Cellular localization of acid carboxypeptidase inAspergillus oryzae

Fresh mycelia ofAspergillus oryzae were shown to have strong acid carboxypeptidase (EC 3.4.12.-) activity. The cellular localization of acid carboxypeptidase was investigated using the broken mycelia and the protoplasts of the fungusAspergillus oryzae IAM 2640. In the broken mycelia, about 40% of the total activity was found in the “cell wall” fraction (2,000×g), with most of the remainder in the soluble fraction (100,000×g supernatant). During the formation of protoplasts, most of the acid carboxypeptidase in the mycelia was solubilized and released. The specific activity of acid carboxypeptidase in the lysed protoplasts was about 10-fold lower than that found in the broken mycelial preparation. These data indicate that most of the acid carboxypeptidase is probably located in the cell surface, which includes the cell wall, the periplasm, and the cell membrane.     

The carbohydrate moiety of the acid carboxypeptidase fromAspergillus saitoi

Acid carboxypeptidase fromAspergillus saitoi is a glycoprotein that contains both N-and O-linked sugar chains. The N-glycanase released high-mannose type oligosaccharides that were separated into eight components on HPLC. One, which had a unique structure of Man₁₁GlcNAc₂, was characterized. Mild alkali treatment of the carboxypeptidase, under conditions that effect -elimination, yieldedd-mannose. Deglycosylation of the carboxypeptidase with endo--N-acetylglucosaminidase and -mannosidase effected the reduction of the molecular mass from 72 kDa to 60 kDa. Partial changes of CD spectra of the native and the deglycosylated enzymes indicate that some conformational changes on the peptide of the enzyme occurred after deglycosylation. Other enzymatic properties, such as catalytic activity, pH, and thermal stability and resistivity to protease digestion, did not appear to change. Tunicamycin halted secretion of the carboxypeptidase extracellularly.     

Characterization of two forms of base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi

Two forms of RNases (RNase ML and RNase MM) from Aspergillus saitoi which are base non-specific and adenylic acid preferential were separated from each other by DEAE-cellulose column chromatography. They are indistinguishable with respect to enzymatic properties such as base preferability, pH optimum, kinetic constants measured with 2',3'-cUMP and 2',3'-cCMP as substrates, and effects of ionic strength, physical properties such as heat stability, isoelectric point and circular dichroism spectra, amino acid composition and immunological property. They only differ in carbohydrate content. The apparent molecular weight determined by SDS-disc electrophoresis was 36,000 for RNase ML and 32,000 for RNase MM. Both RNases were reduced and carboxymethylated, and then digested with trypsin, separately. Glycopeptides were isolated from the both digests by gelfiltration and paper chromatography. The amino acid compositions of glycopeptides obtained from RNase ML (ML TS-IIC) and that obtained from RNase MM (MM TS-IIIC) were the same. The amino acid sequences of both glycopeptides determined by Edman degradation and carboxypeptidase digestion were also the same. The results indicated that RNase ML and RNase MM were the same protein having different sizes of carbohydrate chains at one site on the molecule.     

Palmitoylation of carboxypeptidase D. Implications for intracellular trafficking

Covalent lipid modifications mediate protein-membrane and protein-protein interactions and are often essential for function. The purposes of this study were to examine the Cys residues of the transmembrane domain of metallocarboxypeptidase D (CPD) that could be a target for palmitoylation and to clarify the function of this modification. CPD is an integral membrane protein that cycles between the trans Golgi network and the plasma membrane. We constructed AtT-20 cells stably expressing various constructs carrying a reporter protein (albumin) fused to a transmembrane domain and the CPD cytoplasmic tail. Some of the constructs contained the three Cys residues present in the CPD transmembrane region, while other constructs contained Ala in place of the Cys. Constructs carrying Cys residues were palmitoylated, while those constructs lacking the Cys residues were not. Because palmitoylation of several proteins affects their association with cholesterol and sphingolipid-rich membrane domains or caveolae, we tested endogenous CPD and several of the reporter constructs for resistance to extraction with Triton X-100. A construct containing the Cys residues of the CPD transmembrane domain was soluble in Triton X-100 as was endogenous palmitoylated CPD, indicating that palmitoylation does not target CPD to detergent-resistant membrane rafts. Interestingly, constructs of CPD that lack palmitoylation sites have an increased half-life, a slightly more diffuse steady-state localization, and a slower rate of exit from the Golgi as compared with constructs containing palmitoylation sites. Thus, the covalent attachment of palmitic acid to the Cys residues of CPD has a functional significance in the trafficking of the protein.     

Crystal structures of potent thiol-based inhibitors bound to carboxypeptidase B

This paper presents the crystal structure of porcine pancreatic carboxypeptidase B (pp-CpB) in complex with a variety of thiol-based inhibitors that were developed as antagonists of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recent studies have indicated that a selective inhibitor of TAFIa could enhance the efficacy of existing thrombolytic agents for the treatment of acute myocardial infarction, one of the most prevalent forms of heart attacks. Unfortunately, activated TAFIa rapidly degrades in solution and cannot be used for crystallographic studies. In contrast, porcine pancreatic CpB is stable at room temperature and is available from commercial sources. Both pancreatic CpB and TAFIa are zinc-based exopeptidases, and the proteins share a 47% sequence identity. The homology improves considerably in the active site where nearly all of the residues are conserved. The inhibitors used in this study were designed to mimic a C-terminal arginine residue, one of the natural substrates of TAFIa. The X-ray structures show that the thiol group chelates the active site zinc, the carboxylic acid forms a salt bridge to Arg145, and the guanidine group forms two hydrogen bonds to Asp255. A meta-substituted phenyl was introduced into our inhibitors to reduce conformational freedom. This modification vastly improved the selectivity of compounds against other exopeptidases that cleave basic residues. Comparisons between structures indicate that selectivity derives from the interaction between the guanidine group in the inhibitors and an acidic active site residue. The location of this acidic residue is not conserved in the various carboxypeptidases.     

Soluble and bound acid protease activity of myelin from bovine cerebral white matter and spinal cord

Isolated myelin of bovine spinal cord was found to degrade exogenous myelin basic protein (MBP) at pH 4.4. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Some of the proteolytic activity was soluble at increased ionic strength, some remained bound, withstanding extraction at 37°C for up to 12 hr. While being measurable with exogenous MBP, bound protease degraded neither bound MBP nor any other major intrinsic myelin protein. Both soluble and bound protease activity was completely inhibited by pepstatin A. The patterns of limited proteolysis of MBP they produced were identical. Myelin of cerebral white matter also exhibited soluble and bound acid protease activity which was likewise inhibited by pepstatin A. Protease activity of spinal cord and cerebral myelin is therefore suggested to be due to a cathepsin D-like endopeptidase, present in a loosely and tightly bound form. Both forms increased by 50 to 80% in activity when myelin was isolated from mixtures of white and cortical gray matter. While increased soluble activity of myelin is consistent with binding of cathepsin D of lysosomal origin during the isolation of myelin the tightly bound form might point to a principal mechanism through which exogenous proteins may become attached to the myelin sheath in vivo.     

The isolation and partial characterization of precursor forms of ostrich carboxypeptidase

Ostrich carboxypeptidases A and B were recently purified and characterized. The aim of this study was to isolate and purify, and partially characterize in terms of molecular weight, pI, amino acid composition and N-terminal sequencing, the precursor forms of carboxypeptidases from the ostrich pancreas. Inhibition studies with soybean trypsin inhibitor and activation studies with three proteases (bovine trypsin, bovine chymotrypsin and porcine elastase) were performed on crude ostrich acetone powder and the carboxypeptidase A and B activities were determined. SDS-PAGE was carried out after every incubation to monitor the rate and degree of conversion of a M(r) 66K component to procarboxypeptidase and carboxypeptidase A and B. The precursor forms were purified by Toyopearl Super Q and Pharmacia Mono Q chromatography. All three proteases converted the M(r) 66K component to procarboxypeptidases and carboxypeptidases over a set time interval, with carboxypeptidase A and B activities being detected in the acetone powder. Chymotrypsin was the preferred protease since it exhibited a more controlled activation of the procarboxypeptidases. The amino acid composition of procarboxypeptidase A revealed 525 residues. The N-terminal sequence of procarboxypeptidase A showed considerable homology when compared with several other mammalian sequences. M(r) and pI values of 52K and 5.23 were obtained for procarboxypeptidase A, respectively. This study indicated that ostrich procarboxypeptidase A is closely related to other mammalian procarboxypeptidase A molecules in terms of physicochemical properties.     

Influence of manganese on enzyme synthesis and citric acid accumulation inAspergillus niger

Summary A comparison of citric acid fermentations in manganese-deficient and manganese-containing media showed that manganese strongly influences idiophase metabolism. In the presence of manganese, cell growth increases, sugar consumption is diminished and acidogenesis decreases drastically. An investigation of the key enzymes of glycolysis, the pentosephosphate pathway, TCA-cycle, nitrogen metabolism, and gluconeogenesis indicated that manganese deficiency was accompanied by a repression of anabolic and TCA-cycle-enzymes with the exception of citrate synthase. The activity of this enzyme and the enzymes of glycolysis paralleled the sugar consumption rate. In the presence of manganese, no repression of enzyme synthesis was observed. Activities of 2-oxoglutarate dehydrogenase and isocitrate lyase could not be detected in either case. The results support the hypothesis that manganese deficiency mainly affects the operation of biosynthetic reactions inAspergillus niger, thus leading to an overflow of citric acid as an end product of glycolysis.     

Characterization of a distinct membrane bound dipeptidyl carboxypeptidase inactivating enkephalin in brain

A dipeptidyl carboxypeptidase distinct from the angiotensin converting enzyme (EC 3.4.15.1) was isolated from membrane preparations of rabbit brain. The enzyme cleaved enkephalin at the Gly-Phe bond, releasing either Phe-Leu from Leu-enkephalin or Phe-Met from Met-enkephalin, and also acted on bradykinin, releasing the terminal dipeptide Phe-Arg. In contrast to the converting enzyme, however, this dipeptidyl carboxypeptidase did not act on angiotensin-1, and it did not degrade hippuryl-His-Leu. Chloride ions did not affect its activity, but the enzyme was inhibited by metal chelating agents. The enzyme was not inhibited by captopril (SQ 14225) or by SQ 20881. Kinetic studies indicated a Km for this enzyme of 0.14 mM with Leu-enkephalin and 0.12 mM with bradykinin as substrates. Present data indicate that more than one enzyme is present in brain membrane fractions acting as dipeptidyl carboxypeptidases inactivating enkephalin; these data suggest multiple roles for such enzymes in the regulation of peptide metabolism.