Dipeptidyl peptidase IV (DPP-IV) from pig kidney cleaves analogs of bovine growth hormone-releasing factor (bGRF) modified at position 2 with Ser, Thr or Val. Extended DPP-IV substrate specificity?

The literature reported DPP-IV substrate specificity includes oligopeptides with a penultimate Pro, Hyp or Ala residue. Bovine GRF is a substrate for DPP-IV and is rapidly degraded by the enzyme via removal of its N-terminal Tyr-Ala. Incubation of selected GRF analogs from the [X2,Ala15,Leu27]bGRF(1-29)NH2 series with a porcine-kidney-derived DPP-IV in PBS (pH 7.

Structural alterations in the peptide backbone of beta-amyloid core protein may account for its deposition and stability in Alzheimer's disease.

The structure of beta-amyloid (beta A) from Alzheimer disease brains was examined to determine if post-translational modifications might be linked to the abnormal deposition of this peptide in the diseased tissue. The beta A peptides were isolated from the compact amyloid cores of neuritic plaques and separated from minor glycoprotein components by size-exclusion high-pressure liquid chromatography (HPLC). This parenchymal beta A has a maximal length of 42 residues, but shorter forms with "ragged" NH2 termini are also present.

Alzheimer's disease and control brain contain soluble derivatives of the amyloid protein precursor that end within the beta amyloid protein region.

The 39-43 amino acid beta amyloid protein (A beta) that deposits as amyloid in the brains of patients with Alzheimer's disease (AD) is encoded as an internal sequence within a larger membrane-associated protein known as the amyloid protein precursor (APP). In cultured cells, the APP is normally cleaved within the A beta to generate a large secreted derivative and a small membrane-associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire A beta.

Chloroplast and cytoplasmic enzymes: isolation and sequencing of cDNAs coding for two distinct pea chloroplast aldolases.

Two cDNAs which correspond to two very similar Class I aldolases have been isolated from a pea (Pisum sativum L.) cDNA library. With the exception of one codon they match the experimentally determined N-terminal sequence of a pea chloroplast aldolase.

Purification and characterization of heterodimeric human immunodeficiency virus type 1 (HIV-1) reverse transcriptase produced by in vitro processing of p66 with recombinant HIV-1 protease.

Active recombinant reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) with an amino-terminal extension containing a hexa-histidine sequence has been prepared in milligram quantities in a pure heterodimeric (p66/p51) form by coordinated applications of immobilized metal affinity chromatography (IMAC) and HIV-1 protease treatment. The precursor protein, isolated from extracts of recombinant Escherichia coli by IMAC in a predominantly unprocessed form (p66), migrated on sodium dodecyl sulfate-polyacrylamide gels as a 66-kDa band with minor heterogeneity at lower relative molecular mass. Incubation of this protein with recombinant HIV-1 protease produced a stable heterodimeric RT that was purified in a single step by IMAC.

Chemical synthesis of a biotinylated derivative of the simian immunodeficiency virus protease. Purification by avidin affinity chromatography and autocatalytic activation.

The protease from simian immunodeficiency virus (SIV) was chemically synthesized by automated solid-phase technology as an NH2-terminally extended derivative, capped with biotin. Biotin-linker-(SIV protease (1-99)): the linker segment, Gly-Gly-Asp-Arg-Gly-Phe-Ala-Ala, corresponds to the amino acid sequence preceding that of the protease in the SIV gag/pol precursor polyprotein. Accordingly, the Ala-Pro bond joining the octapeptide linker to the protease constitutes a site naturally cleaved by the protease during viral maturation.

The Hsp56 component of steroid receptor complexes binds to immobilized FK506 and shows homology to FKBP-12 and FKBP-13.

Extracts from human Jurkat cells or from calf thymus contain a 60-kDa protein that is bound to immobilized FK506. As expected, the NH2-terminal sequences of the 60-kDa protein from these two species were found to be nearly the same. We were surprised to discover, however, that the sequence of the human protein was identical to that of Hsp56, a heat shock protein of unknown function that has been shown to be a component of several steroid receptor complexes.

Alzheimer's amyloid precursor protein produced by recombinant baculovirus expression. Proteolytic processing and protease inhibitory properties.

The baculovirus expression system was used to generate recombinant Alzheimer's amyloid precursor (AAP) proteins. Recombinant baculoviruses were constructed, designed to express full-length 695-, 751-, and 770-amino acid forms. Recombinant baculoviruses designed for constitutive secretion were engineered by placing a termination codon between the beta-protein domain and cytoplasmic anchor of the full-length forms.

Actin, troponin C, Alzheimer amyloid precursor protein and pro-interleukin 1 beta as substrates of the protease from human immunodeficiency virus.

We show here for the first time that actin, troponin C, Alzheimer amyloid precursor protein (AAP), and pro-interleukin 1 beta (pro-IL-1 beta), are substrates of the protease encoded by the human immunodeficiency virus (HIV) type-1. As has been seen in other non-viral protein substrates of the HIV protease, the presence of Glu residues in the P2' position appears to play an important role in substrate recognition. Three of the four bonds cleaved in actin, two of the three in troponin C, and all of the bonds hydrolyzed in AAP and pro-IL-1 beta have a P2' Glu residue.

Degradation of growth hormone releasing factor analogs in neutral aqueous solution is related to deamidation of asparagine residues. Replacement of asparagine residues by serine stabilizes.

The incubation of a solution of the human growth hormone releasing factor analog, [Leu27] hGRF(1-32)NH2 at pH 7.4 and 37 degrees, resulted in extensive degradation of the sample. The major degradation products were identified as the peptides [beta-Asp8, Leu27] hGRF(1-32)NH2 and [alpha-Asp8, Leu27] hGRF(1-32)NH2, produced by deamidation of the Asn8 residue.

Ribonuclease A as a substrate of the protease from human immunodeficiency virus-1.

Bovine pancreatic ribonuclease A (RNase) contains two bonds, Met29-Met30 and Tyr92-Pro93 which are representative of sites in the human immunodeficiency virus-1 (HIV-1) gag polyprotein precursors that are cleaved by the HIV-1 protease during viral maturation. Nevertheless, neither native nor performic acid-oxidized RNase is a substrate for the protease. However, RNase derivatives obtained by reduction and S-alkylation with iodoacetate or iodoacetamide undergo cleavage by the HIV-1 protease at a single site, Ala109-alkyl-Cys110, that is distinct from either of the two predicted bonds mentioned above.

Amino acid sequence of a 12-kDa inhibitor of protein kinase C.

The complete primary structure of a bovine-brain-derived inhibitor of protein kinase C has been established. Fragments of the purified protein were obtained by cleavage with cyanogen bromide, Staphylococcus aureus V8 protease, trypsin and chymotrypsin. Subsequent analysis of the resulting fragments by fast-atom-bombardment mass spectrometry and Edman degradation revealed a calculated molecular mass of 11,779 Da with the following 107-amino-acid sequence: [sequence: see text] This inhibitor does not share significant primary structural identity with any other known protein.

Purification and characterization of interleukin 1 receptor level antagonist proteins from THP-1 cells.

Culture medium conditioned by phorbol 12-myristate 13-acetate-differentiated THP-1 cells contained interleukin 1 (IL-1) antagonist activity as measured by inhibition of both IL-1 beta binding to receptors on YT cells and inhibition of IL-1/phytohemagglutinin-stimulated IL-2 synthesis by LBRM-33-1A5 T cells. Based on their ability to compete for 125I-IL-1 beta binding to receptors on YT cells, four distinct antagonist proteins were purified from THP-1 cell conditioned medium using a combination of ion-exchange, hydrophobic interaction, and size exclusion chromatographies. The four proteins had different isoelectric points with molecular masses in the range 22-26 kDa and had similar specific activities for inhibition of IL-1 beta binding to cell surface receptors (Ki values 0.

Amino acid sequence and characterization of a protein inhibitor of protein kinase C.

The complete primary structure has been determined for an inhibitor protein of protein kinase C. The bovine brain-derived inhibitor has a pI of 6 and its N-terminal alanine residue is blocked by acetylation. Fragments obtained by chemical and enzymatic cleavage of the purified inhibitor were analyzed by Edman degradation, fast atom bombardment mass spectrometry, and tandem mass spectrometry.

Biotinylation of reactive amino groups in native recombinant human interleukin-1 beta.

Recombinant human interleukin-1 beta (rIL-1 beta) was chemically modified by a 10-fold molar excess (reagent:protein) of sulfosuccinimidyl 6-(biotinamido) hexanoate (sulfo-NHS-LC-biotin) or sulfosuccinimidobiotin (sulfo-NHS-biotin) under mild conditions. The primary product was purified in each case by cation exchange high performance liquid chromatography (HPLC) and digested with endoproteinase Lys C. Peptide mapping by C18 reverse phase HPLC permitted identification of three sites of biotinylation using both reagents; N-terminal alanine, lysine 93, and lysine 94.

Dimerization and activation of porcine pancreatic phospholipase A2 via substrate level acylation of lysine 56.

The porcine pancreatic phospholipase A2-catalyzed hydrolysis of the water-soluble chromogenic substrate 4-nitro-3-octanoyloxybenzoate shows an initial latency phase similar to the one observed in the hydrolysis of aggregated phospholipids by the same enzyme. We report here that during the latency phase the enzyme undergoes a slow, autocatalytic, substrate-level acylation whereby in a few of the catalytic events the scissile octanoyl group of the substrate, normally transferred to water, is transferred to the epsilon-amino group of lysine 56. The N epsilon 56-octanoylphospholipase shows a strong tendency to dimerize in solution and thus may be separated from the monomeric native enzyme by gel filtration.

A structural model to explain the partial catalytic activity of human prorenin.

Human prorenin, secreted from a Chinese hamster ovary cell line transfected with the cDNA for preprorenin, has been purified in mg quantities by a novel single-step procedure. The method takes advantage of reversible acid activation as a means of generating active prorenin that may be bound and eluted from an affinity column for renin. Analysis of the prorenin so purified revealed that it contained about 80% intact zymogen; the remaining 20% comprised a mixture of various prorenin derivatives truncated in the prosegment, and a small amount of renin.

Evidence for the presence of a carbohydrate moiety in fluorescein isothiocyanate labeled fragments of rat gastric (H+-K+)-ATPase.

Limited tryptic digestion of fluorescein isothiocyanate (FITC)-labeled (H+-K+)-ATPase from rat resting light gastric membranes produced a soluble 27-kDa polypeptide which retained the fluorescence of the parent enzyme. Its production was markedly enhanced in the presence of an amphiphilic detergent, Zwittergent 3-14, which potently inhibits the ATPase activity. This increase is probably due to protection of certain tryptic cleavage sites through conformational changes of the membrane enzyme by the detergent.

Purification and characterization of invertase from a novel industrial yeast, Schwanniomyces occidentalis.

The use of yeast as an expression system for heterologous proteins offers several potential advantages with respect to industrial scale-up and genetics over other expression systems, but suffers from several drawbacks. For example, the secreted proteins of S. cerevisiae, found in the periplasm, are hyperglycosylated and the organism has a limited range of usable substrates.

Atrial peptide inactivation by rabbit-kidney brush-border membranes.

Atriopeptin (AP) 24, containing amino acids Ser103-Tyr126 of the carboxy-terminal portion of the atrial natriuretic peptide prohormone, was degraded rapidly by rabbit kidney brush border membranes. The rate of degradation of AP24 measured by the loss of vasorelaxant activity followed a similar time course to the decrease in peptide peak area measured by high-performance liquid chromatography. Inactivation of AP24 produced peptide fragments which were separated by HPLC.

Inactivation of atrial natriuretic factor by the renal brush border.

Atrial natriuretic factor (ANF), a 28-amino-acid peptide secreted from the mammalian heart, is known to be cleared rapidly from the circulation. In vitro and in vivo studies implicate the kidney as an important site for clearance and subsequent degradation of atrial natriuretic factor. We have observed that atrial natriuretic factor is inactivated rapidly by rabbit kidney brush-border membranes.