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Several bovine spleen enzymes with acid pH optima, some of which hydrolyze bis(p-nitrophenyl)phosphate and therefore fit the definition of "phosphodiesterase IV," were partially separated by isoelectric focusing and ion-exchange techniques. The activities were characterized by zymogram analysis with the aid of p-nitrophenyl and 4-methylumbelliferyl phosphate and phosphonate substrates. A number of these enzymes meet the criteria for phosphodiesterase I or other phosphodiesterases. However, the predominant phosphodiesterase I hydrolyzes the bis(p-nitrophenyl)-and 4-methylumbelliferyl phosphates, p-nitrophenyl and 4-methylumbelliferyl phenylphosphonate, and ATP at the beta-gamma bond, but not p-nitrophenyl or 4-methylumbelliferyl 5'-thymidylate (the usual PDE I substrates). These properties, as well as the pH optimum, distinguish the activity from the previously described, alkaline pH optimum PDE I. A second phosphodiesterase hydrolyzes only the phenylphosphonates. Several other activities, less well described, are apparent on zymograms. None of the phosphodiesterases IV was also a phosphodiesterase II (no hydrolysis of 4-methylumbelliferyl 3'-thymidylate).

A sample consisting of 92 black subjects was examined in this study. According to results of preliminary statistical tests there is a significant relationship between certain genetically determined salivary factors and individual susceptibility to dental disease in the racial group studied. These findings are been validated by the examination of data from several additional studies involving large samples.

Based on data from 76 informative families, linkages between Pa and Pr and between Pr and Db have been established by two-point linkage analysis. In both pairs of loci, there were no significant sex heterogeneity in recombination fractions. Linkage between Pa and Db cannot be established based on two-point analyses, but a significant sex difference in the recombination fraction between Pa and Db was observed. Strong confirming evidence was obtained from three-point analysis to place Pa, Pr, and Db in one linkage group. The most likely order is Pa-Pr-Db, but the relative odds over second order Pr-Pa-Db are small. Haplotype frequencies of Pr, Pa, and Db were obtained based on the phenotypes of the 685 random Caucasians, providing evidence for marked linkage disequilibrium among the three loci.

A new polymorphism, Pc, has been identified in human saliva. Two proteins, Pc 1 and Pc 2, are determined by alleles Pc1 and Pc2, respectively, which show autosomal codominant inheritance. No null phenotype has been encountered in 225 randomly collected salivas. The frequencies of the two alleles differ in the Black and White American populations, with Pc1 and Pc2 being 0.670 and 0.330 in the Black (N = 47) and 0.461 and 0.539 in the White (N = 178) populations, respectively. The alleles are in equilibrium in the two populations and segregation analyses (30 families) do not suggest the existence of a null allele in either population. Of seven polymorphic human salivary proteins determined by genes in the salivary protein complex (SPC), Pc phenotypes show association only with Ps phenotypes. Based on that association, our linkage studies, and the biochemical similarities with other SPC proteins, we tentatively conclude that Pc is a member of the SPC, bringing the total number of genes in that group to 13.

Expression of the dominant allele, Ssps, at the Sex-limited saliva pattern locus in mice results in an alteration of the electrophoretic mobility of a mouse salivary protein from the F (fast) to the S (slow) type [Karn, R.C., et al. (1980). Genetics 94:s52; Karn, R.C., et al. (1982). Biochem. Genet. 20:493]. We now demonstrate that the protein affected binds androgen and has a basic heterodimeric structure with the subunits connected by disulfide bridging. It is produced in the submaxillary gland where the alteration takes place. The change in electrophoretic mobility from F to S appears to be primarily the result of an increase in the molecular weight of the larger subunit since the isoelectric point changes very little. We discuss the possible causes of the increase in molecular weight.

The acidic proline-rich proteins (Pr) showing genetic polymorphism were purified from human parotid salivas by gel filtration and ion exchange chromatography. Molecular weight determinations, amino acid composition analyses, and polypeptide mapping experiments indicate that the Pr 3 protein is a fragment of the Pr 1 protein. Studies of a parotid saliva factor capable of converting Pr 1 to Pr 3 and Pr 2 to Pr 4 indicate that Pr 3 and Pr 4 are generated from Pr 1 and Pr 2, respectively. Evidence suggests that the converting factor is a protease capable of posttranslationally cleaving Pr 1 and Pr 2, the primary or derived products of alleles Pr1 and Pr2.

A genetic analysis of human urinary pepsinogen isozymes is presented. Nine discrete phenotypes were identified in a population survey of 215 unrelated Caucasian individuals. The phenotypes were characterized by differences among the staining intensities of the activated group I pepsinogens, Pg 5, Pg 4, Pg 3, and Pg 2. The genetic studies demonstrated that the codominant expression of four alleles, PgA, PgB, PgC and PgD, at a single genetic locus determined the nine phenotypes identified. Linkage analysis excluded close linkage of the Pg locus with the chromosome 6 markers HLA, GLO1, and Bf.

Isoelectric focusing was performed on parotid salivas selected for their electrophoretic phenotypes of proline-rich acidic salivary proteins. Fractions encompassing narrow pH regions were pooled and examined by polyacrylamide gel electrophoresis. Isoelectric focusing yielded partial purification of major and minor acidic proline-rich proteins which were subsequently compared by immunoelectrophoresis and double immunodiffusion against goat anti-human parotid saliva. Cross-reactivity without spurring between all fractions containing major Pr proteins in both immunoelectrophoresis and double immunodiffusion suggests that these proteins are immunologically very similar or identical.

We report the nucleotide sequence of mRNA for the common electrophoretic isozyme of human pancreatic alpha-amylase, Amy2 A. The sequence was derived from a nearly full-length complementary DNA (cDNA) isolated from a cloned cDNA library. The relatively short 5' untranslated region (15 nucleotides) was determined by primer-extension sequencing. The human Amy2 messenger codes for a 511-residue preamylase polypeptide. An amino-terminal signal peptide of 15 amino acids with an Ala X Gln cleavage site is proposed based on homology to mouse, dog and hog amylases. The Amy2 A mRNA sequence differs from a recently reported human Amy2 sequence. Differences were found at 31 nucleotide positions. The alpha-amylase proteins predicted by the two mRNAs differ at 17 amino acid positions. Relative to the known sequences of other mammalian amylases, most of the differences between the two human Amy2 sequences appear to have occurred as substitutions in the sequence reported by Nakamura et al. (1984). These substitutions predict a protein with a substantially greater net negative charge than that of Amy2 A. We suggest that the two sequences may represent either divergent Amy2 alleles or the expression of non-allelic pancreatic amylase genes.

In situ hybridization of a 3H-labeled probe containing a fragment from PRP-1, a genomic clone with human salivary proline-rich protein gene sequences, revealed significant labeling on the short arm of human chromosome 12 in metaphase preparations from two individuals. Fifty-three percent of metaphases exhibited labeling on one or both chromosomes 12. Additional cells scored at the 850-1,000 band level revealed a significant proportion (52% [32/61] grains, p less than 0.005) of the labeled sites on chromosome 12 to be on band 12p13.2. This probe for a human salivary proline-rich protein gene fragment, probably PMS, is from a cluster of 13 linked genes designated as the human salivary protein complex (SPC). Studies of the DNA of human-mouse somatic-cell hybrids have assigned the SPC to chromosome 12, but have not provided a regional localization (Azen et al, 1985). This paper reports the localization of the SPC to a specific chromosomal band, 12p13.2.

We previously demonstrated polymorphism of a mouse salivary protein which, because of its ability to bind androgen, we designated androgen binding protein (ABP) and its structural gene, Androgen binding protein (Abp). This report describes the purification of salivary ABP and presents the amino acid composition of its subunits. Using an antibody raised against the purified protein, we demonstrated the presence of cross-reactive material (CRM) in mouse parotid, submaxillary, sublingual, and prostate glands by double immunodiffusion. Immunohistochemical detection of proteins on electroblots of polyacrylamide electrophoresis and isoelectric focusing gels, however shows that the prostate CRM is a protein with electrophoretic and isoelectric focusing behavior distinct from that of salivary ABP isoproteins. Because the DBA/2J strain has a variant of salivary ABP, that strain was analyzed to determine if a prostate ABP-CRM variant was present. The approach was hampered by an inability to detect CRM in the prostates of DBA/2J mice. Prostate CRM was detected, however, in some progeny from repeated backcrosses of DBA/2J X C3H/St hybrids to the C3H/St and DBA/2J parental strains. The prostate CRM detected in samples from animals heterozygous for salivary Abp appears to be identical to C3H/St prostate CRM, suggesting that the gene controlling prostate ABP-CRM is related to, but distinct from, Abp. The reason for reduced or absent CRM in the prostates of DBA/2J males is unknown but this finding suggests that there are strain-related differences in the expression of this prostate protein.

The origins and clinical significance of hyperamylasemia during diabetic ketoacidosis are unclear. We have therefore correlated important clinical and laboratory indices of diabetic ketoacidosis with sequential determinations of serum and urine amylase concentrations, amylase/creatinine clearance ratios, and specific amylase isozyme types. Hyperamylasemia occurred in 79% of our patients with diabetic ketoacidosis, often after admission to the hospital. Among these patients, 48% had pancreatic-type, 36% salivary-type, and 16% mixed-type (pancreatic and salivary) hyperamylasemia. There were no correlations between the presence, degree, or isozyme type of hyperamylasemia and most laboratory or clinical characteristics, including gastrointestinal symptoms. Patients with pancreatic-type hyperamylasemia tended to have higher amylase/creatinine clearance ratios, but it was not possible to unequivocably diagnose acute pancreatitis during diabetic ketoacidosis with current routine clinical or laboratory procedures.

We report the identification and partial sequence for a human salivary amylase gene, Amy1. The genomic sequence was compared with known human pancreatic and salivary amylase cDNA sequences and another salivary amylase gene sequence. While most of the intron/exon structure and coding sequences are highly similar to other amylase DNAs, the bulk of the 5' end varies significantly. Major differences in the positions and structures of promoters between human and mouse Amy1 genes suggest that there are important differences between the tissue-specific expressions in the two mammals. Some potential enhancer sequences were identified.

We report on 2 patients who became deeply comatose after transurethral resection of the prostate. Both patients were severely hyponatremic and hyperammonemic but the course of the comas followed serum ammonia concentrations more closely than serum sodium concentrations. The genitourinary irrigant used in both procedures was a 1.5 per cent glycine solution. Serum amino acid analyses in 1 patient suggested that the postoperative hyperammonemia was due to catabolism of glycine absorbed during surgery. The inadequate activation of normal pathways of ammonia metabolism in this patient may have been caused by a partial deficiency of the urea cycle enzyme argininosuccinate synthetase. We believe that hyperammonemia should be considered as a cause of encephalopathy after transurethral resection of the prostate. The 1.5 per cent glycine genitourinary irrigating solution may not be as nontoxic as generally believed.

A patient with an amylase-producing serous cystadenocarcinoma of the ovary had elevated serum and urine amylase levels and high levels of amylase in pleural and ascitic fluids. Serum and urine amylase levels reflected both surgical removal of tumor mass and response to chemotherapy. Tumor homogenates had pronounced amylase activity. Salivary type amylase isozyme patterns were found in electrophoresis of samples from all sources. Ascites tumor cells were successfully cultured and salivary type amylase was found in the culture media throughout 5 passages. The tumor was classified by light microscopy as poorly differentiated serous cystadenocarcinoma. Ultrastructural studies on the tumor were consistent with that diagnosis. Amylase was detected in the cells of the tumor examined by the immunoperoxidase technique.

The age-dependent development of alpha-amylase expression in utero and during the first two years of life is reported. Separation of salivary and pancreatic amylase isozymes in a discontinuous buffered sheet polyacrylamide electrophoretic system, with subsequent densitometry, provides a reliable semiquantitative method of estimating the proportions of salivary and pancreatic amylases in urine and amniotic fluid samples. In the newborn the predominant amylase isozymes seen in the urine are of salivary origin. As the child ages the level of amylase in the urine rises and an increase in the proportion of pancreatic amylase isozymes occurs. Amniotic fluids of late first and early second trimester pregnancies contain salivary isozymes. None of the amniotic fluid samples examined has pancreatic amylase isozymes. These data reflect a differential development of the expression of the two amylase approaches adult levels by 16 months of age. Conversely, the salivary (Amy1) locus is expressed as early as 18 weeks of gestation and remains relatively constant with but a small increase in salivary amylase (units/ml) activity during early development, as the total amylase activity approaches adult values.