa cells were strongly reactive with the antibodies. Moreover after partial tryptic digestion HeLa cells ssDBPs and hnRNPs produced immunoreactive fragments of the same molecular weight and isoelectric point. Extensive structural homologies can thus be evidenced between these two classes of proteins, which share the property of selective binding to single-stranded nucleic acids.

Specific in vitro adenylylation of the simian virus 40 large tumor antigen.

Incubation of the simian virus 40 (SV40) large tumor antigen (T) from either transformed or lytically infected cells with adenosine [8-3H]-, [alpha-32P]-, or [alpha-[35S]thio]-triphosphate in the presence of Mg2+ resulted in its labeling as defined by the appearance of an intact, appropriately immunoreactive band in NaDodSO4/polyacrylamide gels. Radioactivity remained associated with the protein after boiling in buffer containing 3% NaDodSO4, and 2-mercaptoethanol as well as after heating in 0.1 M HCl, 0.1 M NH4OH, or hydroxylamine, but it was dissociated after incubation in 0.1 M NaOH at 37 degrees C. After limited boiling of gel-purified [alpha-32P] ATP + T complex in 5.6 M HCl, o-[32P]phosphoserine was released, and snake venom phosphodiesterase or 0.5 M piperidine treatment of such a complex resulted in the liberation of [alpha-32P]AMP. The reaction proceeded when either purified, soluble T or insoluble, specifically immunoprecipitated antigen was used as substrate. ATP and dATP were the preferred nucleotide substrates by comparison with the other six standard ribonucleoside or deoxynucleoside triphosphates. Partial tryptic digests of T + [alpha-32P]ATP complexes revealed the presence of a single labeled peptide of Mr approximately equal to 12 - 14 X 10(3), and after exhaustive digestion, there was a single radioactive spot in the fingerprint. These data indicate that T can be adenylylated at a specific seryl residue(s) in a limited portion of the protein surface. Furthermore, adenylylation appears to be reversible and to proceed by a pyrophosphorylytic mechanism, since the nucleotide was released from the protein following incubation of adenylylated T with Mg2+, sodium pyrophosphate, and poly(dT).

Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line.

A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The IE94 gene, which maps at coordinates 0.71 to 0.73 in the viral genome, contains a large intron in the 5' leader sequence, and its promoter regulatory region contains novel, multiple-palindromic, repeated elements. Two recombinant plasmids (pTJ148 and pTJ198) containing the 10.5-kilobase-pair HindIII-H DNA fragment from CMV (Colburn) were transfected into mouse Ltk- cells, by either linked or unlinked coselection in hypoxanthine-aminopterin-thymidine medium, together with herpes simplex virus thymidine kinase genes. With both procedures, constitutive synthesis of the IE94 immediate-early protein was detected in pools of Ltk+ cells by immunoprecipitation. Subsequently, we isolated a clonal Ltk+ cell line which expressed the [35S]methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH2p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH2p198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.

Organization of the coiled-coils in the wool microfibril

Two helix-enriched fragments were isolated from a partial tryptic digest of the microfibrillar proteins of wool. From the known sequences of two of the constituent microfibrillar polypeptides, components 7c and 8c-1, one of these fragments was identified as arising from the N-terminal half of the helix-rich region and the other from the C-terminal half of this region. On the basis of the molecular weight in benign and dissociating solvents, crosslinking with dimethylsuberimidate, and amino acid and sequence analyses the N-terminal fragment was shown to be a tetrameric structure, consisting of two segments from each of the components 7 and 8 families. The C-terminal fragment was shown to be a two-stranded coiled-coil containing one chain segment from each of components 7 and 8. The evidence is not compatible with anything but a two-stranded coiled-coil as the basic molecular structure for the wool microfibril and provides experimental proof of a parallel arrangement of the chains. The thermal stabilities of the two fragments and of the parent microfibrillar complex from which they are derived are very similar indicating that the non-helical end regions of the polypeptide chains do not greatly affect the stability of the α-helical rod domain.

Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line.

A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The IE94 gene, which maps at coordinates 0.71 to 0.73 in the viral genome, contains a large intron in the 5' leader sequence, and its promoter regulatory region contains novel, multiple-palindromic, repeated elements. Two recombinant plasmids (pTJ148 and pTJ198) containing the 10.5-kilobase-pair HindIII-H DNA fragment from CMV (Colburn) were transfected into mouse Ltk- cells, by either linked or unlinked coselection in hypoxanthine-aminopterin-thymidine medium, together with herpes simplex virus thymidine kinase genes. With both procedures, constitutive synthesis of the IE94 immediate-early protein was detected in pools of Ltk+ cells by immunoprecipitation. Subsequently, we isolated a clonal Ltk+ cell line which expressed the [35S]methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH2p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH2p198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.

Purification and phosphorylation of fructose-1,6-bisphosphatase from Kluyveromyces fragilis.

Fructose-1,6-bisphosphatase from the yeast Kluyveromyces fragilis was found to have an apparent Mr = 155,000 and to be composed of four Mr = 35,000 subunits. The extent and rate of phosphorylation of fructose-1,6-bisphosphatase (Fru-1,6-P2) by yeast cAMP-dependent protein kinase were dependent on fructose-1,6-bisphosphatase inhibitors, 5'-AMP and fructose 2,6-bisphosphate (Fru-2,6-P2). In the absence of inhibitor, the enzyme was slowly phosphorylated with a maximum incorporation of 1 mol of phosphate/mol of enzyme. The presence of both inhibitors greatly increased the phosphorylation rate with a maximum incorporation of 2 mol of phosphate/mol of enzyme. The presence of only one inhibitor led to an intermediate rate of phosphorylation with 2 mol of phosphate incorporated/mol of enzyme. There was no significant change in enzymatic activity after phosphorylation. The estimated sedimentation coefficient of fructose-1,6-bisphosphatase was lowered by 5'-AMP from 8.2 to 5.7 while Fru-2,6-P2 increased the S value to 8.5. The presence of either Fru-1,6-P2 or Fru-2,6-P2 prevented the 5'-AMP lowering of S value. The susceptibility of enzyme to partial tryptic digestion was not changed by the presence of 5'-AMP. The presence of both Fru-2,6-P2 and 5'-AMP led to the protection of Mr = 35,000 subunit from tryptic digestion while Fru-2,6-P2 alone led to a protection of an Mr = 30,000 peptide fragment. This peptide fragment did not contain the phosphorylation sites. Our results suggest that the rapid regulation of fructose-1,6-bisphosphatase following glucose addition is controlled mainly by enzyme inhibitors.

Molecular architecture of a light-harvesting antenna. Quaternary interactions in the Synechococcus 6301 phycobilisome core as revealed by partial tryptic digestion and circular dichroism studies.

The core of the phycobilisomes of Synechococcus 6301 (Anacystis nidulans) strain AN112 consists of two cylindrical elements each made up of the same four distinct subcomplexes: A (alpha AP beta AP)3; B (alpha AP beta AP)2 . 18.3K . 75K; C (alpha 1APB alpha 2AP beta 3AP) . 10.5K; and D (alpha AP beta AP)3 . 10.5K, where alpha AP and beta AP are the subunits of allophycocyanin, alpha APB is the subunit of allophycocyanin B, and 18.3K, 75K, and 10.5K are polypeptides of 18,300, 75,000, and 10,500 Da, respectively. An 18 S subassembly containing subcomplexes A and B has previously been characterized (Yamanaka, G., Lundell, D. J., and Glazer, A. N. (1982) J. Biol. Chem. 257, 4077-4086; Lundell, D. J., and Glazer, A. N. (1983) J. Biol. Chem. 258, 894-901, 902-908). A ternary core subassembly, containing complexes A, B, and C, was isolated from a limited tryptic digest of AN112 phycobilisomes and characterized with respect to composition and spectroscopic properties. Isolation of this ternary subassembly also establishes that subcomplex D must occupy a terminal position in each of the two core cylinders. Spectroscopic studies of the individual complexes, A-D, of the subassemblies AB and ABC, and of intact AN112 phycobilisomes showed core assembly-dependent changes in the circular dichroism spectra indicative of changes in the environment and/or conformation of the bilin chromophores within the individual subcomplexes. Two terminal energy acceptors are present in the phycobilisome core, alpha APB and 75K. No indication of interaction between the chromophores on these polypeptides was detected by circular dichroism spectroscopy. This result indicates that the bilins on alpha APB and 75K act as independent energy acceptors rather than as exciton pairs.

Heterogeneity in anticatalytic activity of antibody specific for horseradish peroxidase

Rabbit antibodies specific for horseradish peroxidase are heterogeneous in their ability to inhibit enzyme activity. Heterogeneity was demonstrated by fractionation of the total antiperoxidase pool by differential ammonium sulfate precipitation and differential elution of antibody from enzyme affinity columns. Both fractionation methods yielded antibody subpopulations that differed in anticatalytic activity. Some antibody subpopulations decreased enzyme activity almost completely at low molar ratios of antibody to peroxidase. Other subpopulations were not effective inhibitors even at great molar excess. Admixture experiments demonstrated that inefficient antibody pools decreased the anticatalytic effect of highly inhibitory antibody. The degree of inhibition observed with unfractionated antiserum is a reflection of the interaction of various antibody subpopulations with the enzyme. No correlation was found between the immunoglobulin class of antiperoxidase and anticatalytic efficiency in analyses of numerous antisera. The determinant specificity of an antiperoxidase molecule determines its anticatalytic ability. A peptide fragment (mol. wt 22,500) of peroxidase prepared by partial tryptic digestion bount 60 to 70% of the total antiperoxidase in a number of antisera. However, the peptide did not bind inhibitory antibodies.