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We have identified the secretory cleavage site in the Alzheimer amyloid precursor (APP) in a non-transfected neuronal cell line, using cyanogen bromide digests of APP purified from medium conditioned by PC-12 cells which were differentiated to a neuronal phenotype. The results obtained are most consistent with proteolysis of the Lys 16 -Leu 17 bond in the β amyloid peptide, followed by partial removal of Lys 16 by a basic carboxypeptidase.

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (Mr ca. 29,000), and the S-peptide (Mr 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH2-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH2-terminal 60-amino acid peptide.

The amino acid sequence has been determined for the insecticyanin from the hemolymph of the fifth instar larvae of the tobacco hornworm, Manduca sexta. The apoprotein is a single polypeptide chain of 189 amino acids, molecular weight 21,378, containing two disulfide bridges, 9-119 and 42-176. The sequence analysis was performed by automated Edman degradation of reduced and carboxymethylated insecticyanin and fragments generated therefrom by cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus proteinase. Most of the peptides were purified by reverse-phase high-performance liquid chromatography. A purification procedure for the isolation of insecticyanin in high yields and a simple method of determining disulfide linkages are also reported.

The interaction of Pseudomanas putida anthranilate synthetase Component II (AS II) with glutamine, glutamine analogs, and iodoacetamide has been investigated in order to clarify the initial steps in the mechanism for glutamine utilization. AS II is alkylated and irreversibly inactivated by covalent attachment of approximately 1 eg of L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone) or 1 eq of iodoacetamide. Alkylation of AS II by chloroketone involves initial formation of an enzyme-inhibitor complex having a Ki of 28 muM. Alkylation of AS II by iodoacetamide occurs without initial formation of a reversible complex. In both cases glutamine protects against alkylation and exhibits competitive kinetics. When anthranilate synthetase Component I (AS I) is associated with AS II, the second substrate, chorismate, enhances alkylation of AS II by chloroketone. Alkylation of AS II by iodoacetamide is unaffected by AS I and chorismate. These results suggest a role of chorismate-AS I complex to promote binding of glutamine to AS II or to facilitate conversion of an AS II-glutamine complex to the covalent glutamyl-AS II intermediate. This conclusion is supported by the fact that glutaminase activity of AS II, which requires formation of the glutamyl-AS II intermediate, is stimulated by AS I and chorismate.

Native pig kidney fructose-1,6-bisphosphatase, in contrast to the rat liver enzyme, is not a substrate of cyclic AMP-dependent protein kinase. However, the pig kidney enzyme becomes a substrate when phosphorylation is performed in 1.6 M urea, after prior unfolding in 8 M urea. A cyanogen bromide fragment containing the phosphorylation site has been isolated and the amino acid sequence of this 63-residue peptide has been determined. This peptide has the following sequence: Leu-Asp-Pro-Ala-Ile-Gly-Glu-Phe-Ile-Leu-Val-Asp-Arg-Asn-Val-Lys-le-Lys-Lys-Lys- Gly-Ser(P)-Ile-Tyr-Ser-Ile-Asn-Glu-Gly-Tyr-Ala-Lys-Glu-Phe-Asp-Pro-Ala-Ile-Thr- Glu-Tyr-Ile-Glu-Arg-Lys-Lys-Phe-Pro-Pro-Asp-Asn-Ser-Ala-Pro-Tyr-Gly-Ala-Arg-Tyr -Val-Gly-Ser-Met. The amino acid sequence around the phosphorylated serine residue resembles those of other protein substrates of cyclic AMP-dependent protein kinase, but it is completely different from the phosphorylation site found in native rat liver fructose-1,6-bisphosphatase.

A procedure is described for purification of the juvenile hormone carrier protein from the last larval instar of Manduca sexta. Homogeneous carrier protein is obtained after three chromatographic steps in up to 36% yield, based upon the recovery from the first gel permeation chromatography step. The sequence of the N- terminus of the polypeptide chain was determined for 19 of the first 20 residues: l 10 Asp-Gln-Gly-Ala-Leu-Phe-Glu-Pro-Cys-Ser- 11 20 Thr-GIn-Asp-lle-Ala-Cys-Leu-Ser- ?-Ala- Three reagents which modify side chain residues in proteins: methyl acetimidate (amino groups), N- succinimidyl -3-(4- hydroxyphenyl ) propionate (amino groups) and tetranitromethane (phenol groups), were reacted with pure carrier protein. In each case, there was relatively little change in the juvenile hormone binding properties of the modified carrier protein.

A hexameric protein of high (18.8%) aromatic amino acid content has been isolated from the haemolymph of pupae of Hyalophora cecropia. It is the major protein present in the haemolymph, and also occurs in the fat body. It is distinct in its behaviour on polyacrylamide gels and in its antigenic reactivity from the two storage proteins of H. cecropia described earlier by Tojo et al. (1978). The new protein is related to similar proteins reported earlier from several species, all of which share the common property of an unusually high content of aromatic amino acid. We propose the generic name arylphorin for such proteins. Hyalophora arylphorin is composed of six seemingly identical subunits of apparent mol.wt. 73,000. Cross-linking studies confirm the hexameric structure, and pore limiting gradient gel electrophoresis of the native protein is consistent with a mol.wt of about 400,000. It contains about 4% carbohydrate, consisting of glucosamine and mannose in a 1:5 molar ratio. The carbohydrate could not be removed by treatment with endoglycosidase H. Hyalophora and Manduca arylphorins are antigenically similar, but their antibodies did not precipitate Calliphora arylphorin. The concentration of Hyalophora arylphorin in haemolymph was examined during the last larval and the pupal-adult moults. In both cases a 95–100% drop in concentration occurred between apolysis and ecdysis.

The complete amino acid sequence of carboxamidomethylated anthranilate synthetase component II (AS II) from Pseudomonas putida has been determined by analysis of cyanogen bromide fragments, tryptic peptides from the citraconylated protein, and by analysis of subdigests of these peptides. AS II is a single polypeptide chain of 197 residues having a calculated molecular weight of 21,684. Previous studies (Goto, Y., Keim, P. S., Zalkin, H., and Heinrikson, R. L. (1976) J. Biol. Chem, 251, 941-949) identified a cysteine residue required for the formation of an acyl-enzyme intermediate. The protein has 3 cysteine residues at positions 54, 79, and 140. Cysteine-79 was alkylated selectively by iodoacetamide and by the glutamine affinity analogue L-2-amino-4-oxo-5-chloropentanoic acid. Based on this evidence cysteine-79 is the active site residue involved in formation of the acyl-enzyme intermediate. Comparison of the P. putida AS II sequence with that of the NH2-terminal 60 residues of the enzyme from Escherichia coli shows 38% sequence identity.

Fructose-1,6-bisphosphatase from rat liver was phosphorylated with cyclic AMP-dependent protein kinase and [gamma-32P]ATP. Brief exposure of the 32P-labeled enzyme to trypsin removed all radioactivity from the enzyme core and produced a single-labeled peptide. The partial sequence of the 17-amino acid peptide was found to be Ser-Arg-Pro-Ser(P)-Leu-Pro-Leu-Pro-(Ser2, Glx2, Pro2, Leu, Arg2). The kinetics of cyclic AMP-dependent protein kinase-catalyzed phosphorylation of native fructose bisphosphatase were compared with those of rat liver type L pyruvate kinase where the sequence around the phosphoserine is known (Arg-Arg-Ala-Ser(P)-Val; Hjelmquist, G., Anderson, J., Edlund, B., and Engstrom, L. (1974) Biochem. Biophys. Res. Commun. 61, 559-563). The Km for pyruvate kinase (17 microM) was less than that for fructose bisphosphatase (58 microM); the Vmax was about 3-fold greater with pyruvate kinase as substrate. The relationship between the rates of phosphorylation of these native substrates and the amino acid sequences surrounding the phosphorylated sites is discussed.

Silkmoth eggshell (chorion) proteins have been characterized by electrophoresis on sodium dodecyl sulfate and isoelectric focusing polyacrylamide gels; up to 33 and 41 components, respectively, were detected from a single chorion. Some of these components are polymorphic, being absent from chorions of certain animals. A system of nomenclature for all chorion proteins is presented, based on their separation on sodium dodecyl sulfate and isoelectric focusing gels. The chorion is enriched in glycine, alanine, cysteine, and tyrosine and poor in methionine and histidine. The proteins were fractionated into four partially overlapping groups; all four are enriched in the above amino acids, although significant differences exist. Further fractionation by isoelectric focusing of one of the above groups, s/s, yielded seven components, two of which are homogeneous both on sodium dodecyl sulfate and isoelectric focusing gels. The amino acid compositions, molecular weights, and solubility properties of the components share certain features which distinguish s/s as a group from the other three groups. The sequence of the first 67 NH2-terminal residues of a homogeneous protein purified from s/s has been determined. The protein contains a cysteine-rich tail (3 cysteines in the first 18 residues) followed by a 49-residue segment which contains only a single cysteine residue. This latter segment also contains two different tetrapeptide sequences which are each repeated, one twice and the other four times.