Flagellar motility requires the cAMP-dependent phosphorylation of a heat-stable NP-40-soluble 56 kd protein, axokinin

Using NP-40-treated dog sperm as a model, the stimulatory effect of cAMP upon reactivated flagellar motility has been shown to be dependent upon the cAMP-dependent phosphorylation of a heat-stable NP-40-soluble protein of 56 kd. Examination by two-dimensional polyacrylamide gel electrophoresis of NP-40 extract proteins phosphorylated with gamma-32P-ATP revealed a major cAMP-dependent phosphopeptide at 56 kd. This is the only cAMP-dependent phosphoprotein common to NP-40 extracts of all tissues that show cAMP-dependent stimulation of flagellar motility. These cells and tissues include sea urchin, dog, and human sperm, as well as dog trachea and retina. Moreover, this phosphoprotein is absent in nonstimulatory extracts from tissues such as skeletal muscle, brain, and liver. We conclude that the cAMP-dependent phosphorylation of the 56 kd peptide represents a major regulatory component of not only sperm but other types of axonemal motility as well.     

Calmodulin is involved in regulation of cell proliferation.

A chicken calmodulin (CaM) gene has been expressed in mouse C127 cells using a bovine papilloma virus (BPV)-based vector (BPV-CM). The vector-borne genes produce a mature mRNA of the expected size that is present on cytoplasmic polyribosomes. In clonal cell lines transformed by BPV-CM, expression of the CaM gene produced CaM levels 2- to 4-fold above those observed in cells transformed by BPV alone. Increased intracellular CaM caused a reduction of cell cycle length that is solely due to a reduction in the length of the G1 phase. A comparison of six cell lines revealed a linear relationship between the intracellular CaM concentration and the rate of G1 progression. These data provide the first evidence that specific elevation of CaM levels directly affects the rate of cell proliferation.     

Developmental regulation of calmodulin, actin, and tubulin RNAs during rat testis differentiation

Postnatal testis differentiation involves transition through neonatal, pre-meiotic, meiotic, haploid, and mature stages. We have examined the qualitative and quantitative changes in rat testis RNAs that specifically hybridize to cDNAs encoding the cytoskeletal proteins, calmodulin, beta-actin, alpha- and beta-tubulin at ages corresponding to each of these developmental periods. We compared the species and relative levels of specific RNAs from testes of animals engaged in normal spermatogenesis with RNA from germ cell-depleted, Sertoli cell-enriched (SCE) testis. Distinct developmental patterns of expression of the specific RNAs were found with each of the cDNAs in the two animal models. A 2.2 kb (kilobase) actin RNA and a 2.7 kb beta-tubulin RNA are maximal at 5-10 days of age, suggesting these RNAs are required by somatic and germ cells in the postnatal phase prior to puberty. Between 19 and 29 days, when pachytene spermatocytes appear in significant numbers, there is a slight increase in the 2.2-kb actin RNA, but a 4- to 10-fold increase in RNAs hybridizing to cDNAs for calmodulin, alpha- and beta-tubulin. These changes are much less pronounced in the SCE testis than in the normal testis, indicating increases in these RNAs are related to germinal cell maturation. The germ cell-related increase in 1.8-kb beta-tubulin RNA appears to reflect a developmental "switch" in the gene from which the RNA is derived. This hypothesis is based on the observation that the ratio of hybridization of a chicken brain beta-tubulin cDNA versus a rat spleen beta-tubulin cDNA to the 1.8-kb RNA band increases more than 40-fold between 5 and 29 days of age in normal testis, but is constant in SCE testis. These data suggest that a specific beta-tubulin gene is activated in maturing germ cells. Analogously, a 2.1-kb alpha-tubulin RNA is found only in maturing normal testis and increases as spermatids are produced. A 2.0-kb beta-tubulin RNA, not found in normal testes, is maximal in maturing SCE testes, suggesting this RNA is of somatic cell origin. All of the RNA species studied, except the 2.0-kb beta-tubulin RNA, decrease between 5 and 19 days in SCE testes, as Sertoli cell mitotic activity wanes, indicating that their levels may be regulated by the developmental signals that influence mitosis.     

Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm

Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca2+ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KCl from isolated flagella from dog, pig, and sea urchin sperm. All salt-extractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.     

Molecular cloning sequence and distribution of rat calspermin, a high affinity calmodulin-binding protein

Calspermin is a heat-stable, acidic calmodulin-binding protein predominantly found in mammalian testis. The cDNA representing the rat form of this protein has been cloned from a rat testis lambda gt11 library. Sequence analysis of two overlapping clones revealed a 232-nucleotide 5'-nontranslated region, 510 nucleotides of open reading frame, a 148-nucleotide 3'-untranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of a portion of the deduced amino acid sequence with the sequence of a tryptic peptide obtained from the rat testis protein. The lambda gt11 fusion protein was recognized by affinity purified antibodies to pig testis calspermin and bound 125I-calmodulin in a Ca2+-dependent manner. Calspermin cDNA encodes a 169-residue protein with a calculated Mr of 18,735. The putative calmodulin-binding domain is very close to the amino terminus of the protein. This region shows 46% identity with the calmodulin-binding region of rat brain Ca2+/calmodulin-dependent protein kinase II and 32% identity with the equivalent region of chicken smooth muscle myosin light chain kinase. The 5'-nontranslated region reveals significant homology with a portion of the catalytic region of the calmodulin-dependent protein kinase family. Calspermin contains a stretch of 17 contiguous glutamic acid residues in the central region of the molecule. Computer analysis predicts calspermin to be 81% alpha-helix and 14% random coil. Analysis of genomic DNA indicates calspermin to be the product of a unique gene. Northern blot analysis of rat testis RNA reveals a 1.1-kilobase mRNA. This RNA is restricted to testis among several rat tissues examined and could not be identified in total RNA isolated from testes of other mammals. Analysis of cells isolated from rat testis reveals calspermin mRNA to be predominantly expressed in postmeiotic cells indicating that it may be specific to haploid cells.     

Conserved sequences in enzymes of the UDP-GlcNAc/MurNAc family are essential in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase

The UDP-GlcNAc/MurNAc family of eukaryotic and prokaryotic enzymes use UDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-P as acceptors, and generate sugar-P-P-polyisoprenols. A series of six conserved sequences, designated A through F and ranging from 5 to 13 amino acid residues, has been identified in this family. To determine whether these conserved sequences are required for enzyme function, various mutations were examined in hamster UDP- GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT). Scramble mutations of sequences B-F, generated by scrambling the residues within each sequence, demonstrated that each is important in GPT. While E and F scrambles appeared to prevent stable expression of GPT, scrambling of B- D resulted in GPT mutants that could be stably expressed and bound tunicamycin, but lacked enzymatic activity. Further, the C and D scramble mutants had an unexpected sorting defect. Replacement of sequences B-F with prokaryotic counterparts from either the B.subtilis mraY or E.coli rfe genes also affected GPT by preventing expression of the mutant protein (B, F) or inhibiting its enzymatic activity (C-E). For the C-E replacements, no acquisition of acceptor activity for polyprenol-P, the fully unsaturated natural bacterial acceptor, was detected. These studies show that the conserved sequences of the UDP- GlcNAc/MurNAc family are important, and that the eukaryotic and prokaryotic counterparts are not freely interchangeable. Since several mutants were efficiently expressed and bound tunicamycin, yet lacked enzymatic activity, the data are consistent with these sequences having a direct role in product formation.      

Competition between hydrocarbon and barbiturate for spectral binding to hepatic cytochrome P-450. Inferences concerning spin state of the enzyme

The substrates hexobarbital and ethylbenzene have been shown to compete for the spectral binding site of phenobarbital-induced rat hepatic microsomal cytochrome p-450. The two substrates produce different delta Absmax values, and the presence of one substrate does not affect the delta Absmax of the other substrate and vice versa. The respective binding constants for the two substrates are similarly unaffected. The conclusion drawn from these observations is that, over the concentration ranges studied, there is no change in the availability of the enzyme as a result of substrate addition; the difference in delta Absmax apparently being due to varying abilities of different substrates to bring about a spin shift in the enzyme. Evidence is presented to indicate that differences between enzymes from untreated male rats and phenobarbital-treated male rats are attributable to differences in the enzyme itself and not to changes in the nature of the membrane brought about by phenobarbital administration, at least insofar as heat entropy compensation is concerned. The enthalpy-entropy compensation observed in the binding of a homologous series of barbiturates to the microsomal membrane as determined from the membrane concentration dependence of their binding constants is shown to agree surprisingly well with the direct determination performed by Sitar and Mannering.     

Calmodulin is required for cell-cycle progression during G1 and mitosis.

In order to examine the consequences of a transient increase or decrease in intracellular calmodulin (CaM) levels, two bovine-papilloma-virus (BPV)-based expression vectors capable of inducibly synthesizing CaM sense (BPV-MCM) or anti-sense (BPV-CaMAS) RNA have been constructed and used to stably transform mouse C127 cells. Upon addition of Zn2+, cells containing the BPV-MCM vector have transiently increased CaM mRNA and protein levels. Cells carrying the BPV-CaMAS vector transiently produce CaM anti-sense RNA resulting in a significant decrease in intracellular CaM concentration. Increased CaM caused a transient acceleration of proliferation, while the anti-sense RNA induced decrease in CaM caused a transient cell cycle arrest. Flow cytometric analysis showed that progression through G1 and mitosis was affected by changes in CaM levels. These data indicate that CaM levels may limit the rate of cell-cycle progression under normal conditions of growth.