In vivo processing of N-terminal methionine in E. coli (FEBS 08497) H. Dalboge, S. Bayne, J. Pedersen 1 Direct activation of cdc2 with phosphatase: identification of pl 3suc~-sensitive and insensitive steps (FEBS 08481) C. Jessus, B. Ducommun, D. Beach 4 Association of iron-protoporphyrin-IX (hemin) with myosins (FEBS 08496) V. Bhoite-Solomon, G. Kessler-Icekson, N. Shaklai 9 Identification of a new P1 residue mutation (444Arg--,Ser) in a dysfunctional C 1 inhibitor protein contained in a type II hereditary angioedema plasma (FEBS 08498) K.S. Aulak, M. Cicardi, R.A. Harrison 13 An X-ray solution scattering study of the cofactor and activator induced structural changes in yeast pyruvate decarboxylase (PDC) (FEBS 08499) G. Hiibner, S. Krnig, A. Schellenberger, M.H.J. Koch 17 Expression and pharmacological characterization of the human M I muscarinic receptor in Saccharomyces cerevisiae (FEBS 08500) P. Payette, F. Gossard, M. Whiteway, M. Dennis 21 Interaction of benzoquinones with mitochondria interferes with oxidative phosphorylation characteristics (FEBS 08506) D. IV. Makawiti, V.N. Konji, J.O. Olowookere 26 Partial purification and characterization of the human erythrocyte Mg 2+-ATPase: A candidate aminophospholipid translocase (FEBS 08489) G. Morrot, A. Zachowski, P.F. Devaux 29 Interleukin-1 induces rapid and transient expression of the c-fos proto-oncogene in isolated pancreatic islets and in purified flcells (FEBS 08490) J.H. Hughes, M.A. Watson, R.A. Easom, J. Turk, M.L. McDaniel 33 Post-translational processing of prepro-urotensin II (FEBS 08491) J.M. Conlon, D. Arnold-Reed, R.J. Balment 37 Separation of two f17 subunit complexes of brain GTP-binding proteins composed of distinct ~ subunits (FEBS 08493) T. Asano, R. Morishita, T. Kobayashi, K. Kato 41 Reduced levels of ADP-ribosylatable elongation factor-2 in aged and SV40-transformed human cell cultures (FEBS 08492) B. Riis, S.LS. Rattan, A. Derventzi, B.F.C. Clark 45 Transforming growth factor-fl and epidermal growth factor modulate basal and interleukin-6-induced amino acid uptake and acute phase protein synthesis in cultured rat hepatocytes (FEBS 08494) J. Bereta, K. Szuba, IV. Fiers, J. Gauldie, A. Koj 48 Prokaryotic 20fl-hydroxysteroid dehydrogenase is an enzyme of the 'short-chain, non-metalloenzyme' alcohol dehydrogenase type (FEBS 08501) L. Marekov, M. Krook, H. J&nvall 51 Subcellular distribution of ~t and fl subunit proteins of Ca 2 ÷/calmodulin-dependent protein kinase II expressed in Chinese hamster ovary cells (FEBS 08502) T. Yamauchi, S. Sekihara, S. Ohsako 55 A protein conformational change associated with the photoreduction of the primary and secondary quinones in the bacterial reaction center (FEBS 08505) E. Nabedryk, K.A. Bagley, D.L. Thibodeau, M. Bauscher, IV. Mdntele, J. Breton 59 Purification, some properties and nucleotide sequence of 5-carboxymethyl-2-hydroxymuconate isomerase of Escherichia coli C (FEBS 08503) D.I. Roper, R.A. Cooper 63 Expression of catalytically active radish 3-hydroxy-3-methylglutaryl coenzyme A reductase in Escherichia coli (FEBS 08504) A. Ferrer, C. Aparicio, N. Nogubs, A. Wettstein, T.J. Bach, A. Boronat 67 Cholesterol oxidation on fluorocarbon emulsion surface leads to the formation of 7-peroxycholesterol (FEBS 08507) A.T. Beriozov, A.S. lvanov, V.G. lvkov, V.V. Obraztsov, E.M. Khalilov, A.I. Archakov 72 Na,K-ATPase labelled with 5-iodoacetamidofluorescein: E2-E t conformational transition induced by different nucleotides (FEBS 08508) O.D. Lopina, E. V. Pindel, A.A. Boldyrev 75 Disruption of the gene encoding subunit VI of yeast cytochrome be t complex causes respiratory deficiency of cells with reduced cytochrome clevels (FEBS 08514) C.H. Kim, R.S. Zitomer 78 Regulation of ATP hydrolysis in liver mitochondria from ground squirrel (FEBS 08509) G.E. Bronnikov, S.O. Vinogradova, B. V. Chernyak 83 A recombinant snake neurotoxin generated by chemical cleavage of a hybrid protein recovers full biological properties (FEBS 08515) P. Boyot, L. Pillet, F. Ducancel, J.-C. Boulain, O. Tremeau, A. M~nez 87 The caffeine-sensitive Ca 2÷ store in bovine adrenal chromaflin cells; an examination of its role in triggering secretion and Ca 2+ homeostasis (FEBS 08495) T.R. Cheek, A.J. O'Sullivan, R.B. Moreton, M.J. Berridge, R.D. Burgoyne 91 Cyclic nucleotide-activated channels in the frog olfactory receptor plasma membrane (FEBS 08510) S.S. Kolesnikov, A.B. Zhainazarov, A. V. Kosolapov 96 Fusion of negatively charged phospholipid vesicles by ~t-latrotoxin (FEBS 08511) V.K. Lishko, Y.T. Terletskaya, LO. Trikash 99 X-Ray crystal structure of sangivamycin, a potent inhibitor of protein kinases (FEBS 08513) L. Lebioda, P.A. Hargrave, K. Palczewski 102 Characterization of potassium channels in pancreatic ,8 cells from ob/ob mice (FEBS 08512) M. Kukuljan', M.Y. Li, L Atwater 105 Serine phosphorylation of biosynthetic pro-urokinase from human tumor cells (FEBS 08516) M.R. Mastronicola, M.P. Stoppelli, A. Migliaccio, F. Auricchio, F. Blasi 109 Isolation and characterization of a human cDNA clone encoding a novel DNA topoisomerase II homologue from HeLa cells (FEBS 08517) C.A. Austin, L.M. Fisher 115 A protein with homology to the C-terminal repeat sequence of Octopus rhodopsin and synaptophysin is a member of a multigene family in Dictyostelium discoideum (FEBS 08518) ,4.A. Noegel, G. Gerisch, F. Lottspeich, M. Schleicher 118 Complete amino acid sequence of fetal bovine serum acetylcholinesterase and its comparison in various regions with other cholinesterases (FEBS 08520) B.P. Doctor, T.C. Chapman, C.E. Christner, C.D. Deal, D.M. De La Hoz, M.K. Gentry, R.A. Ogert, R.S. Rush, K.K. Smyth, A.D. Wolfe 123 The primary structure of the larger subunit of soluble guanylyl cyclase from bovine lung: Homology between the two subunits of the enzyme (FEBS 08519) D. Koesling, C. Harteneck, P. Humbert, A. Bosserhoff , R. Frank, G. Schultz, E. Bi~hme 128 Prediction of domain organisation and secondary structure of thyroid peroxidase, a human autoantigen involved in destructive thyroiditis (FEBS 08522) J.P. Banga, D. Mahadevan, G.J. Barton, B.J. Sutton, J.W. Saldanha, E. Odell, A.M. McGregor 133 Metal ion co-ordination in the DNA binding domain of the yeast transcriptional activator GAL4 (FEBS 08525) J.F. Povey, G.P. Diakun, C.D. Garner, S.P. Wilson, E.D. Laue 142 NMR studies of the lipid metabolism of T47D human breast cancer spheroids (FEBS 08523) S.M. Ronen, A. Stier, H. Degani 147

Functional cloning in yeast has been used to isolate full-length cDNAs encoding an endo-α-1,5-L-arabinanase from the filamentous fungus Aspergillus aculeatus. Screening of a cDNA library constructed in a yeast expression vector for transformants that hydrolysed AZCL-arabinan identified 44 Saccharomyces cerevisiae clones all harbouring the same arabinanase-encoding cDNA. The cloned cDNA was expressed in A. oryzae and the recombinant enzyme was purified and characterized. The mode of action of the enzyme was studied by analysis of the digestion pattern towards debranched arabinan. The digestion profile obtained strongly suggests that the enzyme is an endo-arabinanase. In addition, the feasibility using Nicotiana tabacum as an alternative host for arabinanase expression was investigated.

Nucleotide sequence analysis shows that Trichoderma harzianum and Penicillium purpurogenum alpha 1,3-glucanases (mutanases) have homologous primary structures (53% amino acid sequence identity), and are composed of two distinct domains: a NH2-terminal catalytic domain and a putative COOH-terminal polysaccharide-binding domain separated by a O-glycosylated Pro-Ser-Thr-rich linker peptide. Each mutanase was expressed in Aspergillus oryzae host under the transcriptional control of a strong cu-amylase gene promoter. The purified recombinant mutanases show a pH optimum in the range from pH 3.5 to 4.5 and a temperature optimum around 50-55 degrees C at pH 5.5. Also, they exhibit strong binding to insoluble mutan with K-D around 0.11 and 0.13 mu M at pH 7 for the P. purpurogenum and T. harzianum mutanases, respectively. Partial hydrolysis showed that the COOH-terminal domain of the T. harzianum mutanase binds to mutan, The catalytic domains and the binding domains were assigned to a new family of glycoside hydrolases and to a new family of carbohydrate-binding domains, respectively.

Recombinant β-1,4-galactanase from Aspergillus aculeatus has been crystallized and characterized by X-ray diffraction. Crystals were obtained in hanging drops by vapour-diffusion under the conditions 30% PEG 400, 0.2 M CaCl2 and 0.1 M Na HEPES buffered to pH 7.5. The crystals diffract to 2.3 Å resolution and belong to one of the orthorhombic space groups I222 or I212121. The unit-cell dimensions are a = 60.42, b = 88.94 and c = 129.08 Å. With one molecule in the asymmetric unit, the corresponding solvent content is ∼48%.

Evolution has favoured microorganisms that produce efficient enzymes with substrate-adapted biocatalytic activities. Progress in molecular techniques, especially expression cloning, molecular screening, protein engineering and in vivo and in vitro shuffling, have paved the way for greater speed and accuracy in cloning enzyme genes from microorganisms and generating versions with improved properties. Recently, two new approaches have been added: screening directly from uncultivated microorganisms and generating additional hits by database mining using bioinformatic tools.

Well diffracting crystals of rhamnogalacturonan acetylesterase from Aspergillus aculeatus have been obtained in two polymorphic modifications despite its heterogeneous glycosylation. The best-diffracting crystals (resolution 1.55 A) are orthorhombic. The limit of the diffraction pattern of the other (trigonal) form is 2.5 A. The ability of the enzyme to crystallize appears to depend on the glycosylation of the protein sample. This aspect has been investigated by mass spectrometry, which also showed that the orthorhombic crystals have the same glycosylation as the protein sample used in the crystallization.

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4.5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1 alpha and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters. Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning.

The crystal structure of the 154-amino-acid form of human basic fibroblast growth factor (hbFGF154), probably representing the intact form of hbFGF as deduced from the open reading frame of hbFGF cDNA, was determined by X-ray crystallography and refined to a crystallographic residual of 19.0% for all data between 20.0 and 2.0 A resolution. Crystals were obtained from recombinant hbFGF154 expressed in E. coli. hbFGF154 has the same overall structure as the N-terminus truncated 146-amino-acid form. The structure has a Kunitz-type fold and is built of 12 beta-strands of which six antiparallel strands form a beta-sheet barrel. In the structure it was possible to locate two additional residues at the N terminus and the last three C-terminal amino-acid residues, which seem to be disordered in all but one of the reported structures of the truncated form of hbFGF. The C-terminal amino-acid residues are part of the last beta-strand through the formation of a hydrogen bond between the main-chain amide group of Ala152 and the carbonyl O atom of Pro28. An apparent phosphate ion is bound within the basic region on the surface of the molecule and has as ligands the side chains of Asn35, Arg128 and Lys133 and two water molecules. A slightly different hydrogen-bonding pattern to the phosphate ion is observed as compared with the sulfate ions in the truncated forms [Eriksson, Cousens & Matthews (1993). Protein Sci. 2, 1274-1284; Zhang, Cousens, Barr & Sprang (1991). Proc. Natl Acad. Sci. USA, 88, 3446-3450]. One molecule of beta-mercaptoethanol forms a disulfide bridge to Cys77.

Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein.

Seventeen full-length cDNAs encoding pectin methyl esterase I (PME I) have been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. Yeast colonies expressing functional PME I were identified on agar plates containing highly esterified pectin, and a cDNA encoding PME I was isolated. The deduced amino acid sequence of PME I is highly similar (74 % identity) to the PME from Aspergillus niger. A full-length cDNA encoding PME I was cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for heterologous expression, purification and characterization of the recombinant enzyme. The recombinant PME I had a molecular mass of 36.2 kDa, an isoelectric point of pH 3.8, a pH optimum of 4.6 and a temperature optimum of 45 degrees C. The authentic PME I was purified from A. aculeatus culture supernatant and subjected to amino acid sequencing. The peptide sequences covered 138 amino acid residues and were in complete agreement with the deduced PME I sequence, Both recombinant and authentic PME I were glycosylated, but the composition of the glycan moieties was different. PME I was able to remove 75-85 % of the methyl groups in highly methylated pectin, and it did not remove acetyl groups from acetylated polysaccharides. When the enzyme was added together with polygalacturonases to pectin, a rapid depolymerization was observed. By comparison, polygalacturonases alone showed a very limited degradation of the methylated substrate. This demonstrates that PME I acts in synergy with polygalacturonases in the degradation of plant cell wall pectin.

The synthetic exochitinase substrate 4-methylumbelliferyl N-acetylglucosamine was used to identify seven full-length exochitinase-encoding cDNAs from a Trichoderma harzianum cDNA library by expression in yeast. The cDNA clones represented transcripts of two exochitinase genes, designated as exc1 and exc2, which cross-hybridized under moderate stringency conditions in genomic Southern blots. The exc1 cDNA encodes a 578 amino acid polypeptide showing 72% similarity to the exc2-encoded 602-residue polypeptide. The deduced exochitinase amino acid sequences were found to be homologous with mammalian and fungal hexosaminidases as well as a bacterial chitobiosidase. The substrate specificity of the recombinant enzymes expressed in S. cerevisiae indicates that the enzymes are N-acetylglucosaminidases releasing single N-acetylglucosamine residues from the non-reducing end of the chitin substrate.

A rhamnogalacturonan acetylesterase (RGAE) was purified to homogeneity from the filamentous fungus Aspergillus aculeatus, and the NH2-terminal amino acid sequence was determined. Full-length cDNAs encoding the enzyme were isolated from an A. aculeatus cDNA library using a polymerase chain reaction-generated product as a probe. The 936-base pair rha1 cDNA encodes a 250-residue precursor protein of 26,350 Da, including a 17-amino acid signal peptide. The rha1 cDNA was overexpressed in Aspergillus oryzae, a filamentous fungus that does not possess RGAE activity, and the recombinant enzyme was purified and characterized. Mass spectrometry of the native and recombinant RGAE revealed that the enzymes are heterogeneously glycosylated. In addition, the observed differences in their molecular masses, lectin binding patterns, and monosaccharide compositions indicate that the glycan moieties on the two enzymes are structurally different. The RGAE was shown to act in synergy with rhamnogalacturonase A as well as rhamnogalacturonase B from A. aculeatus in the degradation of apple pectin rhamnogalacturonan. RNA gel blot analyses indicate that the expression of rhamnogalacturonan degrading enzymes by A. acculeatus is regulated at the level of transcription and is subjected to carbon catabolite repression by glucose.

Several plant cell wall degrading enzymes have been cloned by the expression cloning technique. These enzymes can be used to degrade isolated plant cell wall polysaccharides into oligomers or to extract poly- or oligosaccharides from insoluble and complex plant cell wall material, thereby providing soluble dietary fibre or oligosaccharides with potential beneficial physiological effects. Also the cloned enzymes can be used to control e.g. viscosity in the industrial processing of plant material. This is illustrated by the degradation of various arabinoxylans or arabinoxylan containing plant material with cloned xylanases and by the degradation of rhamnogalacturonans or rhamnogalacturonan containing plant material with cloned rhamnogalacturonases and assessory enzymes.

Proton nuclear magnetic resonance spectroscopy has been used to characterise and compare wild-type fungal and recombinant Coprinus cinereus peroxidase (CIP) and three mutants in which Gly156 and/or Asn157 was replaced by Phe. Analysis of one- and two-dimensional NMR spectra of recombinant CIP was undertaken for comparison with the fungal enzyme and in order to establish a meaningful basis for solution studies of CIP mutants. Proton resonance assignments of haem and haem-linked residues obtained for the cyanide-ligated form of recombinant CIP revealed a high degree of spectral similarity with those of lignin and manganese-dependent peroxidases and extend previously reported NMR data for fungal CIP. The three mutants examined by NMR spectroscopy comprised site-specific substitutions made to a region of the structure believed to form part of the peroxidase haem group access channel for substrate and ligand molecules. Proton resonances of the aromatic side-chains of Phe156 and Phe157 were found to have similar spectral characteristics to those of two phenylalanine residues known to be involved in the binding of aromatic donor molecules to the plant peroxidase, horseradish peroxidase isoenzyme C. The results are discussed in the context of complementary reactivity studies on the mutants in order to develop a more detailed understanding of aromatic donor molecule binding to fungal and plant peroxidases.

We have developed a method for fast and efficient isolation of enzyme genes from filamentous fungi by combining the ability of Saccharomyces cerevisiae to express heterologous genes with the utilisation of sensitive and reliable enzyme assays. A cDNA library from the fungus Humicola insolens was constructed in a S. cerevisiae/Escherichia coli shuttle vector in E. coli. Sub-pools of the library were subsequently screened for enzyme activity in S. cerevisiae. More than 130 clones were identified as positive in either an endo-beta-glucanase or an endo-xylanase assay. Based on a partial characterization of the DNA sequence of the individual clones, they could be grouped into five distinct types of endo-beta-glucanases and three types of endo-xylanases. A representative cDNA from each type was sub-cloned in an Aspergillus vector and expressed in A. oryzae. The new cloning method may be an important alternative to traditional cloning methods based on amino acid sequence information.

Proton nuclear magnetic resonance spectroscopy has been used to characterise and compare wild-type fungal and recombinant Coprinus cinereus peroxidase (CIP) and three mutants in which Gly156 and/or Asn157 was replaced by Phe. Analysis of one- and two-dimensional NMR spectra of recombinant CIP was undertaken for comparison with the fungal enzyme and in order to establish a meaningful basis for solution studies of CIP mutants. Proton resonance assignments of haem and haem-linked residues obtained for the cyanide-ligated form of recombinant CIP revealed a high degree of spectral similarity with those of lignin and manganese-dependent peroxidases and extend previously reported NMR data for fungal CIP. The three mutants examined by NMR spectroscopy comprised site-specific substitutions made to a region of the structure believed to form part of the peroxidase haem group access channel for substrate and ligand molecules. Proton resonances of the aromatic side-chains of Phe156 and Phe157 were found to have similar spectral characteristics to those of two phenylalanine residues known to be involved in the binding of aromatic donor molecules to the plant peroxidase, horseradish peroxidase isoenzyme C. The results are discussed in the context of complementary reactivity studies on the mutants in order to develop a more detailed understanding of aromatic donor molecule binding to fungal and plant peroxidases.

A cDNA library from the filamentous fungus Aspergillus aculeatus was constructed in the yeast expression vector pYES2.0 and used to isolate 57 full length cDNA's encoding beta-1,4-mannanase by expression in S. cerevisiae. The positive clones were identified on agar plates containing 0.2% azurine dyed cross-linked mannan by the formation of blue halos around the colonies. All clones represented transcripts of the same mannanase gene (man1). The gene was sub-cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for overexpression and purification of the enzyme. The recombinant enzyme had a molecular weight of 45 kDa, an isoelectric point of pH 4.5, a pH optimum of pH 5.0 and a temperature optimum of 60-70 degrees.

Two rhamnogalacturonases from the filamentous fungus Aspergillus aculeatus have been cloned and characterized. A cDNA library from A. aculeatus was constructed, and a novel rhamnogalacturonase B was isolated by expression cloning in yeast. For this purpose a new plate screening assay was developed, specific for the detection of rhamnogalacturonase activity. The rhamnogalacturonase A, known from previous reports, was shown not to be expressed in yeast in an active form. Therefore, rhamnogalacturonase A was purified, peptide sequences were obtained, and full-length cDNAs encoding the enzyme were isolated using a polymerase chain reaction-generated product as a probe. Comparison of the deduced primary structures indicates that the two rhamnogalacturonases are structurally different. This is further supported by the finding that polyclonal antibodies raised against native rhamnogalacturonase A do not cross-react with rhamnogalacturonase B. The cloned genes were transformed into Aspergillus oryzae for high level expression. The recombinant enzymes were purified and characterized, revealing significant differences in glycosylation pattern and substrate specificity as well as in pH and temperature optima and stability. Data from the hydrolysis of apple rhamnogalacturonan with the recombinant rhamnogalacturonases suggest that the two enzymes exert their action at different sites in the backbone.

A cDNA coding for cystatin D, a human member of the cystatin protein family, has been cloned after specific amplification of reverse-transcribed parotid gland RNA. After replacing the segment encoding the putative 20-residue signal peptide with one encoding the Escherichia coli OmpA leader sequence, the cDNA was expressed in E. coli. The isolated recombinant protein exhibited Ki values of 1.2 nM and > 1 microM for papain and cathepsin B, respectively. An antiserum raised against recombinant cystatin D recognized a protein in human saliva with electrophoretical mobility identical to that of the recombinant protein. Immunoenzymatic analysis revealed that this cysteine proteinase inhibitor is present in human saliva and tears at concentrations of 3.8 and 0.5 mg/liter, respectively, while it was not detected in seminal plasma, blood plasma, milk, or cerebrospinal fluid. Cystatin D purified from human saliva by immunosorption displayed a heterogeneous N-terminal end, with sequences starting at residues 5, 7, 9, and 11 of the predicted N-terminal portion of the mature protein. On the basis of structural and functional properties, cystatin D represents a novel cysteine proteinase inhibitor possibly playing a protective role against proteinases present in the oral cavity.